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DOI 10.1007/s11051-009-9769-9
RESEARCH PAPER
Received: 23 May 2009 / Accepted: 21 September 2009 / Published online: 4 October 2009
Springer Science+Business Media B.V. 2009
Abstract Our experimental results of using the bacteria. Mg(OH)2 nanoparticles could be added
Mg(OH)2 nanoparticles as an antibacterial agent are directly to wood pulp to make paper sheets, whose
reported in this study. The antibacterial behavior of antibacterial efficiency increased with the increase
Mg(OH)2 nanoparticles in liquid culture and in paper of the nanoparticle amount. The possible mechanism
sheets was investigated. The colony forming units of antibacterial effect of Mg(OH)2 nanoparticles is
(CFU) counting and the headspace gas chromatogra- discussed.
phy (HS-GC) measurement were used to determine
the cell viability. Results indicate that Mg(OH)2 Keywords Antibacterial agent
nanoparticles are effective antibacterial agent against Mg(OH) nanoparticles Metal oxide
Escherichia coli (E. coli) and Burkholderia phytofir- E. coli Environment EHS
mans, and the OH- and Mg2? ions in Mg(OH)2 water
suspension were found not to be the reason for killing
Introduction
C. Dong G. He
State Key Laboratory of Fine Chemicals, School Due to the ubiquity of microorganisms and their ability
of Chemical Engineering, Dalian University to establish themselves, the control of microbial
of Technology, Dalian 116012, China populations is a universal concern. Antibacterial
C. Dong Q. Sun Y. Deng agents are thus of relevance to a number of industrial
Institute of Paper Science and Technology, Georgia sectors including those focused on the environment,
Institute of Technology, Atlanta, GA 30332, USA food, synthetic textiles, packaging, healthcare, medical
care, as well as construction, and home and workplace
J. Cairney
School of Biology, Georgia Institute of Technology, furnishing. Antibacterial agents are broadly catego-
Atlanta, GA 30332, USA rized as organic or inorganic. Both the natural and the
synthetic organic antibacterial agents, such as chitosan
O. L. Maddan and its derivatives, quaternary ammonium salts,
Aqua Resources Corporation, 20 Linwood Drive,
Fort Walton Beach, FL 32547, USA biguanide agents, and pyridines inhibit strongly the
growth of a wide variety of bacteria and fungi
Y. Deng (&) (Sudarshan et al. 1992; Liu et al. 2001). However,
School of Chemical and Biomolecular Engineering, organic antibacterial agents have some shortcomings,
Georgia Institute of Technology, 500 10th Street,
N.W., Atlanta, GA 30332, USA such as low heat resistance, high decomposability, and
e-mail: yulin.deng@chbe.gatech.edu short life expectancy, which have limited their
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2102 J Nanopart Res (2010) 12:21012109
application. As a result, inorganic antibacterial agents pathogens, both locally and between continents. Anti-
have received more and more recognition in the bacterial paper capable of preventing bacterial growth
antibacterial product market (Wang et al. 2003; Fang and eliminating bacterial contaminants is attracting
et al. 2006; Jung et al. 2008). more and more attention. Domtar, a Canada based
Among the inorganic antibacterial agents, silver company, has introduced the first antimicrobial office
and copper are traditionally well-known antibacterial paper available in North America. Designed to protect
materials (Feng et al. 2000; Yoshinari et al. 2001; paper against the growth of bacteria, fungus, mold, and
Jeon et al. 2003). Metal oxide materials, such as mildew, antimicrobial office paper from Domtar is
TiO2, ZnO, and MgO, have also been recognized as specially treated with a silver compound that kills most
antibacterial agents (Sawai et al. 2000; Stoimenov bacteria with which it comes in contact. Japanese
et al. 2002; Yu et al. 2003; Zhang et al. 2007). It is companies, such as Nippon Paper, are also developing
considered that the detected active oxygen species photocatalyst enhanced antibacterial papers using
generated by these metal oxide particles could be the TiO2 nanoparticles. In this article, we report antibac-
main mechanism of their antibacterial activity. terial papers made directly by the addition of Mg(OH)2
However, it is not clear that how the active oxygen nanoparticles to wood pulp during a paper making
is produced and how to improve the active oxygen process. A pronounced inhibition of bacterial growth is
production. As a metal hydroxide material, Ca(OH)2 observed for nanoparticles-containing handsheets.
has been widely used in endodontics as an intra-canal
medication. This strongly alkaline substance has a
high antibacterial activity against oral bacterial and Materials and methods
its effects are highly dependent on the availability of
hydroxide ions in solution. However, the metal Equipment
hydroxide particles itself has the antibacterial activity
has always been ignored. The scanning electron microscopy (SEM), zeta
Compared with other metal hydroxide materials, potential measurement, Brunauer, Emmett and Teller
Mg(OH)2 is a strong base but a sparingly soluble (BET) method, and energy dispersive spectroscopy
inorganic particle with a very low solubility product (EDS) were applied to characterize the size, shape,
constant (5.61 9 10-12). As a suspension in water at composition, zeta potential, and specific surface of
room temperature, Mg(OH)2 functions as a buffer the obtained Mg(OH)2 nanoparticles. The SEM
material with pH of *10.4, is much lower than pH of observation was conducted on a LEO 1530 thermally
*12.5 in Ca(OH)2 solution, so it is often used as an assisted field emission scanning electron microscope
antacid to neutralize stomach acid. In addition, due to machine with an acceleration voltage of 5.0 kV.
its non-toxicity and low cost, Mg(OH)2 is an approved Headspace gas chromatograph (HS-GC) with a HP-
drug and food additive which has been widely used in 7694 Automatic Headspace Sampler and Model HP-
flame-retardant composite formulations (Jiao et al. 6890 capillary gas chromatograph (Hewlett-Packard,
2006), wood pulp bleaching, and cultural heritage CA, USA) was used for detecting the CO2 released by
conservation. Its nanoparticles were also found live E. coli from solutions. The detail HS-GC
effective in the deacidification treatment and protec- measurement procedure will be described later.
tion against cellulose aging (Giorgi et al. 2005).
In this article, Mg(OH)2 nanoparticles were used Mg(OH)2 nanoparticles
as an antibacterial agent. The antibacterial effects of
Mg(OH)2 nanoparticles both in water and in paper Mg(OH)2 nanoparticles were synthesized by Aqua
sheet were studied. Resources Corporation (Florida, USA) using an elec-
In addition to its use as a communication medium, trolytic method with a membrane between anode and
paper is used in personal care products, food packages, cathode. The detail procedure for Mg(OH)2 nanopar-
wallpapers, medical records, bills etc. Since these ticles synthesis was given in the US patent application
products pass through many countries and, literally, (Maddan 2008). As shown in Fig. 1a, the Mg(OH)2
through many hands, a concern has arisen that paper nanoparticles are thin platelets with about 10 nm
packaging could act as a vector for the transmission of thickness and 200300 nm plate size. Table 1 shows
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J Nanopart Res (2010) 12:21012109 2103
Materials
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2104 J Nanopart Res (2010) 12:21012109
study, the CO2 released from E. coli growth in sheets with a target basis weight of 100 g m-2 were
solution was measured using an HP-7694 Automatic produced. The Mg(OH)2 content in the paper was
Headspace Sampler and Model HP-6890 capillary determined by ashing the paper in a muffler oven at
gas chromatograph (Hewlett-Packard, CA, USA) 525 C for 4 h. The actual weight of Mg(OH)2 after
using a thermal conductivity detector (TCD). The paper ashing was obtained after correction of the
detailed method description was reported previously weight loss from the self-dehydration of Mg(OH)2
(Chai et al. 2008). Two milliliter LB medium with or during the ashing process.
without Mg(OH)2 nanoparticles was added to a
22 mL headspace sample vial. A total of 200 lL Antibacterial tests of Mg(OH)2 nanoparticles
E. coli culture with an approximately 107 CFU mL-1 filled paper
was then inoculated into the vial. After mixing, each
vial was immediately sealed by a rubber septum and The process for evaluating inhibition of bacterial
then placed in the headspace sampler at a constant growth by Mg(OH)2 nanoparticles filled paper is
temperature, 37 C under a gentle agitation condition illustrated in Fig. 2. Paper sheets with or without
of 300 rpm to allow the bacteria growth. The nanoparticles prepared as described above were cut
headspace of the vial was periodically sampled into coupons (15 mm 9 8 mm). These coupons were
followed by GC measurement. The headspace sam- immersed into the E. coli culture with an approxi-
pling cycle time is 30 min. mately 107 colony forming units per milliliter
(CFU mL-1) once and allowed to dry at room
Preparation of paper sheets with Mg(OH)2 temperature. They were then placed randomly on a
nanoparticles plastic surface and a plastic cover was placed over
them at room temperature (22 C) overnight (16
The bleached softwood kraft pulp was diluted and 18 h). After submerged individually in 1.5 mL tube
various amounts of Mg(OH)2 nanoparticles were with LB media and vortexed vigorously (10 9 5 s),
added during the paper sheets making. Percol-175 all the paper coupons were taken out from the tubes.
was used for particle retention at dosage of 0.1 wt% At this time, the pH value of each culture was \8.
based on solid weight. The slurry was stirred for 20 s Surviving bacteria were harvested by placing these
at 1,000 rpm. After cursory wet pressing twice, paper tubes at a constant temperature, 37 C under a gentle
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J Nanopart Res (2010) 12:21012109 2105
agitation condition of 300 rpm. In each case, 200 lL Effect of OH- and Mg2? in Mg(OH)2 suspension
of the resuspended bacteria solution was plated on LB on E. coli growth
agar plates which were incubated at 37 C overnight.
CFU number was counted to determine the cell As described above, the Mg(OH)2 nanoparticles
viability. All experiments were replicated thrice. could kill E. coli in solution. However, it is unknown
if E. coli bacteria were killed by dissolved OH-,
Mg2?, or the nanoparticles. This was further exam-
Results ined by comparing the antibacterial efficiency of
Mg(OH)2 nanoparticles with solutions of NaOH and
Effect of Mg(OH)2 nanoparticle suspension MgSO4, respectively.
on different bacterial growth In these tests, the LB medium with 10 mg mL-1
(0.17 mol L-1) uniformly dispersed Mg(OH)2 nano-
In order to determine the Mg(OH)2 nanoparticles particles was made, and the pH value was measured to
acted as bacteriostatic agents (preventing growth of be 10.0. In comparison with Mg(OH)2 nanoparticles,
bacteria but not killing them) or bacteriocidal agents in a separated test, aqueous solution without Mg(OH)2
(killing bacteria), the bacteria were grown in the LB nanoparticles, but with NaOH was used to raise the pH
medium with or without Mg(OH)2 nanoparticles. of LB medium to 10.0. A culture of E. coli
Figure 3a shows the results of bacterial growth with (C108 CFU mL-1) was added to the LB medium,
the same amount of initial bacterial culture in the and the changes in the number of live bacteria were
presence or absence of the Mg(OH)2 nanoparticles monitored over time. The results of CFU counting and
after 12 and 40 h growth. In the absence of nanopar- HS-GC measurement are shown in Fig. 4. The top of
ticles, we observe a confluent growth (colonies Fig. 4 show that the contrast of live population of
aggregate together, usually[106 colonies) of bacteria E. coli in these two cultures is remarkable. Bacteria
in growth media after 12 and 40 h. However, in the inoculated into LB medium with Mg(OH)2 nanopar-
presence 10 mg mL-1 Mg(OH)2 (0.17 mol L-1), ticles significantly decreased after 7 h and totally
only few 100 live bacteria remain after 12 h and no killed within 24 h, while they remained high after 24 h
live bacteria were present after 40 h. A soil bacte- growth in the LB/NaOH medium. The CO2-headspace
rium, Burkholderia phytofirmans shows even greater assay (the bottom of Fig. 4) is in good agreement with
sensitivity to the presence of Mg(OH)2 nanoparticles, the above observation. It clearly demonstrates minimal
and there was no bacteria survive after 24 h in the CO2 evolution from E. coli growth in the presence of
presence of 1 mg mL-1 Mg(OH)2 (Fig. 3b). These Mg(OH)2 nanoparticles after 4 h, when the control
results indicate that Mg(OH)2 nanoparticles are culture (LB only) was beginning to multiply, and no
bactericidal, and in this case, they kill bacteria. evidence of any respiration after 10 h, implying that no
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Fig. 4 a Change of
bacteria numbers with time
in different cultures of LB
medium with 0.17 mol L-1
Mg(OH)2 nanoparticles or
LB medium of pH 10.0
adjusted with NaOH
solution. b CO2 released
during E. coli growth in a
glass cell measured with
HS-GC for four different
LB solutions (LB medium;
0.17 mol mL-1 Mg(OH)2
in LB medium solution,
pH = 10.0; NaOH in LB
medium solution,
pH = 10.0; 0.17 mol mL-1
MgSO4 in LB medium
solution)
150
Control
125
CO2 formed (GC singnal)
Mg(OH)2
100 NaOH
75 MgSO4
50
25
-25
0 2 4 6 8 10
Incubation time (h)
live bacteria remained in the vial after 10 h exposure to It is almost identical to the control one, which
the nanoparticles. Compared the curves of LB/NaOH indicates that Mg2? has no effect on bacterial growth.
with the control culture, we can find that the slopes of
the E. coli growth curves in these two cultures vary Preparation of paper sheets with Mg(OH)2
from each other, which means the bacterial in LB/ nanoparticles
NaOH culture grew slower than in the control culture.
Therefore, both the results of CFU counting and HS- The SEM pictures of the wood fibers from paper
GC measurement indicate that the OH- in Mg(OH)2 sheets without and with Mg(OH)2 nanoparticles are
suspension has no effect on E. coli viability. compared in Fig. 5a which shows that the Mg(OH)2
Meanwhile, the 0.17 mol mL-1 MgSO4/LB solu- nanoparticles are distributed uniformly on the surface
tion, which has the same molar concentration of of the wood fibers. Meanwhile, the ashing results of
Mg2? as the LB/Mg(OH)2 suspension, was also the samples also show a good result. The particle
investigated for its antibacterial activity. The CO2 retention ratio, as shown in Fig. 5b, proves that the
evolution curve from E. coli growth is shown in good retention ratio of Mg(OH)2 nanoparticles on
Fig. 4b. fibers (higher than 75%) was achieved. That means it
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J Nanopart Res (2010) 12:21012109 2107
90
85
Retention ratio (%)
80
75
70
65
60
0 1 2 3 4 5 6 7
Mg(OH) 2 nano particles content (%)
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suspension of Mg(OH)2 nanoparticles in water could Mg(OH)2 nanoparticles was measured to have a stable
function as a buffer material with pH of *10.4. In this pH of 10.0. We used NaOH to adjust the LB medium to
study, the LB medium with 10 mg mL-1 dispersed the same pH of 10.0 and did the antibacterial test. As
shown in Fig. 4a and b, although E. coli still grew in
NaOH LB-culture solution at pH of 10, its growth rate
is much slower than neutral LB solution. In contrast, all
E. coli was killed at the same pH, but with Mg(OH)2
nanoparticles suspension. Mendonca et al. (1994)
described that the treatment of E. coli in NaHCO3
NaOH buffer (pH = 10) at 37 C did not damage cell,
growing equally well on both selective and non-
selective media. In this article, our results agreed well
with their results.
Besides the antibacterial effects obtained in LB/
Mg(OH)2 nanoparticles suspension, good antibacte-
rial activity was also achieved when Mg(OH)2
nanoparticles were incorporated into paper as inor-
ganic filler particles. In order to confirm that E. coli
was killed on dry paper rather than in solution, the
E. coli contaminated papers were contacted with
water under vertex agitation for 1 min to transfer
small number of E. coli to water for further testing
12,000 (Fig. 2). It was found that the Mg(OH)2 nanoparticles
10,000
were not directly transferred to water by above
E. colis urvivors
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J Nanopart Res (2010) 12:21012109 2109
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