Authors Accepted Manuscript

Development of nitrocellulose membrane filters

impregnated with different biosynthesized silver
nanoparticles applied to water purification
Jorge G. Fernndez, Csar A. Almeida, Martn A.
Fernndez-Baldo, Emiliano Felici, Julio Raba,
Mara I. Sanz
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S0039-9140(15)30280-0
http://dx.doi.org/10.1016/j.talanta.2015.08.060
TAL15920

To appear in: Talanta

Received date: 1 June 2015
Revised date: 25 August 2015
Accepted date: 26 August 2015
Cite this article as: Jorge G. Fernndez, Csar A. Almeida, Martn A. FernndezBaldo, Emiliano Felici, Julio Raba and Mara I. Sanz, Development of
nitrocellulose membrane filters impregnated with different biosynthesized silver
nanoparticles
applied
to
water
purification, Talanta,
http://dx.doi.org/10.1016/j.talanta.2015.08.060
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Development of nitrocellulose membrane filters impregnated with different

biosynthesized silver nanoparticles applied to water purification

Jorge G. Fernndez, Csar A. Almeida, Martn A. Fernndez-Baldo, Emiliano Felici, Julio

Raba, Mara I. Sanz*

INQUISAL, Departamento de Qumica. Universidad Nacional de San Luis, CONICET.

Chacabuco 917. D5700BWS. San Luis, Argentina.

*Author to whom correspondence should be addressed: (e-mail) [email protected] (Tel)

+54-2652-425385; (Fax) +54-2652-43-0224. INQUISAL, Departamento de Qumica.
Universidad Nacional de San Luis, CONICET. Chacabuco 917. D5700BWS. San Luis,
Argentina.

ABSTRACT

Bactericidal water filters were developed. For this purpose, nitrocellulose membrane filters
were impregnated with different biosynthesized silver nanoparticles. Silver nanoparticles
(AgNPs) from Aspergillus niger (AgNPs-Asp), Cryptococcus laurentii (AgNPs-Cry) and
Rhodotorula glutinis (AgNPs-Rho) were used for impregnating nitrocellulose filters. The
bactericidal properties of these nanoparticles against Escherichia coli, Enterococcus
faecalis and Pseudomona aeruginosa were successfully demonstrated. The higher
antimicrobial effect was observed for AgNPs-Rho. This fact would be related not only to
the smallest particles, but also to polysaccharides groups that surrounding these particles.
Moreover, in this study, complete inhibition of bacterial growth was observed on
nitrocellulose membrane filters impregnated with 1 mg L-1 of biosynthesized AgNPs. This
concentration was able to reduce the bacteria colony count by over 5 orders of magnitude,
doing suitable for a water purification device.

Keywords: Silver nanoparticles; Nitrocellulose membrane filter; Water purification;

Bactericidal filters

1. Introduction

Globally, eighty percent of gastrointestinal infectious and parasitic diseases are due
to the use of non-potable water. Poor hygiene and lack or malfunctions of health services
are some of the reasons for which diarrhea remains a major health problem in developing
countries. Contaminated water and food are regarded as the main vehicles involved in the
transmission of bacteria, viruses or parasites. Most pathogenic microorganisms contained in
the water are removed in the early stages of treatment for water purification. However,
water disinfection is necessary as one of the last steps to prevent drinking water is harmful
to our health. In special situations, silver salts are used to maintain the bacteriological
quality of drinking-water [1]. However, high levels of silver content in water could cause
possible human health effects [2]; so it must be taken with special care.

In the last years, the biosynthesis of metallic nanoparticles has been proposed as an
environmental friendly alternative to chemical and physical methods [3]. These last
methods, usually, employ hazardous and toxic chemicals such as reducing agents, organic
solvents, or non-biodegradable stabilizing agents which are potentially dangerous to the
environment and biological systems [4].

Recently, microbiological methods for synthesis of metallic nanoparticles have

attracted an ever-growing research interest [5-9]. These biological methods are a green
chemistry approach that interconnects nanotechnology and microbial biotechnology. On the
other hand, among the metallic nanoparticles, silver nanoparticles (AgNPs) have received
much attention in various fields, such as antimicrobial activity [10], therapeutics [11], biomolecular detection [12], silver nanocoated medical devices [13] and optical receptor [14].

Hence, biogenic approach, in particular the usage of microorganisms has offered a reliable,
simple, nontoxic and environmental friendly method [15-17].

According to the World Health Organization [18], many people worldwide do not
have access to clean and potable water sources. The greatest water borne threat to human
health is bacterial contamination of drinking water sources, leading to outbreaks of several
diseases [19]. Recently, considerable interest has arisen in the use of nanotechnology for
water purification [20]. In particular, nanomaterials can be used for small-scale or point-ofuse systems for water systems [2, 21] that are not connected to a central network and for
emergency response following disasters. Such systems should be cheap, highly portable,
non toxic, easy to use and distribute and require low energy input.

In the present study, we have incorporated different kind of biosynthesized AgNPs

obtained from Aspergillus niger, Cryptococcus laurentii and Rhodotorula glutinis into
nitrocellulose membrane filter. The antibacterial effectiveness of developed filters was
evaluated in contaminated water samples with Escherichia coli, Enterococcus faecalis and
Pseudomona aeruginosa. To our best knowledge, until now, has not been found
bibliography to report the use of biosynthesized AgNPs incorporated to nitrocellulose
membrane filter used to purify drinking water.

2. Materials and methods

2.1. Silver nanoparticles used and their characteristics

In the present work were used commercial AgNPs (Sigma Chemical, St. Louis, MO,
USA) obtained by chemical synthesis and different extracellular biosynthesized AgNPs

obtained from A. niger (NRRL 1419), C. laurentii (BNM 0525) and R. glutinis (BNM
0524). The nanoparticles were synthesized and characterized in Industrial Microbiology
Laboratory of Universidad Nacional de San Luis (Data unpublished). Characteristics are
shown in Table 1.

Table 1

2.2. Microorganisms and culture

Water borne pathogens such as E. coli (ATCC 8739), P. aeruginosa (ATCC 9027)
and E. faecalis (ATCC 29212) were used as testing organisms. E. coli measures
approximately 0.5 m in width by 2 m in length, while E. faecalis measures 0.6 m in
width by 2 m in length and P. aeruginosa measures 0.5 m in width by 1.5 m in length.
To prepare bacterial culture, bacteria were added to 100 mL of sterilized nutrient broth and
agitated on a shaker (120 rpm) at 37 C during 24 h. The bacteria were separated from the
nutrient broth by centrifuging for 10 min at 2800 rpm and then suspended in sterilized tap
water and diluted to approximately 105 colony-forming units (CFU) mL-1.

2.3. Impregnation of nitrocellulose membrane filters with AgNPs

The characteristics of nitrocellulose membrane filters employed (Sartorius Stedim

Biotech) were: pore size 0.45 m, thickness 130 m, bubble point 2.4 bar and diameter 47
mm. Prior to application, AgNPs were diluted with deionized water to achieve the

appropriate concentrations. Solutions of the following concentrations were prepared: 1 mg

L-1, 0.5 mg L-1 and 0.1 mg L-1. The nitrocellulose membrane filters were sprayed with 250
L of the different solutions of AgNPs. Then, the filters were dry at 60 C for 30 min and
cooled at room temperature by 4 hour (Figure 1). The filters were characterized by
scanning electron microscopy and energy dispersive X-ray spectroscopy (SEMEDX)
using a LEO 1450 VP. This instrument equipped with an energy dispersive X-ray
microanalyzer (EDAX Genesis 2000) and a Si(Li) detector allowed the analytical electron
microscopy measurements. The samples were sputter coated with gold.

Figure 1

2.4. Antimicrobial activity of nitrocellulose filter impregnated with Ag NPs

The antimicrobial activity of the nitrocellulose membrane filters impregnated with

AgNPs was measured by membrane filter method using APHA procedures [22]. Different
solution with microorganisms were filtered through nitrocellulose membrane filter with the
different AgNPs and subsequently incubated at 37 C for 48 h. Three replicates were
performed for each sample. The retention capacity of the filter was also analyzed in the
filtered liquid using the plate count method [22]. The culture of E. coli, E. faecalis and P.
aeruginosa were performed in Chromocult Coliform Agar (Merck KGaA), Azide Agar
(Merck KGaA) and Pseudomonas Agar P (Britania) respectively. In addition, control
growth of different microorganisms was performed using filters without AgNPs.

2.5. Silver content in effluent

The silver loss from nitrocellulose membrane filter impregnated with AgNPs was
determinate in the filtrate by ICP-OES [BAIRD (Bedford, MA, USA) ICP 2070]. The 1 mCzerny Turner monochromator had a holographic grating with 1800 grooves mm -1. The
328.068 silver wavelength was used and systems were expressed as peak height, which was
corrected against the reagents blank.

3. Results and Discussion

3.1. Characterization on nitrocellulose paper impregnated with Ag NPs

Nitrocellulose filter impregnated with different AgNPs were characterized. The

aggregation of AgNPs in the nitrocellulose paper was studied by SEM. Figure 2 shows
SEM characterization of the nitrocellulose filters with different AgNPs and water (negative
control). There, it can be seen that the adhesion of the chemical nanoparticles (Sigma
Chemical, St. Louis, MO, USA) in filter fibers was less successful than the nanoparticles
biosynthesized. Compounds surrounding the biosynthesized nanoparticles would be
responsible of this phenomenon. Functional groups would interact with functional groups
of nitrocellulose. It is possible that an interaction would occur between the carbonyl groups
of proteins coated to the biosynthesized AgNPs and the nitrate groups of nitrocellulose
filters (Figure 1). The macromolecules bind to nitrocellulose is not well understood, but
both electrostatic and hydrophobic interactions have been suggested as possible binding
mechanisms. According to van Oss et al. [23], the mechanism of binding of

macromolecules to nitrocellulose by hydrophobic interaction is demonstrated by a

minimum contact area of 100 and minimum value of free energy of adhesion (G) of 1.5 kT being required for stable fixation to the matrix. An example of this is SDS-protein
complexes, which are negatively charged, bind to negatively charged nitrocellulose
membranes [24]. Additional support for the hypothesis that hydrophobic interactions play a
dominant role in the binding of proteins to nitrocellulose membranes comes from a study
that compared the binding of antibodies and other proteins to nitrocellulose membranes in
acidic, neutral and basic buffers [25].

In Figure 3, the different deposited AgNPs on the nitrocellulose filters was also
evidenced using EDS (energy dispersive spectrometry). The presences of Ag peaks were
observed in greater proportion in the nitrocellulose filters with biosynthesized AgNPs
(Figure 3b, 3c and 3d) than commercial AgNPs (Figure 3a). These results confirmed
micrographs obtained by SEM.

In order to analyze the dispersion of the nanoparticles on the nitrocellulose filters,

an Ag mapping was performed (Figure 4). As can be observed, the higher silver loading
retained is observed in Figure 4b, 4c and 4d; in agreement with that seen in Figures 2 and 3.

Figure 2

Figure 3

Figure 4

3.2. Antibacterial effect of AgNPs in nitrocellulose filter

E. faecalis and E. coli are considered to be indicator organisms of fecal

contamination. Moreover, in costal marine waters, enterococci are the preferred fecal
indicator [26]. On other hand, P. aeruginosa is an opportunistic pathogen and is capable of
growing in water and colonizing various surfaces including water pipes [27].Thus, these
bacteria were selected to evaluate the effectiveness of the system.

After filtration, the filtered liquids were analyzed by plate count method [28]. In all
cases the bacterial growth was negative, since the bacteria were retained on the filter also in
the case of nitrocellulose filter without nanoparticles. But the control nitrocellulose
membrane filter (without silver particles) did not show any inhibitory activity against the
pathogen bacteria when the count of pathogens was performed with the retained by the
filters. Table 2 show the degree of inhibition of filters impregnated with the different
AgNPs. The bacterial growth is represented with the plus sign (+), and growth inhibition is
shown with a minus sign (-). It is noted that the inhibition of E. coli, P. aeruginosa and E.
faecalis was depended on the concentration and the origin of AgNPs.

Filters with nanoparticles from R. glutinis and C. laurentii were more effectives
than filters with nanoparticles from A. niger or chemicals (Sigma Chemical, St. Louis, MO,
USA), against all bacteria. But, filters with nanopaticles from A. niger were more effectives
against P. aeruginosa, than filters with chemical nanoparticles.

The increased antimicrobial activity by biosynthesized AgNPs incorporated to

filters, could be given by the polysaccharides arabinogalactan, arabinan and
galactoglucomannan confirmed by Fourier transform infrared spectroscopy (FTIR) analysis
(data not shown). These polysaccharides could contribute to the enhancement of the
antibacterial activity of the biosynthesized AgNPs due to its ability to anchor the microbial
cell wall and then penetrate it, thereby causing structural changes that affect the
permeability of the cell membrane and cause cell death [29, 30]. In addition, the size and
surface area of the nanoparticles are closely related since with decreasing size increases the
surface area of biosynthesized AgNPs leaving a greater number of atoms exposed on the
surface, which will be available interactions with cells [31, 32]. For example, the smaller
particle size of AgNP-Rho (see Table 1) contributes to greater bactericidal activity.
Moreover, this also justifies the best antimicrobial activity of AgNP-Asp regarding AgNPCom. Although both have similar particle sizes, the polymers associated with the
biosynthesized AgNPs allowed a better bactericidal activity.

According to US EPA standards, for a water purification device to meet, it must

reduce the bacteria colony count by over 5 orders of magnitude [33-37]. In this context,
biosynthesized AgNPs inhibited bacterial growth at level of concentration equal 1 mg L-1.
Moreover, AgNPs-Rho obtained the same results with a concentration of 0.5 mg L-1.

It is necessary to emphasize that the tested AgNPs have bactericidal effects resulting
not only in inhibition of bacterial growth but also in killing bacteria. This irreversible
inhibition of bacterial growth is desirable to prevent bacterial colonization of drinking
water sources. In addition, all the impregnated filters preparations were perfectly stable for
at least 8 month.

Table 2

3.3. Leaching of silver ion released from AgNPs

The content of silver was analyzed in the effluent water by ICP-OES. The
maximum average concentration of Ag+ leached from Ag NPs in the liquid filtrate were
16.10-5 mg L-1, 2 .10-5 mg L-1, 7.10-5 mg L-1 and 1.10-5 mg L-1 for AgNPs-Com, AgNPsAsp, AgNPs-Cry and AgNPs-Rho respectively. According to World Health Organization
(WHO) [1], higher levels of silver, up to 0.1 mg L-1 (a concentration that gives a total dose
over 70 years of half the human with no observed adverse effect level of 10 g), could then
be tolerated without risk to health. In all case; levels of silver were below the limit
recommended by WHO in relation to human health.
Furthermore, the average concentration of Ag+ in the liquid filtrate of commercial
AgNPs was an order of magnitude higher than biosynthesized AgNPs. Probably the major
adherence to the biosynthesized AgNPs could be due to the protein and carbohydrate
groups, encouraging a greater interaction with nitrocellulose membrane filter.

3.4. Stability of AgNPs and porosity of the nitrocellulose membrane filter

In order to establishing the stability of filters impregnated with AgNPs, a tap water
solution was prepared with E. feacalis at a concentration of 105 CFU mL-1. Different levels
of the solution (0.5, 1, 3, 5 and 7 L) were filtered at a flow of 25 mL min-1. The filters used

were impregnated with AgNPs-Asp. Subsequently, the filters were incubated for 48 hours.
No growth was observed on any of the filters. These results showed a significant stability
of impregnated filters, making them cheap and reliable for purifying water.

Another test was conducted to evaluate if AgNPs will affect the porosity of the
nitrocellulose membrane filter. In this context, the different membrane filters were analyzed
by bubble point method, to detect the largest pores in a membrane filter. The analysis
was performed using Bubble Point Tester "it-BP-HP (measure range 200-7500 mbar),
wetted with water. According to the results observed in Table 3, the pores sizes were
slightly reduced by applying of different nanoparticles, especially by biosynthesized
nanoparticles. However, using ANOVA test, there were no significant differences (p <0.05)
between the groups with respect to commercial nitrocellulose membrane filter without
nanocomposites.

Table 3

4. Conclusions

In general, biosynthesized AgNPs had a higher antimicrobial activity than

commercial AgNPs. The coated of polysaccharides on the surface of the biosynthesized
AgNPs contributed to a better antimicrobial activity. The content of silver particles in the
effluents water was below the limit recommended by WHO. The higher antimicrobial
effect was observed for AgNPs- Rho, which is related not only to the smallest particles, but
also to polysaccharides groups that surrounding the particles. Moreover, in our study,

complete inhibition of bacterial growth was observed on nitrocellulose membrane filters

impregnated with 1 mg L-1 of biosynthesized AgNPs. This concentration was able to reduce
the bacteria colony count by over 5 orders of magnitude, doing suitable for a water
purification device.

Acknowledgements

The authors would like to thank Universidad Nacional de San Luis, the Agencia
Nacional de Promocin Cientfica y Tecnolgica, and the Consejo Nacional de
Investigaciones Cientficas y Tcnicas (CONICET) for providing the financial means and
laboratory facilities.

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Highlights
A new method including nanotechnologies was used for water treatment.
An impregnated filter with commercial and biosynthesized AgNPs was used.
Antibacterial effectiveness was evaluated in water samples according to WHO.
Biosynthesized AgNPs reduced the bacteria colony count.
Antimicrobial activity could be given by polysaccharides over silver nanoparticles.

Rhodotorula
glutinis

Aspergillus
niger

Criptococcus
laurentii

*Graphical Abstract (for review)

AgNPs

Table 1

Table 1. Principal characteristics of AgNPs used

Origin

Size
(nm)

Chemical

40 4

Aspergillus niger

40 5

High concentration of proteins and low

concentration of carbohydrates

Cryptococcus
laurentii

35 10

High concentration of carbohydrates type

arabinogalactan. Low concentration of proteins.

Rhodotorula
glutinis

15 8

High concentration of carbohydrates type

arabinan and galactoglucomannan

Compounds surrounding nanoparticles

None

Table 2

Table 2. Bacterial growth exposed to different concentration and type of AgNPs.

Ag NPs-Aspa

Ag NPs-Rhoa

Ag NPs-Crya

Ag NPs-Coma

0.5

0.1

0.5

0.1

0.5

0.1

0.5

0.1

E. coli

E. faecalis

P. aeruginosa

Concentration expressed as mg L-1

Table 3

Table 3. Study of pore size change of the nitrocellulose membrane filters by AgNPs, using
the bubble point method.

Type of membrane filter Bubble point (bar)

Without nanoparticles
2.480.01
+ AgNPs-Com
2.550.01
+ AgNPs-Asp
2.670.03
+ AgNPs-Cry
2.730.02
+ AgNPs-Rho
2.740.01

Figure 1

Figure 1. Scheme for possible interaction mechanism between the carbonyl groups of
proteins coated to the biosynthesized AgNPs with the nitrate groups of nitrocellulose filters.

Figure 2

(a)

(b)

(c)

(d)

(e)

Figure 2. SEM characterization of the nitrocellulose filters with different AgNPs and water
(negative control). (a) AgNP-Com, (b) AgNP-Asp, (c) AgNP-Cry, (d) AgNP-Rho and (e)
negative control.

Figure 3

(a)

(b)

(c)

(d)

(e)

Figure 3. EDS spectra of the nitrocellulose filters with different AgNPs. (a) AgNP-Com,
(b) AgNP-Asp, (c) AgNP-Cry, (d) AgNP-Rho, and (e) a negative control.

Figure 4

(a)

(b)

(c)

(d)

Figure 4. Silver mapping on nitrocellulose filters with different AgNPs. (a) AgNP-Com,
(b) AgNP-Asp, (c) AgNP-Cry and (d) AgNP-Rho. The bright areas correspond to AgNPs.