Materials Science & Engineering C 92 (2018) 663–672

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Materials Science & Engineering C

journal homepage: www.elsevier.com/locate/msec

ZnO-SiO2 nanohybrid decorated sustainable geopolymer retaining anti- T

biodeterioration activity with improved durability
⁎
Manas Sarkara,1, Moumita Maitib,1, Soumen Maitic,d, Shilang Xua, , Qinghua Lia
a
Institute of Advanced Engineering Structures and Materials, College of Civil engineering and Architecture, Zhejiang University, China
b
College of Biosystems Engineering and Food Science, Zhejiang University, China
c
CENIMAT/I3N, Faculdade de Ciências e Tecnologia, FCT, Portugal
d
Present address: St Thomas college of Engineering & Technology, India

A R T I C LE I N FO A B S T R A C T

Keywords: Geopolymer, consists of industrial by-product fly ash, and alkaline activator, possesses similar strength along
ZnO-SiO2 nanohybrid with durability like conventional cement composite, is an alternative construction substantial of Portland cement
Geopolymer in current scenario. Corrosion of the concrete materials resulted mainly from the chemical degradation. Besides
Anti-microbial activity chemical degradation, biogenic-deterioration is also another alarming issue especially in the sewer systems,
Durability
bridge piers, several pipelines and offshore platforms and the need to act on it is long-standing. In this study,
Mechanical and structural behaviour
application of zinc oxide–silica nanohybrid based sustainable geopolymer (GMZnO–Si) has been investigated for
the development of a sustainable, anti-biodeteriorate cementitious material having significant mechanical
strength and durability. Initially, zinc oxide nano-rods (ZnO NRs) have been synthesized and spherical silica
nanoparticles were decorated on the surface of ZnO NRs. The ZnO-SiO2 composite was characterized by various
techniques (FTIR, XRD, FESEM, EDS, TEM, and XPS). Ambient temperature cured GMZnO–Si mortar was further
explored in terms of mechanical strength, durability, mechanistic anti-microbial (E. coli, S. aureus, A. niger)
influences. Mechanical properties of GMZnO–Si are found significantly higher than that of control samples. MIC,
MBC, and MFC results demonstrate enhanced anti-microbial efficacy of GMZnO-Si. Inner permeability assay,
reactive oxygen species generation and microscopic images of cell wall rupture and DNA damage studies sup-
ported the detailed understanding of anti-microbial activities. These experimental findings suggest that in-
corporation of ZnO-SiO2 hybrid in geopolymer will pave the way for biodeterioration resistant concrete with
enhanced mechanical and structural behaviour.

1. Introduction up the time rate and improve the geo-polymerization process, resulting
greater compressive strength [6,7]. To achieve high compressive
In construction industry, Geopolymer have useful significance for strength and durability at low curing temperature numerous protocols
their distinct energy proficiency, lower environmental impact with have been adopted, amongst which usage of various reinforced fibres,
highly desirable structural engineering properties and mechanical steels, metals, nanoparticles, additives, chemicals in geopolymer con-
strengths [1–3]. Additionally, low budget and 75% less greenhouse gas crete are the commonly accessed routes. [8–10].
production as compared to the Portland cement ensue their broader Corrosion, an alarming concern for the ancient or aged geopoly-
usage window [4]. Geopolymer are generally inorganic polymeric, meric structures, is largely attributed to harmful chemicals and mi-
ceramic-like polysialate materials and mainly come from industrial crobial attacks in them. Microbiologically (bacteria/fungi) induced
waste-products such as fly ash, clays, slag granulated blast-furnace and deterioration strictly compromises the structural integrity of any infra-
even agricultural-wastes like rice husks [5]. Along with aforesaid ad- structures and its protection has a great impact in different industries
vantages, geopolymer have also bottlenecks regarding brittleness, set- such as sewer pipes systems, gas pipelines, subsea pipelines, bridge
ting times, curing temperature, and life-times, etc. For fly ash-based piers, storage tanks, and offshore platforms, toilets, hospital-floors walls
geopolymer mortar, the curing temperature and curing time play pi- etc. [11–15]. Such damages to the aforesaid systems entail considerable
votal role as higher temperature and longer curing time help to speed risk and necessitate very expensive repairing. Physical deterioration of

⁎
Corresponding author.
E-mail address: [email protected] (S. Xu).
1
Authors are equally contributed.

https://doi.org/10.1016/j.msec.2018.07.005
Received 29 September 2017; Received in revised form 25 June 2018; Accepted 1 July 2018
Available online 06 July 2018
0928-4931/ © 2018 Published by Elsevier B.V.
M. Sarkar et al. Materials Science & Engineering C 92 (2018) 663–672

old structure is generally resulted from the growth and movement of stirring span the solution was left for aging for 2 h. Further the solution
microbial which further ensue crack or fissures and mechanical was centrifuged with ethanol and D.I. water. Finally, the product was
breaking. Aesthetic deterioration and chemical damages further in- annealed at 450 °C prior to for their various characterizations.
crease porosity and thereby weakens the structures. More than 50% of Fourier transformed infra-red (FTIR) spectroscopy was performed
the failures in underground concrete structures are occurred mainly due from 400 to 4000 cm−1 wavelengths to analyse the bending and
to formation of biofilm and excretion of metabolites agents [16]. Fur- stretching vibrations of synthesized nano structured samples. The
thermore, presence of bacterial/fungal contamination in high volume crystallinity of the as-synthesized sample was characterized by X-ray
over concrete surface for a long period of time makes an adverse impact diffractometer (XRD, D8 Advanced Bruker) with Cu-Kα radiation of
on human health. wavelength 1.5406 °A at 55 kV and 40 mA. The sample was examined at
Scientist and researchers have implemented different techniques 2θ from 10° to 80° and identified by referring to data of Joint
against microbe induced corrosion and used several defensive materials Committee on Powder Diffraction Standards (JCPDS). The surface
like epoxy resins, polyurethane, acrylic, silane, siloxane and polymers morphology and elemental analysis were evaluated using field emission
to the corresponding damage controlling [17,18]. However, usage of scanning electron microscope (FESEM) and energy dispersive spectro-
different anti-microbial agents such as nanomaterials and bactericidal scopy (EDS) respectively. Morphological behaviour was further con-
or fungicidal admixtures in this regard are less cultivated. These ma- firmed by High Resolution Transmission Electron Microscopy (HRTEM;
terials and coatings have huge propitious as they can hinder the growth JEOL, JEM 2100). The X-ray photoelectron spectroscopy (XPS) mea-
of microbes. Although they have not much effect on strength en- surements of ZnO NRs and ZnO-SiO2 hybrid samples were carried out
hancement. using AXIS Supra (Kratos) instrument with a monochromatic Mg-Kα X-
In this context, present exertion is designed to develop ZnO-silica ray source.
hybrid based geopolymer. Initially effort has been given to synthesize
rod shaped ZnO nano-rods (NRs) with silica NPs attachment on their 2.3. Preparation of mortar mixtures (GMSi and GM ZnO–Si)
surfaces. The ZnO-SiO2 nanohybrids have been characterized by FTIR,
XRD, XPS, FESEM equipped with EDS and TEM techniques. Usage The geopolymeric mortar samples (GMSi, GMZnO–Si) were prepared
perspective of ZnO-SiO2 nanohybrid based geopolymer have examined according to the following manners:
by observing durability changes with respect to control samples and
sustainability with anti-microbial activities against the bacterial/fungal (12 M) NaOH + Na2SiO3 + SiO2 NPs + Fly Ash + Sand = GMSi
strains (E. coli, S. aureus and A. niger). Durability of the as synthesized
samples was accessed via rapid chloride ion penetration test, sulphate (12 M) NaOH + Na2SiO3 + ZnO − SiO2 nanohybrid + Fly Ash + Sand
resistance test, water absorption tests. To explore the mechanistic ap- = GMZnO–Si
proach of the synthesized cementitious materials for anti-microbial
effect, microbial growth kinetics, MIC, MBC, MFC, inner permeabili- NaOH and Na2SiO3 were mixed at gyrometric ratio 1:2. The SiO2
zation test, reactive oxygen species (ROS) detection, fluorescence mi- NPs and ZnO-SiO2 nano hybrids were mixed with this solution dis-
croscopic analysis, morphological investigation, DNA damage study tinctly to make activators (ASi and AZnO-Si) for GMSi and GMZnO–Si
have been conducted also. mortar samples respectively. The activator to fly ash ratio was fixed at
0.75 and the fly ash, sand ratio was taken 1:3. The amount of SiO2 NPs
2. Materials and methods and ZnO-SiO2 nanohybrid in their respective activator solutions (ASi or
AZnO-Si) was 6% (w/w) of fly ash used. The conventional mortar (CM)
2.1. Ingredients samples were prepared by using Ordinary Portland Cement (OPC),
sand, water only. The water to cement ratio was affixed at 0.45 and the
Low calcium class F dry fly ash, locally available sand (specific cement to sand ratio was taken 1:3. The samples were removed from
gravity 2.52, water absorption 0.50%, and fineness modulus of 2.38), the mould after 24 h, kept in ambient temperature and tested after
alkali activator fluid (mixture of sodium hydroxide, sodium silicate and different days (3, 7 and 28 days) of air curing.
deionized water) have been used as basic ingredients of geopolymer
mortar. For control cement mortar, Ordinary Portland Cement (43 2.4. Mechanical strengths and durability experiments
grade OPC) and deionized water (D.I.) have been used. Nutrient Broth
(NB) media and Sabouraud Dextrose (SD) media ingredients like pep- The standard mortar specimens of dimension
tone, beef extract, yeast extract, NaCl, agar (Hi-media Pvt. Ltd., India), (70.6 mm × 70.6 mm × 70.6 mm) were prepared to determine the
zinc acetate (Merck Germany), carbonic acid, E. coli (MTCC 1652 compressive strengths after 3, 7 and 28 days by using 1000 kN Instron
strain), S. aureus (MTCC 96 strain) bacteria and A. niger (MTCC 1344) machine [19]. The mortar bars (0.5 cm × 0.5 cm × 20 cm) and cy-
fungi have been used. Zinc nitrate (Zn(NO3)2, 6H2O), hexamine lindrical specimens (100 mm diameter and 200 mm height) were con-
[(CH2)6N4], tetraethyl orthosilicate (TEOS), colloidal nano silica and ducted for the flexural strength and split tensile strength respectively
ammonia are of analytical grade and used without further purification [20,21]. The bond test specimens consist of mortar cubes
were used. (150 mm × 150 mm × 150 mm) with a single reinforcing bar (de-
formed and mild) embedded vertically along a central axis in each
2.2. Synthesis and characterization of zinc oxide-silica nanohybrid specimen. In this experiment, deformed (Fe-500) and plane (Fe-250)
bar were used for each category of concrete specimens [22].
In a typical procedure, equimolar Zn(NO3)2, 6H2O and (CH2)6N4 The ultrasonic pulse velocity through the CM, GMSi and GMZnO-Si
were dissolved in 80 ml D.I. water to form a 0.04 M solution. Thereafter samples were measured according to ASTM C597-02 [23]. Rapid
the solution was kept in a Pyrex bottle and heated in a regular la- chloride ion penetration test (RCPT) was conducted for the durability
boratory oven at 120 °C for 6 h. Finally, the bottle was naturally cooled assessment of cylindrical specimens (10 cm diameter × 5 cm height) of
down and the product was collected by filtering and washing with each category mortar samples. The prepared samples were subjected to
copious amount of D.I. water. For the synthesis of ZnO-silica hybrid, RCPT by impressing 60 V after 28 days of curing as per ASTM C1202
5 ml TEOS was dissolved in 40 ml ethanol via sonication followed by [24]. Water absorption capacity of standard mortar cubes (dimension:
1 ml D.I. water and 2 ml ammonia solution (25%) were dropped into 50 mm × 50 mm × 50 mm) were determined as per modified Neville's
the solution in stirring condition one after another. Thereafter the pre- method and the sulphate resistivity test was performed as per the gui-
synthesized ZnO was added to solution and stirred for 5 h. After the dance of ASTM C 1012 [25,26].

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Fig. 1. Characterization of synthesized nanohybrid by (A) FTIR; (B) XRD; (C) FESEM of ZnO NR; (D) ZnO-SiO2 nanohybrid; (E) EDS; (F) HRTEM.

2.5. Anti-biogenic deterioration (anti-microbial) study λ = 620 nm. Time dependent killing was also determined by plating
the culture from the treated and control samples in agar plate (1.5%)
Mortar samples from each category (GMSi, GMZnO–Si and CM) were after different period of incubation (0, 2, 4, 6, 8, 12, 24 h) at 37 °C. The
initially immersed in 1 N carbonic acid solutions to reduce the pH numbers of bacterial colonies were tallied after 24 h. In case of growth
(< 8.0) and further used for anti-microbial study. kinetics of fungal strain, well-grown A. niger in Sabouraud Dextrose
broth/Agar (SDB/SDA) medium (10% Mycological peptone, 40%
Dextrose, 15% Agar, pH 6) at 37 °C was treated with GMSi and GMZnO–Si
2.5.1. Microbial growth kinetics study powder samples. The growth kinetic was investigated by measuring OD
Growth kinetics of microbes (S. aureus, E. coli and A. niger) in pre- of SDB at λ = 595 nm at different time intervals. This wavelength is
sence of CM, GMSi and GMZnO–Si were investigated distinctly. The well- usually preferred since the absorbance is only owing to the turbidity
grown fresh culture of each bacterial strains were taken separately and deviation consequently of the variance in the number of existing cells.
washed with PBS buffer. The bacterial cells were diluted in nutrient The growth of inhibition was calculated by the zone of diameter (in
broth (NB: 0.1% beef extract, 0.5% NaCl, 0.2% yeast extract, 0.5% SDA) according to the following formula:
peptone at pH 7.0) to make final cell concentration of 104 CFU ml−1.
About 0.2 mg ml−1 (2 × MIC) of each powder samples (CM, GMSi and Percentage inhibition =
(C − T) × 100
GMZnO–Si) were added to each inoculated broth distinctly. The broth C (1)
samples were incubated at 37 °C. The untreated cell cultures were
considered as control for each strain. The kinetics of each strain in the where, C = colony diameter (mm) of the control (GMSi); T = colony
liquid cultures were estimated by optical density (OD) measurements at diameter (mm) of the test sample (GMZnO–Si).

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2.5.2. Determination of MIC, MBC and MFC test

The Minimum Inhibitory Concentration (MIC) was detected by the
different concentration of geopolymer specimens via using the batch
culture process. The mortar powders (GMSi, GMZnO–Si and CM) at dif-
ferent concentration (0.1% to 5.0% w/v) were added to the bacterial/
fungal growth medium (cell concentration of 107 CFU ml−1) separately
and inoculated at 37 °C on a rotary shaker. The growth inhibitions of
bacterial and fungal strains were measured at λ = 620 nm and
λ = 595 nm by Shimadzu UV–vis-NIR (UV-3101-PC spectro-
photometer) respectively. About 99% mortality for gm (+ve) and gm
(−ve) bacterial and fungal strain were determined to observe the anti-
microbial efficacy of those specimens. Minimum bactericidal con-
centration (MBC) or minimum fungicidal concentration (MFC) is de-
fined as the minimum concentration of GMZnO–Si that kills 99.9% of the
bacterial or fungal strain. The presences of viable micro-organisms were
observed and the lowest concentrations causing bactericidal/fungicidal
effect were reported as MBC/MFC for microbial growing inhibitory
concentrations. The experiment was implemented by plating (NA plate
for the bacteria and SDA plate for the fungi) the culture with highest
quantities above the MIC respectively. Inoculation of these bacterial
and fungus agar plates was done for 24 h and 72 h at 37 °C respectively.

2.5.3. Inner permeabilization assay

Membrane integrity and inner membrane permeability assay were
studied by the treatment of bacteria (E. coli PUC 19 strain) with CM,
GMSi and GMZnO–Si. A single colony was inoculated in fresh nutrient
broth containing IPTG, an inducer for the expression for β-galactosidase
enzyme. The ampicillin nutrient agar plate containing overnight well
Fig. 2. XPS survey scan (A) Zn 2p (B, C) of ZnO NR and ZnO-SiO2 hybrid. grown culture was washed with distilled water and equal amounts of
the cells were treated with GMZnO–Si and incubated at 37 °C for 4 h.
Thereafter, the bacterial culture was added with ONPG (o-nitrophenyl-

Fig. 3. (A) Compressive (B) flexural and split tensile strength (C) bond strength of plane bar and (D) bond strength of deformed bar.

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Fig. 4. (A) UPV (B) RCPT test (C) water absorption (D) slump test.

Table 1 λ = 490 nm for excitation and emission at λ = 520 nm by using

Sulphate resistance test. Fluorescence Spectrophotometer (Motic Image plus 2.0 software). The
Sample Initial mass (g) Final mass (g) % Increment in mass ROS level is directly proportionally related to the intensity of the
fluorescence [28]. 1 ml of each microbial strains (control and treated)
Control 673 ± 0.87 712 ± 0.56 5.79 were taken in 10 ml dye-solution and incubated at 37 °C for 1/2 h. The
GMSi 696 ± 0.61 721 ± 0.18 3.59
solution was prepared by 10 ml SG in DMSO (1:100 v/v) and PI in
GMZnO-Si 706 ± 0.36 724 ± 0.32 2.55
distilled water (1 mg ml−1). After incubation, a few drops of each
Number of sample of each category (n) = 5. sample-solution were mounted immediately over coverslips and ana-
lysed by the fluorescence microscope for this experiment.
β-D-galactoside) for 15 min and absorbance was recorded at
λ = 420 nm.
2.5.5. Morphological investigation for microbial strains by FESEM
A certain volume of microbial growth media (NB for bacteria and
2.5.4. Reactive oxygen species (ROS) detection and fluorescence SDB for fungi) and powder mortar samples (GMSi and GMZnO–Si) were
microscopic analysis added individually to 5 ml broth of each strain resulting the final con-
The freshly prepared pure log-phased cultures of S. aureus, E. coli centration of 1 mg ml−1 samples and microbial concentration of
and A. niger were taken separately in their respective media and su- 108 CFU ml−1.
peroxide radical generation were measured according to standard The mid-exponential phased microbial growth medium and the si-
method [27]. The bacterial cells containing fresh media (concentration milar cell concentration were inoculated in the presence of GMSi and
104 CFU ml−1) were inoculated and treated by GMSi and GMZnO–Si GMZnO–Si separately at 37 °C. The cell pellets were collected by cen-
(~MIC values) at 37 °C for 1 h distinctly. For A. niger, the SD media trifugation and washed by D.I. water and fixed by 2% glutaraldehyde
(104 CFU ml−1) was inoculated and treated with GMSi and GMZnO–Si at on silicon platelet (Plano, Wetzlar, Germany). The samples were taken
37 °C for 6 h. The bacterial/fungal cell pellets were washed several through several ethanol dehydration techniques and stained by 3%
times with phosphate buffer (pH 7.0) and treated with 10 mM DCFHDA uranyl acetate in 25% ethanol. After that the specimens were rinsed by
for 30 min distinctly. 0.1 M sodium phosphate buffer solution (pH 7.0) for morphological
The SYBR Green (SG) and propidium iodide (PI) were conducted to investigation through field emission scanning electron microscope
measure the ROS levels as PI specifically stains only dead cells and SG (INSPECT F50 SEM, The Netherlands).
stains both live and dead cells. The measurement was done at

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Table 2
MIC, MBC and MFC values.
Bacteria/fungi GMZnO-Si (mg ml−1)

MIC MBC MFC

E. coli 0.10 0.15 –

S. aureus 0.12 0.20 –
A. niger – – 0.25

Table 3
Zone of inhibition.
Sample Concentration Zone of inhibition (in mm)

GMZnO-Si 0.2 ml 7
GMSi 0.2 ml 0

2.5.6. The DNA damage study

The DNA damage study was investigated (in-vitro) by treatment of
GMZnO-Si with chromosomal DNA of each microbial strain distinctly.
The well grown culture of each pure microbial strain was taken sepa-
rately. The cell pellets were collected by centrifugation and washed
several times with phosphate buffer (pH 7.0). The bacterial DNAs
(chromosomal) and fungal DNA were isolated by using TIANamp bac-
terial kit and NORGEN Biotek kit respectively. About 1 μg of GMZnO-Si
powder sample was added to the pure DNA (10 μl) and incubated for
15 min. After that, 3 μl ethidium bromide (10 mg ml−1) was mixed to
7 μl each DNA and transferred to sealable centrifuge tubes. After a short
spin, the DNA solution was loaded in 1% low melting agarose gel. The
DNA was visualized under gradient with UV light by trans-illuminator.

2.6. Statistical analysis

Whole experimental set up was repeated twice and data were

averaged and given mean ± S.D. The statistical analysis of the data
obtains from each group (control, treated) were performed by one-way
analysis of variance (ANOVA) along with all pair wise Multiple
Comparison Procedures (Holm-Sidak method) by using Sigma Stat
(version 3.2).

3. Results and discussion

Fig. 5. Mortality studies of microbial strain (A) E. coli; (B) S. aureus; (C) A. niger. 3.1. Characterization of zinc-silica nano hybrid

FTIR analysis was conducted as it is most convenient approach to

Fig. 6. Agar plate/SDA images of microbial strain (A) E. coli (B) S. aureus (C) A. niger.

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Fig. 7. (A) Inner membrane permeabilization assay (B) ROS generation (C) fluorescence microscopic images of E. coli (i: controlled and iv: treated); S. aureus (ii:
controlled and v: treated); A. niger (iii: controlled and vi: treated).

study the surface chemical structure of the sample. Fig. 1A displays the presence of the NR and NPs.
FTIR spectrum of synthesized ZnO-SiO2 hybrid. The absorption peak at Energy-dispersive X-ray spectroscopy was further carried out to
around 3400–3500 cm−1 can be recognised for the stretching vibra- investigate the chemical composition of the hybrid and the corre-
tions of structural hydroxyl (OeH) groups [29]. The peak at about sponding result is shown Fig. 1E. EDX profile suggested strong peak of
1631 cm−1 can be assigned to the bending vibration of adsorbed water Si, O and Zn, only which is an indicative of main constituents on the
and a strong absorption band between 480 cm−1 and 420 cm−1 can be surface of the hybrid are silicon, oxygen and zinc. Here also no other
credited to the stretching modes of ZneO [29]. In addition, the peak at impurity related peaks were observed which corroborates the previous
464 cm−1 is corresponding to the bend vibration of SieOeSi. The results.
abovementioned absorption peaks in FTIR spectra confirms the pre- For precise identification of elemental composition and their oxi-
sence of these functional group and thereby successful preparation of dation state XPS analysis of the pristine ZnO and hybrid sample was
ZnO-SiO2 [29]. carried out. Referencing the C 1 s peak appearing at 284.6 eV, all the
The crystallinity and the phase of the synthesized ZnO-SiO2 hybrid spectra were charge corrected and corresponding results are shown in
was assessed through powder XRD investigation and the pattern is Fig. 2. XPS survey scan analysis of pure ZnO sample reveals the peaks
depicted in Fig. 1B. The well-defined peaks in the spectra can be in- corresponding to Zn, O and C, where additional C related peak results
dexed to the stable hexagonal wurtzite structure of ZnO with lattice from atmospheric carbon. After the formation of hybrid additional
parameters of a = 0.325 nm and c = 0.521 nm (JCPDS No. 36–1451). peaks related Si 2 s and 2p were appeared. Absence of any other peak in
These peaks are associated with the (100), (002), (101), (102), (110), the survey spectrum highlights the phase purity of the samples and
(103), (200), (112) and (201) reflection planes of wurtzite ZnO substantiates the XRD result. Careful comparison of Zn 2p spectra for
(Fig. 1B). In addition, a very small hump was observed at the angle of the pristine ZnO and hybrid samples suggest a shift in peak position
~20°, representing the existence of the amorphous silica in the hybrid. (Fig. 2B and C). Zn 2p peak in the hybrid is found to be shifted by
Absence of any other impurity related peaks in the XRD profile sug- ~1.4 eV in higher binding energy side than ZnO nano-rods sample.
gested high purity of the hybrid. Decoration of the ZnO NR by silica Such type of shift in binding energy after hybrid formation was also
does not change the position of the ZnO characteristics peaks which observed by other study [31]. Furthermore, this peak positioning also
indicated that SiO2 does not significant influence on the crystallinity of indicates that the Zn is in Zn2+ state within ZnO1−x environment [32].
the NRs [30].
ZnO NRs with varying diameter are obvious from the FESEM image 3.2. Mechanical strength and durability
(Fig. 1C). These highly faceted nano-rods possess very smooth side
surfaces. Such high hexagonal faceting is characteristic of wurtzite The compressive strengths of the GMZnO–Si mortar samples are sig-
structure of ZnO. After the hybrid formation, very regular spherical nificantly higher (Fig. 3A). The result depicts that ZnO-SiO2 nano-
shaped silica NPs (~12 ± 3 nm) is decorated on the surface of the ZnO composite in the mortar matrices are able to deliver maximum com-
NRs (Fig. 1D). A close inspection also suggested uniform distribution of pressive strength than silica NPs treated GMSi samples (Fig. 3A). Similar
the silica NPs. Furthermore, the surface of the NR appeared uneven behaviours are also observed on flexural strength and split tensile
after the hybrid formation. HRTEM image of the hybrid shown in strength for GMZnO–Si samples (Fig. 3B). The presence of ZnO NRs in
Fig. 1F, mimics the FESEM image almost identically and confirms co- interlocking orientation and the attached silica particle can react with

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Fig. 8. Microbial cell wall degradation by FESEM (A and B) E. coli control and treated; (C and D) S. aureus control and treated; (E and F) A. niger control and treated.

the surrounding binders and that further make a polymer sodium alu- result suggests the increment in velocity is ensued due to presence of
minosilicate in the composite. This long-chain silicate polymer in the ZnO-SiO2 which reduces of pores and micro fissures. The formulation of
matrix finally increased the mechanical strength. According to the other the ZnO-NPs may contribute to strengthening of geopolymer composite
studies, the sodium aluminosilicate (N-A-S) gel mainly responsible for reinforced as well as more compactness helps to reduce the porousness
the normal alkali activated geopolymers [33–35]. The consumption of as reflected from the better results of UPV and Compressive strengths. It
NaOH associated to the high reactivity of ZnO-Silica hybrid, provide the is also observed that less amount of chloride ions was passed through
strength increment to GMZnO–Si. Bond strength between reinforcement the geopolymer (GMZnO-Si) matrix than CM in case of RCPT study
(Plane or deformed bar) and the surrounding cementitious matrices for (Fig. 4B). This hybrid is also found to increase the sulphate resistant
all samples are depicted in Fig. 3C and D. From the figure, significantly activity of the mortar samples (Table 1). In comparison, the Fig. 4C
higher bond strength for GMZnO–Si sample as compare to the GMSi and exhibits GMZnO–Si samples have lower water absorption and higher
CM is obvious. apparent density in respect to other samples (CM, GMSi). The slump test
The ultrasonic pulse velocity (UPV) through the GMZnO–Si and GMSi was conducted for the workability of these samples, shown in Fig. 4D
samples are represented in Fig. 4A with respect to their control samples. where the results give the better workability possesses for GMZnO-Si
Much improved velocity for GMZnO-Si mortar sample than GMSi and CM samples. The fluidity is improved in GMSi specimens than CM samples
validates the better compactness of the matrices of GMZnO-Si. The matrix and however the proper workability is achieved for GMZnO–Si during
of GMZnO-Si mortar is more able to transmit the ultrasonic pulses; such production of mixes. With high surface area and reactivity silica

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These observations also demonstrate that the MBC for GMZnO-Si treated
bacterial cells are not > 4 times their respective MIC values, while MFC
value is about 0.25 mg for A. niger (Table 2). The results suggest that the
nano composites are bactericidal rather than bacteriostatic.
Optical density of GMZnO–Si treated E. coli is increased in inner
membrane permeabilization assay (Fig. 7A) as compared to Control and
GMSi treated samples. Micro-organisms have negative charge on the cell
wall while ZnO-SiO2 carry a positive charge which create an “electro-
static” attraction on contact surfaces. Once the contact is made, the
microbe is oxidized and dead instantly [39]. The level of ROS in terms
of oxidative stress for the control cells were considered as 100% and the
intensity of GMZnO–Si treated cells were about 5 times higher with
corresponds to the control for S. aureus, E. coli and A. niger samples
(Fig. 7B). Such bacterial growth prevention can be elucidated on the
basis of high ROS generation. Autoxidation of NADH dehydrogenase II
in the respiratory system causes the ROS production in microbes [40].
Increased ROS by disturbing the proper transportation in plasma
membrane rises the inner membrane permeabilization action, as bac-
Fig. 9. DNA damages in-vitro; lane 1 and 2: E. coli control and treated; lane 3 teria/fungi are sensitive to ZnO NRs. Presence of sufficient amount of
and 4: S. aureus control and treated; lane 5 and 6: A. niger control and treated.
ZnO nano rods in GMZnO-Si inhibit the growth of the micro-organisms.
The fluorescence microscopic images reveal that control cells and
nanoparticles attract the water molecules more and diminish the free GMSi treated cells (S. aureus, E. coli and A. niger) are intensely stained
liquid which hampers the fluidity development. In GMZnO–Si matrices, with SYBR Green dye, stains both live and dead cells whereas GMZnO-Si
the amount of unreacted silica NPs are very lower as most of the silica treated cells are found to be PI positive, stains only dead and membrane
attached on the surface of the zinc oxide. compromised cells due to loss of the plasma membrane integrity
(Fig. 7C). It is already documented that highly reactive oxygen species
3.3. Mechanistic approach for anti bio-deterioration (i.e., ·OH, ·O2−, and H2O2) are released on the surface of ZnO nano-
particles [41]. The superoxides and hydroxyl radicals cannot able to
Lack of maintenance as well as relative humidity, influence the penetrate into the cell membrane due to their negative charges, found
microbes to deteriorate any building structure as it provides adequate on the outer surface of the microbes whereas H2O2 molecules are able
quantity of the water molecules to the microbes. In general, sulphur- to pass through the bacterial cell wall, subsequently leading to injuries
oxidizing microbes and other alkaline-tolerant microbes colonise at and destroy, and finally triggering cell death [39]. After ZnO NRs
concrete surfaces. Day after day, through neutralisation or biogenic treatment, the NR particles are adsorbed at the surface of the left over/
oxidation of sulphur and carbonation process, the alkalinity of the killed bacteria blocking additional anti-microbial activity. The presence
structures become lower due to the production of weak acids (i.e., of ZnO NRs in the growth media carry on releasing peroxides covering
carbonic acid, thio-sulphuric acid etc.). Such low alkalinity provides the the entire surfaces of the dead bacteria/fungi and this continuous per-
suitable environment to initiate the growth/colonisation of micro-or- oxide release leads to higher bactericidal/fungicidal efficacy [42,43].
ganisms (neutrophilic and acidophilic) [36]. Microbial growth further The morphological analysis by FESEM shows the significant mem-
allows the permeation of harmful ions (Cl−, SO42−), moisture and brane damage and disruption in GMZnO–Si treated cells after 8 h of in-
forms concrete corrosion agents like gypsum and ettringite [37,38]. cubation in respect to GMSi treated E. coli and S. aureus cells (Fig. 8A–D)
Such types of conversion of concrete to corrosion agents weaken the respectively. Similar occurrence is also observed in case of A. niger
structural integrity of concrete, and eventually lead to structural failure. (Fig. 8E, F). ZnO NRs can attach to the cell wall of micro-organisms by
To prevent bio-deterioration, the incorporation of ZnO-SiO2 nano- direct or electrostatic interaction and interrupt the integrity or damage
composite (6% by weight) in the fly-ash based geopolymer mortar has of the membrane. Lack of electron/proton motive force and acceptance
been made. The composite exhibited a significant anti-microbial effi- of toxic radicals induce the cell wall damage after ZnO-cell wall inter-
ciency as well as augmentation of the strength and excellent durability. action [44]. The DNA was fragmented after GMZnO-Si induction for all
E. coli, S. aureus and A. niger have been used as models for inspecting specimen in-vitro study (Fig. 9A). The control DNA of E. coli, S. aureus
the anti-microbial activities of GMSi and GMZnO-Si samples as they are and A. niger are seen in lane 1, lane 3, lane 5 whereas damaged (smear)
nosocomial pathogens. The growth kinetic patterns of microbial strains DNAs are in lane 2, 4, and lane 6 respectively. Due to the formation of
are represented in Fig. 5. The result reveals the population of ex- the free radicals, the toxicity of the ZnO NRs trigger to kill or stop the
ponentially growing microbial strains (E. coli, S. aureus and A. niger) growth of micro-organisms through disruption of the cellular compo-
were reduced by 99% after 8 h, 6 h and 4 days by GMZnO–Si respectively. nents like nucleic acids. The DNA replication is interrupted, and in-
GMZnO–Si usually delay the lag-phase of the growth cycle and increasing tercellular ATP production is depleted by the penetration of ZnO NRs
the generation time of the micro-organisms. So that, it takes additional into the cell resulting cell death [45].
time to complete cell division. The Fig. 5C exhibits the fungal growth
inhibition (OD at λ = 595 nm), reveals that the GMZnO–Si has ability to 4. Conclusion
kill or stop the fungal growth significantly within 72 h. The anti-mi-
crobial effect is shown by plate culture of microbial growth after 24 h In summary, spherical shaped silica nanoparticles are attached on
treatment in presence of GMZnO–Si and GMSi samples (Fig. 6). Several the surface of the ZnO nano-rods to prepare the ZnO-SiO2 nanohybrid.
numbers of bacterial colonies are found in GMSi treated samples By incorporating this nanohybrid into fly ash based geopolymer, a
whereas less number of colonies are appeared in GMZnO–Si. On other sustainable building composite is realized which exhibited high me-
hand, zone of inhibition (~7 mm) is also observed for GMZnO-Si treated chanical strength along with potential capacity to prevent the biogenic
fungal strain (Table 3, Fig. 6C). These findings indicate that GMZnO-Si deterioration. Plausible mechanism towards anti-microbial activity
has significant anti-microbial property. The MIC, MBC and MFC values using GMZnO-Si has been established via ROS generation, cell wall de-
are represented in Table 2 which indicates very small amount of GMZnO- gradation and DNA damage studies. Such applicability of GMZnO-Si
Si (0.2 mg) can eliminate the (> 99%) bacterial and fungal populations. could provide valuable understandings to develop a new microbial

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M. Sarkar et al. Materials Science & Engineering C 92 (2018) 663–672

incidence free cementitious composite by avoiding the bio-degrada- Cube Specimens), (2002).
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The authors are gratefully acknowledged to Mr. S. Thakur, Junior
Mortars Exposed to a Sulphate Solution, ASTM International, West Conshohocken,
Research Scholar, Jadavpur University, India, and also acknowledge the PA, 2015.
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