Date: Jan 07, 2018

"Bhupendra K Pushkar"
To:
bhupendra.pushkar@gmail.com
From: "Int. J. of Nanoscience" ijn@wspc.com
Subject: Your Submission
Ref.: Ms. No. IJN-D-17-00037
NITRATE REMOVAL USING NANOMATERIAL
International Journal of Nanoscience

Dear Dr. Pushkar,

Reviewers have now commented on your paper. You will see that they are advising that you revise your
manuscript. If you are prepared to undertake the work required, I would be pleased to reconsider my
decision.

For your guidance, reviewers' comments are appended below.

If you decide to revise the work, please submit a list of changes or a rebuttal against each point which is
being raised when you submit the revised manuscript.

To submit a revision, go to http://ijn.edmgr.com/ and log in as an Author. You will see a menu item call
Submission Needing Revision. You will find your submission record there.

Yours sincerely

Junhui He, Ph.D.

Managing Editor
International Journal of Nanoscience

Reviewers' comments:
Reviewer #2: This paper describes the synthesis Fe oxide nanoparticles and nanocomposite of Fe/CNT and
uses them to remove nitrate from environmental contaminated sites. The morphology, compositions and
physical properties were characterized by SEM, XRD,
UV-Visible spectrophotometer and FTIR. The manuscript is very well structured and nicely presented.
However there are some questions in the manuscript, so I don't think it is suitable to be published in present
form.
1. The figure of FTIR spectra is unclear! High magnification image should be used. Commented [p1]: Difficult for me to put high magnification
2. The specific surface area, pore volume and pore size are important parameters to affect adsorption image. Kindly suggest
properties of the adsorbent, it should provide the textural properties of the adsorbents using BET method.
Commented [p2]: In the present work BET work has not been
3. The regeneration properties of the adsorbent are very important, how to recycle the adsorbent after
done so I am finding difficulty in giving proper explanation for the
adsorption?
same. Kindly give your valuable suggestion
4. What is the mechanism of the adsorbent for nitrate removal? It will be better if the authors present the
adsorption mechanism using a schematic figure. Commented [p3]: Explanation to this question is elaborated in
the discussion section. For our understanding I have highlighted it
with grey colour.
Commented [p4]: I have added a generalised explanation
[Highlighted in blue in text] for the raised question. the explanation
for mechanism of nitrate removal need some more studied to be
done. Kindly give your suggestions on the same.
 Nitrate Removal Using Nanomaterial

Abstract: The focus of the present study was to develop an efficient nanomaterial for nitrate removal
from environmental contaminated sites. Fe oxide nanoparticles and nanocomposite of Fe/CNT was
prepared by cannon ball flower extract as reducing agent. Their morphology, compositions and
physical properties were characterized by scanning electron microscope (SEM), X-ray diffractometer
(XRD), UV-Visible spectrophotometer, fourier transform infrared spectroscopy (FTIR).The nitrate
removal efficiency of the three nanomaterial i.e. Fe nanoparticles, CNT and Fe/CNT nanocomposite
was determined. The nanocomposite of Fe and CNT was found to be more efficient in nitrate removal
as compared to Fe oxide and CNT individually. The effective duration for nitrate removal by
nanomaterial was 3 hour after that there was desorption of the nitrate from the nanomaterial. 52.83,
56.14 and 77.3% of nitrate was removed by Fe oxide, CNT and Fe/CNT nanocomposite at 100 mg/l
nitrate concentration and 6 mg/ml of the nanomaterial dose. The statistical analysis of the nitrate
removal using nanomaterial was performed.

Keywords: Nitrate, nanotechnology, green synthesis, characterization, adsorption etc.

Introduction:

Extensive use of nitrogenous fertilizer in agricultural field results in nitrate accumulation in water
resources. The nitrate enter the water system through various sources like N- containing organic and
inorganic fertilizers, animal manure, elevated atmospheric N deposition, improper processing of
industrial wastewater, septic tank discharge, leaking of sewers, spreading of the sewage sludge and
seepage of the waste from landfills (Xue, Dongmei, et al, 2015,Ghafari et al., 2008 and Pastén-Zapata,
Ernesto, et al, 2014).Nitrate present above the threshold limit in the water resources is leading cause
of the environmental pollution (Passy, Paul, et al, 2012 and Zhang, Yan, et al, 2014). The nitrate
pollution supports the blooms of toxic algae, eutrophication of the water system, and may also lead
to theextinction of the species from thewater system. Nitrate at higher concentration affects the
whole ecosystem (Xue, Ying, et al, 2016). According to the World Health Organization (WHO)
maximum contaminant level of nitrate in the water is around 10 mg/ml which is very common in the
developing countries (Gu, Baojing, et al, 2013).

Increased level of nitrate in the water resources has been linked to adverse health effect. After
entering the human body nitrate gets converted into nitrite which reacts with haemoglobin to form
methaemoglobin (Su, Xiaosi et al, 2013). Nitrate contamination can also lead to cancer through
theformation of carcinogenic N-nitrosamines compounds, other effects include spontaneous abortion
and non-Hodgkin's lymphoma (Lockhart, K. M. et al, 2013 and Kapil, Vikas, et al, 2013).
 Due to the increasing concentration of nitrate in the water resources and in turn its adverse effects
on human and several approaches have been used in the past for nitrate removal such asflocculation,
ion exchange, reverse osmosis, and electrodialysis(Zhou, Minghua et al, 2009). However, these
methods were costly, not much effective, and also difficult to conduct at large scale (Zhou, Minghua,
et al, 2007,Samatya et al., 2006andLi, Miao, et al, 2010).Hence,development of cost effective, highly
efficient, scalable method for nitrate removal from the environment is necessary.Waste water
treatment using the emerging nanotechnolgical approachesoffers several advantage in comparison to
other techniques (Qu, Xiaolei et al, 2013,Baruah, Sunandan et al, 2016 and Qu, Xiaolei, et al,
2012).Nanotechnology is theapplication of the nano-sized material in the various fields (Bhupendra
Pushkar et al, 2016). Nanotechnology is found very promising in all the fields including pollution
treatment. Previous research studies has demonstrated the usefullness of nanomaterial in nitrate
removal from waste water (Kassaee, M. Z., et al, 2011; Daud etal., 2015).

Therefore, in the present study, we have synthesised Fe nanoparticles, CNT (procured from Rinkesh
Kurkure, Birla College, Kalyan), and nanocomposite of Fe nanoparticles and CNT and demonstared
their effectiveness in nitrate removal. CNT is excellent nanomaterial,chosen because of their special
properties and widespread application. CNT is porous in structure with high surface area, CNTs can
serve excellent substrates for preparing functional hybrid nanomaterials (Wang, Hui, et al, 2012). The
combination of Fe nanoparticles and CNT provide anadvanced class of nanocomposite (Gupta, V. K.,
and ArunimaNayak, 2012). The magnetic properties of Fe nanoparticles help in separation and also in
adsorption while, CNT with large surface area increases adsorption efficiency of nanomaterial. Hence,
we had compared the efficiency and effectiveness of Fe, CNT and their composite for nitrate removal
from waste water that can be used widespread for nitrate remediation of polluted waste water.

Materials and methods:

1. Green synthesis of Fe nanoparticles-The Fe nanoparticles were synthesized using a procedure

reported by Wang, Ting, et al., 2014 with little modification. The cannon ball flower petals were
washed twice with distilled water and cut into small pieces. The flower extract was prepared by
boiling the petals pieces in the distilled water at 80˚C for 1 hour. The concentration of flower was
200.0 g/L. The debris of petal was separated through vacuum filtration. The Fe nanoparticles were
synthesized using flower extract as reducing agent and FeSO4 as Fe source. The flower extract and 0.1
M FeSO4 were mixed at volume ratio of 2:1 at room temperature. The color of the mixture was
turned into intense black that indicated the formation of Fe nanoparticles. Nanoparticles were
heated in the crucible, to get the purified Fe nanoparticles.
Synthesis of nanocomposite-The nanocomposite of CNT/Fe oxide was prepared as reported
previously with some modification (Qu, Song, et al, 2007). All glassware were cleaned with freshly
prepared aqua regia. The carbon nanotubes (CNTs) were further purified by dispersing the CNT in
concentrated nitric acid at 60°C for 12 h. After nitric acid treatment CNTs were dried at 110°C for 4 h.
 The CNT/Fe oxide nanocomposite was prepared in situ coprecipitation method in presence of Fe 2+in
alkaline pH. The 1.0 g of purified CNTs were suspended in 200 ml of solution containing 1.7 g (4.33
mmol) (NH4)2Fe(SO4)·6H2Ounder N2 atmosphere. The 10 ml of flower extract was added to the above
solution drop wise after the solution was sonicated for 10 mins at 30 W, 40 kHz. The pH of the final
solution was maintained in range of 11-12. The reaction was carried out for 30 mins under constant
mechanical stirring to allow the complete formation of nanoparticle crystals. The precipitate was
separated under magnetic field. The nanocomposite was washed thrice with absolute alcohol. The
nanocomposite was heated in crucible to get the final purified nanocomposite.
2. Characterization of Fe nanoparticles and nanocomposite-The nanomaterial were characterized for
selected physical and chemical properties. The formation of Fe nanoparticles from the flower extract
was confirmed by measuring its absorption in theUV-Visible range of thespectrum. The absorption of
nanoparticles was read at MultiscanGo Nanodrop Thermofisher. The morphology of the nanomaterial
was determined using scanning electron microscope (FEI INSPECT F50) at National centre for
Nanoscience & Nanotechnology, University of Mumbai. The X-ray diffraction (XRD) was performed on
aShimadzhu 7000 instrument, with the X-ray diffractometer using Cu Kα radiation (λ=1.54Å). The
crystalline phase was determined at scan rate of6.0000 deg/min and at the 40 kV and 30 mA of
accelerating voltage and applied current, respectively. The FT-IR spectra of the Fe nanoparticles were
recorded in the range of 7500–300 cm−1 with a resolution of 4 cm−1.
3. Nitrate adsorption study: The batch experiment to study the nitrate adsorption was performed in 100
ml screw cap bottles covered with aluminium foil. The reaction bottles were covered to avoid any
photocatalytic activity of the nanomaterials and, to study the nitrate adsorption efficiency of the
nanomaterial. The adsorption study was performed at100 mg/l concentration of nitrate. The dosage
of the nanomaterial was 6 mg/ml of reaction volume. The test solutions containing the nanomaterial
were placed on an orbital shaker at 150 rpm at room temperature. The nitrate removal was analysed
by withdrawing the sample from reaction mixture till 5 hours at a time interval of 1 hour. The
concentration of nitrate in the solution after the reaction with nanomaterial was quantified as
reported by Wang, Ting, et al, 2014. The percentage adsorption of nitrate by nanomaterial was
calculated using equation 1 (Mehrabi, Novin, et al, 2015).

% Nitrate removed = (Ci - Cf)/Ci X100 ...................(1)

Where Ci is initial concentration of nitrate and Cf final concentration of nitrate (mg/l).

Adsorption isotherms: In order for designing an adsorption process it is necessary to have information about
the adsorption
Statistical Analysis: All the studies have been conducted in three replications and performed atleast
five times to get accurate data. The data has been represented as mean ± SD, ANOVA has been
prepared and probability values P < 0.05 has been considered statistically significant.

Results and discussion:

1. Fe nanoparticle and nanocomposite- The Fe nanoparticles and nanocomposite were synthesised
using cannon ball flower extract. The colour of the FeSO4 solution was turned into intense black
colour after mixing the flower extract which visually indicated the formation of the Fe nanoparticles.
The nanoparticles in the mixture attracted toward magnet that further confirms the formation of the
Fe nanoparticles with ferromagnetic properties.
2. Characterization of the nanomaterials- The absorbance of the nanoparticles was read in UV-Visible
range by keeping flower extract as blank. As presented in fig1, the solution of the Fe nanoparticles
showed a sharp and narrow peak at 304 nm confirmed the formation of the Fe nanoparticles from
cannon ball flower extract. The narrow peak at 304 nm indicates the monodispersed Fe
nanoparticles. The nanoparticles with similar size have same plasmon bandwidth and absorb in the
same wavelength while, the polydispersed nanoparticles with different size have different bandwidth
and absorb in wider wavelength range and give broad peak.

Fig 1: Spectrum of the green synthesised Fe nanoparticles in UV-Visible region.

The SEM microgram of the Fe nanoparticles, CNT and nanocomposite have represented in fig2. The
spherical Fe nanoparticles were observed in the SEM microgram with an average size of 80 nm. The
morphology of the CNT was conformed using SEM analysis. The CNTs were seen to be randomly
aligned. The width of the CNT (fig 2b) was 35.57 nm. The Fe nanoparticles and CNT in the
nanocomposite was seen to be homogenously mixed and associated with each other. The SEM image
of the Fe/CNT nanocomposite confirmed the efficiency of the flower extract as efficient reducing
agent for synthesizing nanomaterial. The SEM image of the nanocomposite is given in the fig 2c.
 Fig 2: Scanning electron microgram of the a) Fe nanoparticles, b) CNT and c) nanocomposite.

The XRD diffraction pattern was used to determine the nature and structure of the Fe nanoparticles.
The XRD data confirm the crystalline nature of the Fe nanoparticles. The diffraction peaks of value
28.411°, 33.247°, 35.727°, 40.562° and 62.810° have evidenced the hematite (Fe2O3) nature of
nanoparticles. The average size of the nanoparticles was calculated using the Debye-Scherrer formula
i.e. Ds = 0.9λ/β cosθ. The size of the Fe nanoparticles was found to be around 21.28 nm. XRD
diffraction pattern is given in fig 3a and b.

Fig 3: X-ray diffraction pattern of the Fe nanoparticles.

 The absorbance of the Fe nanoparticles in the infrared region before and after (fig 4), its heating was
studied to determine the surface elemental phase. The absorption of the material in at 1696 cm-1
and 1595 cm-1 before and after heating indicate the C=O vibration. The presence of the band stretch
from 7500 cm-1 to 6700cm-1 in the before heating sample indicated the intermolecular hydrogen
bonding due to OH vibration while, the intensity of this peak increased after heating showed the
increase in the OH vibration. The heating procedure removed the unwanted elements present on the
Fe nanoparticles, therefore, enhances the absorption by OH groups.

Fig 4: FTIR absorption of the Fe nanoparticles a) before heating and b) after heating.

3. Nitrate adsorption study: The adsorption of the nitrate by the Fe nanoparticles, CNT and
nanocomposite was determined. All three nanomaterial could adsorb nitrate from the solution in
defined time. The percentage adsorption of nitrate by different nanomaterial was calculated as per
the equation 1 and tabulated in table 1.

Table 1: Percentage nitrate adsorption by Fe nanoparticles, CNT and nanocomposite.

Sr. No. Contact time Nitrate adsorption
Fe nanoparticles(%) CNT(%) Nanocomposite (%)
1 1 hour 16.01 7.36 1.23
2 2 hours 38.72 22.69 61.16
3 3 hours 52.83 56.14 77.3
4 4 hours 50 55.62333 76.45
5 5 hours 46.47 54.67 74.84

Fe Nanoparticles
CNT
85
Nanocomposite
80
75
70
65
60
55
Percent Nitrate removal

50
45
40
35
30
25
20
15
10
5
0
-5
1 2 3 4 5

Time (Hour)

Fig 5: Percentage adsorption of the nitrate by Fe nanoparticles, CNT and nanocomposite.

The graphical representation of the nitrate removal by Fe nanoparticles, CNT and nanocomposite are
given in fig 5. Three different nanomaterials could efficiently adsorb nitrate but the combination of Fe
nanoparticles and CNT in the form of nanocomposite found to be more efficient in nitrate adsorption
than nanomaterial used individually. The nitrate adsorption by three nanomaterial was fast till 3
hours and then the plateau was observed. It was reported previously in other studies also (Chen,
Shiao-Shing, 2004).The nanocomposite could adsorb 77.3% of nitrate in 3 hours. The maximum Formatted: Highlight

adsorption occurred in a period of three hours whereas, after that desorption start. All three
nanomaterialnanomaterials showed maximum adsorption till three hour and after 3 hours of reaction Formatted: Highlight

time the adsorbed nitrate seemed to be desorbed from nanomaterial. With this observation it can be
concluded that nanomaterials can be further regenerated and can be recycled for the next set of
nitrate removal experiment. The weak interactions like hydrophobic, ionic or van der waals force
between the nanomaterial and nitrate might be the probable reason for this particular observation.
The nanocomposite could remove higher concentration of nitrate as compared to material reported
previously by Bhatnagar, Amit et al, 2010,Chen, Shiao-Shing, 2004.
 The above synthesized Fe/CNT nanocomposite can be used for nitrate removal from the various
nitrate contaminated site. Further studies like adsorption isotherm need to be done to elucidate the Formatted: Highlight

complete mechanism for nitrate removal using Fe/CNT nanocomposite. Also before application to Formatted: Highlight

field other parameters need to be considered which can interfere in the nitrate adsorption by
nanocomposite.

Conclusion: In summary, the Fe nanoparticles and Fe/CNT nanocomposite have been prepared first
time with cannon ball flower extract. The green method of nanomaterial synthesis is environmentally
benign and safe.The results from the current study strongly suggest the higher efficiency of the
Fe/CNT nanocomposite to remove nitrate as compared to Fe nanoparticle and CNT individually. All
three nanomaterial could remove nitrate but Fe nanoparticles and CNT in Fe/CNT nanocomposite
work synergistically to remove nitrate. The nanomaterial was found to do the task in very short time
of 3 hours. The nanomaterial can be utilized to nitrate removal from the polluted sites.

Acknowledgement: The authors are thankful to Mr. Rinkesh Kurkure for providing CNT for
experimental study.

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