Separation and Purification Technology 162 (2016) 61–67

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Separation and Purification Technology

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

Removal and regrowth inhibition of microalgae using visible light

photocatalysis with ZnO nanorods: A green technology
Priyanka Sathe a,b, Myo Tay Zar Myint a, Sergey Dobretsov b,⇑, Joydeep Dutta a,c,⇑
a
Chair in Nanotechnology, Water Research Center, Sultan Qaboos University, PO Box 17, Al-Khoudh, Muscat 123, Oman
b
Department of Marine Science and Fisheries, College of Agricultural and Marine Sciences, Sultan Qaboos University, PO Box 34, Al-Khoudh, Muscat 123, Oman
c
KTH Royal Institute of Technology, Functional Materials Division, Materials and Nano Physics Department, ICT School, Isafjordsgatan 22, SE-164 40 Kista Stockholm, Sweden

a r t i c l e i n f o a b s t r a c t

Article history: Algal biofouling can be a major problem during membrane filtration processes reducing membrane
Received 9 June 2015 efficiency. Removal of microalgae by visible light photocatalysis using zinc oxide (ZnO) nanorods was stud-
Received in revised form 14 January 2016 ied in this work. ZnO nanorods were grown on polypropylene support substrates. The treatment unit was
Accepted 3 February 2016
constructed by incorporating ZnO nanocoated substrates in a glass tube. Anti-algal activity of the treat-
Available online 4 February 2016
ment units were tested using green microalga, Dunaliella salina, of 107 cells/mL concentration, which is
higher than the concentration of cells during algal blooms. Nearly total algal cell inactivation was achieved
Keywords:
within 2 h of continuous visible light illumination in the presence of nanocoated support substrates, as
Anti algal
Pre-treatment
determined by flow cytometry analysis (98%) and trypan blue staining (95%). Uncoated support substrate
Zinc oxide under light illumination did not lead to algal cell mortality (1.7%). Complete inhibition of any regrowth of
Nanorods algal cells treated with nanocoated substrates was confirmed as no significant changes in the total number
Photocatalysis of cells were observed even after 2 weeks of incubation of the treated culture. The anti-algal activity of ZnO
nanorods was attributed to the formation of reactive oxygen species (ROS) through photocatalytic pro-
cesses. ZnO nanorod coated substrates used in the treatment units could be a suitable green method to
control membrane fouling in water treatment plants avoiding the utilisation of harmful chemicals.
Ó 2016 Elsevier B.V. All rights reserved.

1. Introduction Membrane biofouling occurs due to the attachment and subse-

Water on earth is a valuable but finite resource. In developing
countries more than 1.2 billion people lack access to clean and safe
drinking water [1,2]. In arid regions, available drinking water is
even more of a problem due to the scarcity of fresh water
resources. Desalination technologies that alter saline water to fresh
water have high potential to satisfy growing water demand which
has led to massive reliance on membrane technologies for water
desalination [3]. Membrane fouling is a major problem encoun-
tered in filtration processes, and is a major limitation that under-
mines the practical application of membranes for desalination
and wastewater treatment [4,5]. Fouling of membranes in
desalination plants compromises the efficiency of the treatment
processes leading to higher production costs due to increased
energy consumption [6].
⇑ Corresponding authors at: Functional Materials Division, Materials and Nano
Physics Department, ICT School, KTH Royal Institute of Technology, Isafjordsgatan
22, SE-164 40 Kista Stockholm, Sweden (J. Dutta), Department of Marine Science
and Fisheries, College of Agricultural and Marine Sciences, Sultan Qaboos Univer-
sity, PO Box 34, Al-Khoudh, Muscat 123, Oman (S. Dobretsov).
E-mail addresses: sergey@squ.edu.om (S. Dobretsov), joydeep@kth.se (J. Dutta).
quent growth of microorganisms such as bacteria and microalgae
[7,8]. Algae are difficult to eliminate with conventional pre-
treatment methods. Also algal cells have tendency to deposit on
the membrane surface which can reduce membrane lifetime [9].
Seasonal algal blooms can also affect water quality through the
release of algal toxins and production of unfavourable odour ren-
dering water unsuitable for drinking [10]. Thus, removal of algae
is important for improved performance of membranes in water
treatment plants. There are several microalgal species known to
produce blooms that can even be potentially hazardous if they pro-
duce toxins [11]. Most reports available in the literature are related
to the treatment of Microcystis aeruginosa [12] but few studies have
considered other bloom forming species such as Chlorella or Duna-
liella. The microalga Dunaliella salina is an organism that is com-
monly observed in salt lakes and marine environments which
has tolerance towards variable salt concentrations [13].
Common practice to control biofouling involves the usage of
high concentrations of chlorine. Longer contact of chlorine with
organic matters in water leads to the formation of carcinogenic
substances known as trihalomethanes [14]. In addition, chlorine
breaks down natural organic matter to biodegradable products

http://dx.doi.org/10.1016/j.seppur.2016.02.007
1383-5866/Ó 2016 Elsevier B.V. All rights reserved.
62 P. Sathe et al. / Separation and Purification Technology 162 (2016) 61–67
offering an additional nutrient source to remaining viable cells
after the treatment, leading to transient effectiveness of chlorina-
tion [15]. Hence, multiple exposures of chlorine are required for
effective treatment of fouling in membranes. Due to the problems
accompanied with chlorination, numerous efforts have been made
to develop novel, less toxic yet effective techniques for the preven-
tion of membrane biofouling [16] including the use of ozone or
ultraviolet (UV) treatments [14]. Also conventional biocide appli-
cations generates waste which have been demonstrated to lead
to environmental, ecological and toxicological problems apart from
being uneconomical [17].
Zinc oxide (ZnO) is a well-known semiconductor with a wide
ranging application as a catalyst [18]. It is established that ZnO is
a suitable visible light active photocatalyst capable of degrading
harmful organic molecules [19,20] and to arrest bacterial growth
[21,22] through the generation of reactive oxygen species (ROS)
under visible light irradiation [23,24]. ZnO nanorod coatings have
significant advantages over ZnO nanoparticles due to increased
stability, lower toxicity and no added processing steps for the
removal of nanoparticles from the treated water [25]. Moreover,
it is feasible to modify any support material by ZnO nanorods
growth due to low temperature (below 100 °C) synthesis process.
The purpose of the present study is to investigate activity of
ZnO nanorod coated supports as a pre-treatment technology for
the removal of the marine microalga D. salina. Specific objectives
of the study include investigation of anti-algal activity of the nano-
coated polypropylene substrate under continuous visible light irra-
diation and monitoring regrowth of algal cells after photocatalytic
treatment.
2. Experimental
ZnO nanorods coatings were prepared by a two-step process. In
the first step, a seed layer of ZnO nanoparticles were deposited on
commercially available non-woven polypropylene support sub-
strate (CUNO, CP 110, Oman). ZnO nanorods were then grown on
these seeded substrates by utilizing simple hydrothermal route
as described hereunder.
2.1. Synthesis of ZnO nanoparticles
Sol–gel synthesis of ZnO nanoparticles in ethanol medium was
conducted by following a protocol reported earlier [26]. Briefly,
4 mM zinc acetate dihydrate (Zn (CH3COO)2
2H2O) and 4 mM
NaOH (MERCK, Germany) were prepared in 20 mL each of absolute
ethanol solution under rigorous stirring. Then, the prepared zinc
acetate solution was diluted with 20 mL of ethanol. Subsequently,
freshly prepared NaOH solution was added drop wise under con-
tinuous stirring at room temperature. The mixture was then
hydrolysed in a temperature controlled water bath at 60 °C for
2 h. The resultant ZnO colloidal solution was transparent, consist-
ing of nanoparticles of 4–7 nm [27] (Supp. Info. Fig. S1).
2.2. ZnO nanoparticle seeding
Prior to hydrothermal growth of ZnO on the substrates, a pre-
treatment step was introduced using the synthesized ZnO
nanoparticulate colloid. The polypropylene substrates were modi-
fied following a procedure reported by Baruah et al [28]. First, the
substrates were treated with 1% dodecanethiol solution in ethanol
followed by heating at 100 °C for 15 min. The thiolated substrate
surfaces help in the firm attachment of ZnO nanoparticles to
polypropylene substrate. Subsequently, the substrates were dipped
in colloidal suspension of ZnO nanoparticles for 15 min. The seeded
polypropylene substrates were then dried at 150 °C for 15 min.
This process was repeated five times in order to achieve a proper
coverage of ZnO nanoparticles on the substrates and then dried
and stored in a dehumidified chamber (25% RH) until further use.
2.3. Hydrothermal synthesis of ZnO nanorods
ZnO nanorods were grown in a sealed chemical bath containing
an equimolar solution (10 mM) of zinc nitrate hexahydrate and
hexamethylenetetramine (hexamine) (Sigma–Aldrich, USA) kept
in an oven maintained at 90 °C. The precursor solution was chan-
ged every 5 h in order to replenish the depleted zinc ions [29,30]
and the growth process was carried out for 15 h. Subsequently
the samples were thoroughly rinsed with deionized (DI) water
and kept in an oven at 90 °C overnight for drying.
Two different types of substrates were used throughout the
experiment: uncoated substrates (only support substrates: non-
woven polypropylene support substrate without ZnO nanorod
coatings) and nanocoated substrates (support substrates: non-
woven polypropylene support substrate coated with ZnO
nanorods).
2.4. Characterization of nanocoated substrate
The modification of support substrates were studied using Four-
ier transform infrared spectroscopy (FTIR) (PerkinElmer Frontier 1,
USA) spectrometer (Supp. Info. Fig. S2). Surface morphology of ZnO
nanorods on support substrates were characterized by JEOL JSM-
7200 (Japan) field emission scanning electron microscope (FESEM)
working at 20 kV (working distance = 8 mm). ZnO crystal structure
was studied by using X-ray diffraction (XRD) (Rigaku Miniflex 600,
Japan) working at 40 kV, 15 mA with a scanning speed and step
size of 10 deg/min and 0.02° respectively, for 2h values from 20°
to 80°. Zeta potential of the support substrates and nanocoated
substrates were measured by streak potential technique using
electro kinetic analyser (Anton paar, SurPASS, Austria). Specific
surface area (m2/g) measurements were conducted using NMR
relaxation technique in a Xigo Nanotools equipment, with ethanol
as the solvent [31,32]. In this system the spin magnetic moments
of hydrogen nuclei are probed. A perturbation in the applied static
magnetic field changes the total nuclear magnetization, whose
relaxation back to equilibrium is registered as a function of time
(comprising of longitudinal relaxation time T1, defining the decay
of spin alignment and the transverse relaxation time T2, defining
the decay of precession). In this work, transverse relaxation time
(T2) of the solvent in contact with pore surface was analysed by
Acorn area software to extract the specific surface area (SSA) of
the substrate in contact with the solvent. Active surface area
(m2) was subsequently calculated from the SSA by multiplying
SSA (m2/g) with weight of the support (g) substrate used for the
experiment. Surface wettability measurements were carried out
before and after photocatalysis for both uncoated and nanocoated
support substrates using Theta Lite attention tensiometer (Biolin
Scientific, Sweden). Water contact angle was measured at five ran-
dom locations on each substrate using 5 lL DI water droplet and
average values are presented with standard deviation. All other
measurements were carried out in triplicates at room temperature.
2.5. Algal culture
Batch cultures of D. salina (Culture Collection of Algae and Pro-
tozoa, UK, CCAP 19/18) were grown in seawater of 35 ppt (parts
per thousand) salinity and enriched with nutrients as described
by Guillard and Ryther [33]. Cultures were grown in aerated Erlyn-
mer flasks with continuous light irradiation of 150 lmol photons
m2 s1 at 30 °C. Growth rates were determined by the change in
cell number over time by counting using the haemocytometer
 P. Sathe et al. / Separation and Purification Technology 162 (2016) 61–67
(Sigma, USA). Initial algal cell concentration used for the experi-
ment was 6  107 cells/mL. A very high cell concentration was cho-
sen for the experiments which was 10,000 times higher than that
have been reported in algal blooms [34].
2.6. Photocatalytic inactivation of algae
Mini-reactors were constructed comprising of uncoated
polypropylene substrates as controls and ZnO nanorod coated
polypropylene substrates enclosed in a glass test tube (internal
diameter 1.5 cm and length 13 cm). Same weight (0.7 g) of
nanocoated and uncoated substrate was used to fabricate the
mini-reactors. 20 mL of fresh algal culture was added to the mini
reactors consisting of nanocoated and uncoated substrates and
kept overnight in the dark 0 W/m2 (0 lmol/m2 s) to saturate the
substrate surface with target algal cells. Later algal cultures in both
the mini reactors were replaced with 20 mL of fresh algal culture
prior to carrying out photocatalysis experiments. Photocatalysis
was carried out by irradiating algal suspension in contact with
each type of substrate using continuous visible light irradiation
for duration of 2 h with dual halogen lamp (24 V, 250 W). The dis-
tance between mini reactor and light source was maintained to
about 5 cm as shown in (Fig. 1). Light intensity measurement
was performed over the surface of the sample with light intensity
metre (ISO-TECH ISM 410, Taiwan). Light intensity measured over
the surface was observed to be 530 W/m2 (1549.70 lmol/m2 s).
Also second set of mini reactors were kept in dark 0 W/m2
(0 lmol/m2 s) and used as a dark control. After 2 h of photocataly-
sis process, approximately 5 mL aliquot of algal suspension was
withdrawn from each mini reactor for the investigation of anti-
algal activity. Reaction duration of 2 h was fixed depending upon
optimum activity of treatment unit observed at different times
(Supp. Info. Fig. S3).
2.7. Anti-algal activity measurement
2.7.1. Flow cytometry measurements
For the estimation of anti-algal activity, flow cytometry mea-
surements (FCM) were performed using BD FACSAriaTM III (BD Bio-
sciences, USA). FCM enables the counting, sorting or studying
single cell with different features or physiological states on the
basis of quantification of scattered and fluorescent light signals.
A special feature of the algae is the presence of photosynthetic pig-
ments exhibiting strong auto fluorescence that can be detected by
FCM detectors [35]. Flow cytometry acquisition was performed
using approximately 1 mL of algal suspension aliquoted at the
end of 2 h photocatalytic process. Chlorophyll a and b auto fluores-
cence was used as a measure of cell viability. Chlorophyll was
Fig. 1. Schematic representation of experimental setup used for investigation of anti-algal activity of treatment unit based on zinc oxide nanorod coated support substrates.
63
excited at 488 nm and emitted light was detected with red-
orange filter (660–695 nm). Total 10,000 cells were counted in
each flow cytometry acquisition.
2.7.2. Vital staining with trypan blue
Cell viability was assessed by trypan blue (Sigma Aldrich, USA)
staining using the haemocytometer (Sigma Aldrich, USA) and an
optical microscope (Carl Zeiss, Germany). For trypan blue staining,
the treated algal suspension was mixed with 0.4% (v/v) trypan
blue solution and visualized under a microscope using
haemocytometer.
% Cell mortality ¼ ðNumber of dead cells=Total number of cellsÞ
 100:
2.7.3. Monitoring algal cell re-growth after photocatalytic treatment
Algal cell suspensions from each type of mini reactors were col-
lected after 2 h of photocatalytic treatment and monitored for algal
cell re-growth for up to 2 weeks. Collected cell suspensions were
transferred to aerated Erlynmer flasks and maintained under static
conditions ideal for algal cell growth and maintained as described
in Section 2.5. Cell viability was tested after 24 h, 1 week and
2 weeks with flow cytometry and vital staining with trypan blue
as described in previous sections.
2.8. Toxicity testing
Toxicity of Zn2+ ions against D. salina cells was investigated
using zinc chloride (ZnCl2) (Sigma–Aldrich, USA) solutions in fil-
tered seawater. In this experiment, 1.5 mL algal cell suspension
was added to 64 wells of a multiwell plate containing 1 mL of
400 lg/L of ZnCl2 solution [24] or seawater (control). After 2 h, cell
viability was assessed with trypan blue staining and the results
were averaged.
2.9. Statistical analysis
In experiments 1 and 2 differences in number of viable cells
after treatment with nanocoated and uncoated substrates were
compared using Student’s t-test. In experiment 3, the effect of
the photocatalytic treatment and culture time of the re-growth
of algal cells was investigated by 2-way ANOVA. Differences due
to photocatalytic treatments were further investigated by Dun-
nett’s test. Before the statistical analysis, the normality of the data
was verified by the Shapiro-Wilk’s test. All calculations were per-
formed using Statistica version 11.0 (Stat Soft, USA) software. In
all cases, the threshold for significance was 5%.
64 P. Sathe et al. / Separation and Purification Technology 162 (2016) 61–67

3. Results and discussion

3.1. ZnO nanorod growth on support substrates

Fig. 2(A–C) shows scanning electron micrographs (SEM) of ZnO

nanorods on the support substrates (nanocoated). It is evident that
the ZnO nanorods are evenly distributed on the polypropylene sup-
port substrates with an average length and width of 1.5 ± 0.4 lm
and 100 ± 7 nm respectively (Fig. 2C). The width, length and den-
sity of the nanorods on the substrates are usually dependent on
the synthesis conditions, such as, different seeding processes, con-
centration of precursor solutions used in hydrothermal process as
well as growth time [29,36]. Thus the effective surface area avail-
able for photocatalysis is a function of the width and length of
the nanorods as well as the density of the nanorods covering the
Fig. 3. XRD pattern of typical uncoated polypropylene support substrates and ZnO
support substrates [20].
nanorod coated polypropylene support substrates (JCPDS card No. 36-1451).
Fig. 3 shows the comparison of XRD pattern of nanocoated and
uncoated substrates. XRD of nanocoated substrates showed hexag-
onal wurtzite structure of ZnO nanorods confirmed with 2h values Table 1
which are typically located at 31.8, 34.4, 36.2, 47.4, 56.5, 62.8 and Active surface area (m2) estimation of support substrates with and without ZnO
67.8 corresponding to (1 0 0), (0 0 2), (1 0 1), (1 0 2), (1 1 0), (1 0 3) and nanorods coatings. Active surface area (m2) was calculated by multiplying specific
surface area (m2/g) by weight of the support (g) substrate used for the experiment.
(1 1 2) crystal planes respectively (JCPDS card No. 36-1451) [26,29].
The active surface area of uncoated and nanocoated substrates Type of sample Weight Specific surface area Active surface area
used in this work are summarized in Table 1. It was calculated (g) (m2/g) (m2)

from the specific surface area and weight of the substrate. Three
independent measurements were carried out and average
value ± standard deviation (SD) is reported as an active surface
area of the sample. The higher surface area of the nanocoated sub-
strate (57% higher than uncoated substrate) is due to the additional
surface contribution of the ZnO nanorods.
3.2. Photocatalysis
Anti-algal activity of ZnO nanorods on D. salina microalga was
investigated through the photocatalysis process. All algae contain
chlorophylls and inactive degradation product of chlorophyll is
known as pheophytin which is found in damaged and dying cells.
Algal cells containing more chlorophylls than pheophytin are con-
sidered as viable cells [37]. Depending on concentration of these
pigments, dead and live cells can be differentiated and counted
with flow cytometry. From our experimental results, the percent-
age of viable cells significantly (Student’s t-test: p < 0.0001)
decreased (from 98% to 2%) after photocatalysis in the mini reactor
containing ZnO nanocoated support substrates (Fig. 4A). In com-
parison, the mini reactor containing uncoated substrate did not
show any significant mortality upon light irradiation (Student’s
t-test, p > 0.05).
The results of the flow cytometry experiment were supported
by trypan blue vital staining of algal cells. Algal mortality signifi-
cantly increased (Student’s t-test: p < 0.0001) up to 95% in the
presence of ZnO nanocoated substrates (Fig. 4B). On the other
hand, uncoated substrate did not show significant algal cell death
Uncoated 0.35 0.93 0.33 ± 0.02
substrate
Nanocoated 0.35 1.49 0.52 ± 0.03
substrate
upon light irradiation (Student’s t-test, p > 0.05). Moreover, under
dark conditions, algal mortality remained unchanged upon using
either of the uncoated or the ZnO nanocoated substrates.
Previous studies demonstrated that ZnO nanorods inhibited
growth of Gram-positive and Gram-negative pathogens upon irra-
diation with visible light [38,39]. Similarly, antifouling activity of
ZnO nanorods coatings against marine bacteria and larvae of bry-
ozoan was established in a previous work [24]. The present study
using two different methods of flow cytometry and vital staining
has demonstrated that number of viable algal cells significantly
decreased as a result of photocatalytic treatment.
In seawater, ZnO nanorods dissociate slowly and release Zn2+
ions [24]. Toxicity of Zn2+ ions [40,41] cannot attribute to the
observed reduction in number of viable D. salina cells during the
experiments. Our experiments with Zn2+ ionic solutions at concen-
trations similar to ones produced during photocatalytic treatments
have shown no mortality of D. salina cells after 2 h of photo-
irradiation (data are not shown). Similarly, the bryozoan Bugula
neritina larvae were not killed by concentrations of Zn2+ ions pro-
duced during photo-irradiation [24]. Also, pH of the test solutions
was monitored with algal cells before and after photo-irradiation.
In all the samples exposed to the uncoated and ZnO nanocoated

Fig. 2. Scanning electron micrograph (SEM) of typical ZnO nanorods grown on polypropylene support substrates (A) SEM of uncoated polypropylene substrate used as
support for ZnO nanorod growth (B) SEM of ZnO nanorods grown on a single fibre strand of support substrate (C) Cross sectional view of ZnO nanorods.
 P. Sathe et al. / Separation and Purification Technology 162 (2016) 61–67
Fig. 4. Effect of uncoated substrate (US) and ZnO nanocoated substrate (NS) in
presence of light (530 W/m2) or in dark (0 W/m2) conditions on Dunaliella salina
cells after 2 h of photo-irradiation. Data are presented as mean ± SD of 3 replicates.
⁄
– Data are significantly different (Student’s t-test: p < 0.0001) (A) Viable cell
percentage (number of viable cells over total number of cells in %) is determined by
flow cytometry measurements. (B) Viable cell percentage is determined by staining
with vital dye trypan blue. (For interpretation of the references to colour in this
figure legend, the reader is referred to the web version of this article.)
substrates, pH remains relatively constant within a range of 8.2–8.4
ruling out possibilities of pH dependent variations in cell viability.
These results conclusively demonstrate that the reduction in over-
all algal cell viability upon photocatalytic treatment in the presence
of ZnO nanorods was not due to changes in pH or toxicity of Zn2+
ions. Thus, the observed algal mortality can be attributed to the
reactive oxygen species (ROS) produced during photocatalytic pro-
cesses [22,42].
ZnO is a wide bandgap semiconductor (bandgap energy
3.37 eV), which absorbs light in the ultraviolet region. However,
by creating the surface defects in ZnO nanorods, it can be tuned
to absorb visible light. Hydrothermally grown ZnO nanorods pos-
sess mid-band defect states or quasi-stable energy states in the
form of oxygen vacancies and zinc interstitials, which shift its opti-
cal absorption from the ultraviolet to the visible region [43]. Thus
upon irradiation with visible light of photon energy (hm) P band-
gap energy of semiconductor materials, electron–hole pair genera-
tion takes place. These generated electron and holes are highly
reactive producing hydroxyl radicals (OH), superoxide anions
(O2 ) and hydrogen peroxide (H2O2) that damage the microbe cell
walls [22] (Fig. 5).
The presence of hydroxyl group on the polar facet of ZnO nanor-
ods as well as in surrounding medium and with the contribution of
dissolved oxygen in the medium, it is easy way to generate hydro-
xyl radicals and ROS. It has been reported that the fatty acid chain
can be affected by the free radicals deforming the lipids which
gives rise to a permanent rupture of cell membranes followed by
DNA damage [21,44]. Moreover, higher concentration of ROS spe-
cies could degrade polysaccharides, impact the cell walls, and
65
Fig. 5. Schematic representation of mechanism of microbial cell immobilization by
ZnO nanorods under visible light irradiation. Generated free electrons interact with
oxygen forming superoxides while the holes convert hydroxyl ions into highly
reactive hydroxyl radicals (OH). Reactive oxygen species (ROS) damages microbial
cell walls resulting in their inactivation.
degrade extracellular polymeric substances of cells, leading to
microbial cell inactivation [41].
Previously, it was shown that ZnO nanorods are capable of
modulating adhesion of macrophage [45] and bacterial cells
[25]. Within our experimental conditions, there is a high possibil-
ity that algal cells can get adsorbed on the surface of ZnO nano-
coated substrate. It has been previously reported that bacteria
loosely attach to ZnO nanorods coated surfaces [24]. In our study,
algal cells were found also to loosely attach to the surface of the
ZnO nanocoated substrates (Fig. 7) but can be easily removed.
Upon coating the support substrate with ZnO nanorods, which
is natively hydrophilic, slight decrease of water contact angle
(WCA) from 139° ± 2° to 119° ± 3° was observed (as shown by ⁄
in Fig. 6A) However, WCA reduced dramatically from 116° ± 3°
to 49° ± 2° for nanocoated substrates upon exposure to algal
medium under illumination. In this case, two possible scenarios
can be envisaged for the increased hydrophilicity (Fig. 6B): (1)
accumulation of negatively charged algal cells on ZnO nanorods
coated surface leading to higher surface energy; (2) increased
surface coverage of the substrate surface by adsorption of algal
cells leading to increased hydrophilicity [46,47]. However, for
uncoated substrates, water contact angle remained obtuse main-
taining its hydrophobicity (WCA 141° ± 5° to 119° ± 4°). There-
fore, reasonable explanation of this phenomenon is the
selective algal adsorption on hydrophilic ZnO nanocoated sub-
strate surface over hydrophobic uncoated substrates. Loosely
attached algal cells can be easily removed from the surface of
the nanocoated substrate by rinsing with water (data not shown).
The attachment of algal cells on nanocoated substrates after pho-
tocatalysis process is clearly seen as lysed algal cells in electron
micrographs (Fig. 7A, B). It was observed that there was no
significant change in the morphology of ZnO nanorods after
photocatalysis due to algal cells attachment (Supp. Info. Figs. S4
and S5).
In order to justify if the cell mortality is irreversible, possible
algal re-growth after photocatalytic treatment with ZnO nano-
coated substrate was monitored for up to two weeks. A consistent
and long term prevention of algal growth was observed. (Fig. 8)
Two-way ANOVA showed that both photocatalytic treatments
(ANOVA: F1,17 = 610.862, p < 0.0001) and time period (ANOVA:
F2,17 = 73.882, p < 0.0001) significantly affected density of algal
culture independently, as well as in combination (ANOVA:
F2,17 = 72.790, p < 0.0001). It is observed that the total number of
cells as determined by flow cytometry remains reasonably constant
for algal cells treated with nanocoated substrates, confirming the
complete inactivation of algal cells within 2 h of photocatalytic
66 P. Sathe et al. / Separation and Purification Technology 162 (2016) 61–67

Fig. 6. (A) Effect of algal cell adsorption on uncoated substrate and nanocoated substrate with measurement of surface wetting (water contact angle) before and after
treatment. (B) Schematic representation of charge distribution over nanocoated substrate surface before and after photocatalysis process.⁄ – decrease of Water Contact Angle.

Fig. 7. SEM images of (A) the algal cells immobilized on ZnO nanocoated substrate surfaces after 2 h of photocatalysis process (B) Magnified image of lysed cells on
nanocoated substrate surface after 2 h of photocatalysis.

Fig. 8. Total concentration of Dunaliella salina algal cells treated with mini reactor consisting of uncoated and nanocoated support substrates as determined by (A) flow
cytometry and (B) vital staining with Trypan blue. Total cell concentration was monitored over the time period of 2 weeks. Cells treated with uncoated substrate showed
significant increase (ANOVA, Dunnett p < 0.05) in number of cells over time period of 2 weeks. (For interpretation of the references to colour in this figure legend, the reader is
referred to the web version of this article.)

treatment. In cultures treated with uncoated substrates, re-growth 4. Conclusions

of algal cells was observed and algal cell numbers also increased
over time (Fig. 8A).
Vital staining by trypan blue confirmed these results obtained
by flow cytometry. The total number of algal cells treated with
ZnO nanocoated and uncoated support substrate is significantly
different (ANOVA, Dunnett’s test: p < 0.0001) as determined by try-
pan blue staining. Algal cells treated with ZnO nanocoated sub-
strates showed reasonably constant cell numbers over 2 weeks
confirming high algal inactivation efficiency and consistent perfor-
mance of the treatment unit consisting of ZnO nanorods coated
support substrates against algal growth (Fig. 8B).
We have conclusively demonstrated that nanocoated support
substrates are effective in reducing the density and viability of
marine green microalga D. salina. Up to 98% algal cell mortality
was observed upon visible light treatment for 2 h in the presence
of ZnO nanorod coated substrates attributed to the hydroxyl radi-
cals and superoxides produced during the photocatalysis process.
Long term anti-algal properties of water treated with ZnO nano-
coated substrates were demonstrated by monitoring algal cell
growth of treated culture suspension over a period of two weeks,
where no algal re-growth was observed. This suggests that the
 P. Sathe et al. / Separation and Purification Technology 162 (2016) 61–67
treatment unit comprising of non-woven polypropylene substrates
coated with ZnO nanorods prevents algal fouling in marine envi-
ronments and can be potentially used as a pre-treatment units in
water treatment plants.
Acknowledgements
The authors would like to acknowledge financial support of the
Chair in Nanotechnology, The Research Council (TRC). SD work was
supported by The Research Council (TRC) grant RC/AGR/FISH/16/01
and internal SQU grant IG/AGR/FISH/15/02. Authors also would like
to thank Ms. Lesya Pronoza for her help in toxicity experiments.
The authors would like to thank Ms. Muneera al-Shidhani (CAARU)
for her assistance in flow cytometry data analysis. The authors
would like to thank CAARU for the usage of Scanning electron
microscopy and Flow cytometry facility.
Appendix A. Supplementary material
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.seppur.2016.02.
007.
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