Combined photocatalytic degradation of pollutants and inactivation of waterborne pathogens using solar light active α/β-Bi2O3

Article abstract of DOI:10.1016/j.colsurfa.2021.126214

A solar light active composite of α/β-Bi₂O₃ was synthesized using a chemical-free solid-state reduction method. The obtained composite was characterized by X-ray diffraction, UV–vis spectroscopy, field emission scanning electron microscopy, and zeta potential. Initially, to validate the photocatalytic effectiveness, the obtained α/β-Bi₂O₃ composite was used to degrade indigo carmine dye. Then, the inactivation of E. coli and S. aureus waterborne pathogens was performed on solid and in liquid media. On solid agar media, a significant inhibition zone was observed for both bacterial strains. Similarly, in liquid culture, these strains E. coli and S. aureus were reduced from 1 × 10⁶ CFU/mL to a few CFU/mL, after 240 min of photocatalytic exposure. Furthermore, mixed wastewater of indigo carmine and E. coli/S. aureus were tested to study the combined photocatalytic mechanism against the organic dye and microorganisms. Overall, the obtained results suggested the efficacy of α/β-Bi₂O₃ towards visible light inactivation of bacteria even in combination with other pollutants, highlighting the great potential of the advanced photocatalytic process for combined treatment of organic pollutants and pathogens.

Full text of DOI:10.1016/j.colsurfa.2021.126214

Colloids and Surfaces A: Physicochemical and Engineering Aspects 615 (2021) 126214
Contents lists available at ScienceDirect
Colloids and Surfaces A: Physicochemical and
Engineering Aspects
journal homepage: www.elsevier.com/locate/colsurfa
Combined photocatalytic degradation of pollutants and inactivation of
waterborne pathogens using solar light active
α
/β-Bi O
2
3
a
a,
a
b,
Najeebullah Channa , Tanveer A. Gadhi *, Rasool Bux Mahar , Alessandro Chiad o` *,
b
b,c
Barbara Bonelli , Alberto Tagliaferro
a
U.S. Pakistan Center for Advanced Studies in Water (USPCASW), Mehran, University of Engineering and Technology, Jamshoro, 76062, Pakistan
Department of Applied Science and Technology, Politecnico di Torino, Corso Duca degli Abruzzi 24, 10129, Torino, Italy
Faculty of Science, Ontario Tech University, 2000 North Simcoe Street, Oshawa, Ontario, L1G 0C5, Canada
b
c
H I G H L I G H T S
G R A P H I C A L A B S T R A C T
•
•
•
The
α
/β-Bi
2 3
O composite was synthe-
sized with a scaleable thermal reduction
method.
α/β-Bi
Carmine
photocatalysis.
2
O
3
effectively degraded Indigo
organic pollutant by
The composite was able to inhibit E. coli
and S. aureus pathogens via produced
ROS.
•
•
The live/dead bacterial staining validate
the biocidal effect of the composite.
The effective photocatalysis of mixed
solutions of dye and bacteria was
proved.
A R T I C L E I N F O
A B S T R A C T
A solar light active composite of
Keywords:
α/β-Bi O was synthesized using a chemical-free solid-state reduction method.
2
3
Antibacterial
The obtained composite was characterized by X-ray diffraction, UV–vis spectroscopy, field emission scanning
Waterborne pathogens
electron microscopy, and zeta potential. Initially, to validate the photocatalytic effectiveness, the obtained
α
2 3
/β-Bi O
α
/β-Bi
2 3
O composite was used to degrade indigo carmine dye. Then, the inactivation of E. coli and S. aureus
Photocatalysis
Wastewater
waterborne pathogens was performed on solid and in liquid media. On solid agar media, a significant inhibition
zone was observed for both bacterial strains. Similarly, in liquid culture, these strains E. coli and S. aureus were
6
reduced from 1 × 10 CFU/mL to a few CFU/mL, after 240 min of photocatalytic exposure. Furthermore, mixed
wastewater of indigo carmine and E. coli/S. aureus were tested to study the combined photocatalytic mechanism
against the organic dye and microorganisms. Overall, the obtained results suggested the efficacy of
α
/β-Bi
2 3
O
towards visible light inactivation of bacteria even in combination with other pollutants, highlighting the great
potential of the advanced photocatalytic process for combined treatment of organic pollutants and pathogens.
*
Corresponding authors.
E-mail addresses: tanveer.uspcasw@admin.muet.edu.pk (T.A. Gadhi), alessandro.chiado@polito.it (A. Chiad o` ).
https://doi.org/10.1016/j.colsurfa.2021.126214
Received 30 November 2020; Received in revised form 21 January 2021; Accepted 24 January 2021
Available online 1 February 2021
0
927-7757/© 2021 Elsevier B.V. All rights reserved.

N. Channa et al.
Colloids and Surfaces A: Physicochemical and Engineering Aspects 615 (2021) 126214
1
. Introduction
2. Material and methods
The anthropogenic and industrial activities produce in their waste-
2.1. Materials
water (WW) high loads of organic and inorganic compounds, such as
dyes, resins, metals, nutrients and pathogenic bacteria, as well as other
by-products, classified as new emerging contaminants, like drugs and
antibiotics [1,2]. The presence of these pollutants resulted in
cross-contamination to surface water bodies with organic and inorganic
compounds alongside pathogens. For such a mixed stream, pathogens’
removal needs immediate attention due to the risk of waterborne dis-
eases and the possibility of microorganisms to develop antibiotic resis-
tance [3,4]. Many treatment systems and techniques are available to
remove pathogens from drinking water or WW, such as chlorination and
ultraviolet (UV) light [5,6]. Chlorination is mostly applicable to all
water systems, but it produces toxic by-products, and some of them are
carcinogens [7]. UV disinfection methods are widely used because of
their efficacy with a targeted attack on the DNA or RNA of the bacterial
cells. However, the UV treatment has some limitations, such as the high
costs, the energy requirements, and the reduced interactive exposure for
bulk applications [8,9].
3 3 2
Precursor salt of bismuth (Bi(NO ) :5H O), triethylamine (TEA), p-
benzoquinone (BQ), isopropanol (IP) and Indigo Carmine (IC) were
purchased from Sigma-Aldrich, Italy, and used as received. Ethanol and
sodium chloride (NaCl, 99.5 %) were purchased from Daejung, China.
For the bacterial analysis, Luria-Bertani (LB) agar and broth and Tryp-
tone Soya broth (TSB) were purchased from Oxoid, England. The LIVE/
DEAD® BacLight™ Bacterial Viability Kit was purchased from Thermo
Fisher Scientific, USA, for the live/dead cell staining.
2.2.
α
2 3
/β-Bi O synthesis
The
α
/β-Bi
2 3
O composite was synthesized in bulk powder form,
using a previously reported method that employed thermal decompo-
sition of Bi(NO
3
)
3
⋅5H
2
O [17]. In brief, a measured quantity of Bi
◦
(NO ) ⋅5H O salt was directly heated at 150 C for 30 min in the muffle
3
3
2
furnace i.e. to evaporate the moisture content. Afterward, the temper-
◦
Heterogeneous photocatalysis is an advanced oxidation process
considered a green approach. Indeed, it applies the irradiation and
activation of photocatalytic materials via UV/solar light to generate
reactive oxygen species (ROS) for the degradation and treatment of
contaminants, and some studies reported as well a bacterial inactivation
using photocatalytic metal oxide nanoparticles (e.g. titanium, copper,
and zinc-based) [10–12]. In principle, ROS attacks the cell wall/-
membrane and results in bacterial cells’ damage as revealed for instance
by live/dead cell viability using fluorescence microscopy [10,11]. In a
nutshell, all of these works highlight that there is a constant need for
efficient and stable photocatalytic active materials that could reduce the
water pollution problem and all of its consequences, including various
diseases resulting from pathogenic bacteria [11]. However, most of the
mentioned studies focused on either antimicrobial activity or the
removal and degradation of organic/inorganic pollutants alone.
Considering the mixed nature of WW, rare work has been done on
evaluating simultaneously the photocatalytic antibacterial activity and
organic pollutant degradation i.e. when the bacteria and organic com-
pounds are found together and interfering with each other.
ature was increased to 250 C and kept for 2 h. Finally, the resultant salt
◦
was calcined at 550 C for 2 h and allowed for ambient cooling inside
the furnace to obtain the thermally reduced and calcined bulk
composite.
α/β-Bi O
2
3
2.3. Characterization
The as-calcined powder morphology was investigated through a
MERLIN ZEISS field-emission scanning electron microscopy (FESEM).
The calcined powders were analyzed using X-ray diffraction (XRD) to
investigate both the crystalline structure and phase’s composition. The
UV–vis diffuse reflectance spectra were recorded by using a Varian Cary
5000 spectrophotometer (Agilent Technologies). The Tauc plots of the
Kubelka Munk function were used to calculate the energy band gap (Eg)
value. The Brunauer ꢀ Emmett ꢀ Teller (BET) specific surface area of
the obtained powder was measured through N adsorption at 77 K on a
2
Micromeritics Tristar-II instrument. The zeta potential was measured
through Malvern-Zetasizer, at neutral pH.
Bismuth-based materials are widely studied for the photocatalytic
2.4. Photocatalytic removal of indigo carmine
degradation of organic compounds [13–15]. Bismuth oxide (Bi
six polymorphic crystalline structures i.e. and : the
, β, δ, γ,
2
O
3
) has
and β
α
ε
ω
α
To confirm the synthesized
α/β-Bi O photocatalytic activity,
2
3
phases were mostly investigated for photocatalytic applications [15]. It
initially, the degradation of the IC dye was evaluated under a white LED
lamp (Phillips, emission spectrum ranging from 430 to 800 nm) with an
2 3
has been reported that Bi O has better solar/visible light efficiency due
2
to the wide absorption spectrum in the visible region and optimum
optical energy bandgap, if compared to other metal oxides. For instance,
irradiance of 100 W/m , placed 45 cm far from the dye solution. The IC
dye concentration was kept at 10 ppm, within a solution of 50 mL; the
compared to pristine TiO
higher under visible light [16].
In the present study, the use of solar light active
2
, the Bi
2
O
3
photocatalytic activity is two times
α/β-Bi O loading in the dye solution was 1 mg/mL (1:1). In the
2
3
beginning, the suspension was stirred for 30 min in the dark for
adsorption-desorption equilibrium before exposure to irradiation. The
dye solution’s absorbance spectra were recorded with different irradi-
ation time intervals using a UV–vis spectrophotometer (Shimadzu 1800)
by taking an aliquot of 3 mL centrifuged at 10,000 rpm for 3 min. The
powder sample and aliquot were returned to the vial to preserve the
same powder and solution amount. The amount of IC dye removed
during the treatment was estimated from the decrease of the absorbance
at 610 nm, corresponding to the principle peak in the spectra of the dye.
In order to confirm the dependence of ROS on the photocatalytic
degradation of the dye, such tests have been repeated in the presence of
known ROS scavengers/quencers (4% w/v) in the IC dye solution: TEA
α/β-Bi
2 3
O com-
posite, earlier reported for degradation of organic compounds [17], was
investigated for bacterial inactivation alone or combined with an
organic dye, to simulate a real mixed WW. To the best of our knowledge
this is one of the first attempt for the combined degradation of organic
pollutants and pathogens. The
2 3
α/β-Bi O composite was synthesized by
using a chemical-free solid-state thermal reduction method. The syn-
thesized bulk composite was characterized by X-ray diffraction (XRD)
for the analysis of crystal phase and composition, by UV–vis spectros-
copy for the optical properties, by scanning electron microscopy to
assess the morphological structure and zeta potential for the surface
charge properties. The obtained composite was initially tested for the
photocatalytic degradation of indigo carmine (IC) dye under visible light
and afterward evaluated for the antibacterial potential against two
bacterial strains i.e. the Gram-positive S. aureus and the Gram-negative
E. coli, by using the plate count method and fluorescence microscopy.
+
•
2
¡
for quenching h , BQ for the reactive oxygen (O ) and IP for the OH-
radicals.
2.5. Preparation of bacterial culture
Two bacterial strains were used to analyze the antibacterial activity
by
2 3
α/β-Bi O : E. coli (ATCC 8739), a Gram-negative considered an
2

N. Channa et al.
Colloids and Surfaces A: Physicochemical and Engineering Aspects 615 (2021) 126214
indicator of contaminations by bacteria in fresh/seawater and S. aureus
2.7. Fluorescence microscopy
(
ATCC 25923), a Gram-positive pathogenic bacterial strain generally
found not only in WW but even in hospital infections. E. coli was cultured
in LB broth (Oxoid) [18], whereas S. aureus was inoculated into TSB
During photocatalytic exposure, along with the bacterial density
reduction analysis, the withdrawn samples were analyzed for live/dead
bacterial cell staining by using the LIVE/DEAD® BacLightTM Bacterial
Viability Kit and fluorescence microscopy. This kit includes the SYTO 9,
a dye that stains green on a fluorescence microscope in a live bacterial
cell, and another dye, propidium iodide (PI) stains red in case of a dead
bacterial cell. The method for fluorescence analyses was followed as per
the guidelines provided by the viability kit supplier. In brief, 1 mL of the
(
Oxoid) [19,20]. Afterward, both bacterial strains were grown overnight
◦
in a shaker at 37 C and 120 rpm.The day after, 50 mL of both strains’
broth culture were placed in the centrifuge tubes and centrifuged at
5
000 rpm for 15 min. Bacterial biomass was separated and washed
several times with sterile 0.85 % NaCl solution [21], then diluted in the
same saline solution to get a final bacterial suspension of 50 mL pre-
6
pared at the concentration of 1 × 10 colony forming unit per mL
treated suspension (i.e. a mixture of bacterias and
after 0, 30, 60, 120, and 240 min, and centrifuged at 10,000 × g for
0 min, then the supernatant was drained. Further, the obtained bac-
α
2 3
/β-Bi O ) was drawn
(
CFU/mL).
1
2
2
.6. Photocatalytic bacterial inactivation
terial biomass was washed with a sterile washing buffer solution,
centrifuged again, and resuspended and vortexed with 1 mL of 0.85 %
.6.1. Antibacterial tests on solid media
NaCl. Then, 3 L of the mixed dye solution (PI: SYTO 9-1:1 (v:v)), was
μ
The antibacterial tests of E. coli and S. aureus were conducted using a
added to the resuspended solution, vortexed for thorough mixing, and
incubated for 15 min in dark conditions and at room temperature. Af-
modified Kirby–Bauer test, by placing the powders directly on agar
without any filter disk in between. The bacterial inoculum (100 L) at
μ
terward, 5 L of the stained suspension was pipetted onto a sterilized
μ
6
the concentration of 1 × 10 CFU/mL was spread on LB agar plates for
both strains. The test was then performed in the dark or under the same
white LED lamp used for dye degradation (see 2.4). In brief, 10 mg of
glass slide and analyzed on a Zeiss fluorescence microscope (Zeiss Axio
Scope. A1 Carl Zeiss Germany). The FITC and Texas Red filters were
used for acquiring the stained live and dead cells images, respectively.
The acquired images of live and dead cells were analyzed using ImageJ
1.50d to assess the percentage of live (green stained) and death (red
stained) bacterial cells.
α
/β-Bi
2 3
O powder was placed in defined circular spots on the bacteria
◦
inoculated petri dishes and then incubated overnight at 37 C under
dark conditions or irradiated with the LED lamp. The zone of inhibition
was calculated based on the method reported in [22].
3
. Results and discussion
2
.6.2. Antibacterial tests in liquid media
For photocatalytic evaluation of the inactivation of the same bacte-
3.1. Characterization of synthesized
α
2 3
/β-Bi O
rial strains in liquid cultures, 50 mg of
α
/β-Bi
2 3
O powder was added to
5
0 mL of the above-mentioned bacterial suspension at a concentration of
At first, the obtained powder was characterized using XRD, UV–vis
spectroscopy, FESEM, and zeta potential to investigate the composition
of the crystal phase and the optical, morphological, and surface charge
properties. Afterward, it was tested for photocatalytic removal of IC dye
and bacteria.
6
about 1 × 10 CFU/mL. The resulted suspension (of
α
2 3
/β-Bi O powder
and bacteria) was stirred in the dark for 30 min. Afterward, the photo-
catalytic response was observed with and without the presence of
◦
α
2
/β-Bi O
3
under the LED lamp at about 25 C, following the same con-
ditions used for dye degradation (see 2.4). A “dark control” (the bacte-
rial suspension incubated with the nanomaterial in the dark), was also
3.1.1. XRD
◦
included. 100
μ
L of microbial suspensions were drawn from each tested
The XRD pattern of the as-synthesized powder sample at 550 C and
condition after 30, 60, 120, 240 min, and were serially diluted. Finally,
commercial
α
-Bi
2
O
3
and β-Bi
2 3
O powders are shown in Fig. 1A. Most of
1
00
μ
L of each serially diluted samples were spread on the respective
the peaks correspond to monoclinic-
α-Bi
2
O
3
, with principal peaks at
◦
◦
agar plates and incubated overnight at 37 C for analyzing the reduction
in bacterial growth respective to different treatment time. Moreover, to
analyze the stability and reuse potential of the recovered bismuth-based
27.06, 27.52, 33.9 (JCPDS card no. 01-071-0465). Additionally, some
◦
2 3
peaks due to tetragonal β-Bi O at 27.96, 41.36, 46.22 and 55.45
(JCPDS card no. 01-078-1793) were also found, showing the occurrence
of a mixed composition of two different phases i.e.
and β with the
formation of an composite material of i.e. with around 20 %
/β-Bi
proportion of β-phase [17]. Most of the XRD peaks with minor intensity
are ascribed to -Bi as revealed after detailed analysis through the
PDXL2 software and comparison to XRD patterns of the commercial
-Bi powders.
It is known that when the metastable β-Bi
from high temperatures to ambient conditions, it is usually transformed
into - Bi unless some dopants, such as Tantalum or Niobium, are
nanomaterial, the tested
α
/β-Bi
2
O
3
powder was recovered, washed,
α
dried, and reused up to 3-cycles for antibaterial tests on solid media (as
reported above).
α
2 3
O
α
2 3
O
2
.6.3. Photocatalytic degradation of mixed pollutants and pathogens in an
artificial WW
For the photocatalytic evaluation of an artificial WW i.e. containing
IC dye and E. coli or S. aureus, the stock solution was prepared by adding
ppm of IC in sterilized 0.85 % NaCl. The overnight grown culture of
α
2 3
O
2 3
O phase is cooled down
5
α
2 3
O
E. coli and S. aureus was centrifuged to separate the pellets from the
broth. Then, the separated pellets were washed with 0.85 % NaCl so-
lution and resuspended in 50 mL of the prepared stock solution of IC to
make two mixed WW solutions i.e. one of IC and E. coli, and another of IC
and S. aureus. The initial concentration of bacteria in the mixed WW was
introduced to stabilize the metastable β-phase at room temperature
[23–25]. In one of our studies [26], we established that the Nitrogen
present in the precursor salt has a β-Bi
formation of
/β-Bi
the precrursor salt (Bi(NO
2
O
3
stabilizing role. Indeed, the
can be associated with the decomposition of the
⋅5H O) at increased temperature, with a
. With the complete decomposition of
α
2 3
O
3
)
3
2
6
maintained at around 1 × 10 CFU/mL. For photocatalytic evaluation,
consequential loss of NO and O
2
◦
the
α/β-Bi
2
O
3
powder was added in the mixed WW, and the obtained
NO above 540 C [26], some traces of N could remain in the bulk syn-
slurry was initially stirred in the dark for 30 min. Afterward, the irra-
diation was started, and the samples were collected after different
exposure time, centrifuged, and analyzed for removal of IC. The
collected samples were serially diluted for bacterial reduction analysis
through the plate count method and live/dead cell staining using fluo-
rescence microscopy (see 2.7). All the experiments and analyses were
performed at least twice for the reproducibility of obtained results.
thesized Bi
2
O
3
, stabilizing some of the β-Bi
2
O
3
at room temperature (i.e.
) and resulting in the for-
/β-Bi [17,23].
after most of the transformation in
α
-Bi O
2 3
mation of a composite heterostructure of
α
2 3
O
3.1.2. Optical properties
Fig. 1B shows the diffused reflectance UV–vis spectroscopy (DRS)
analysis of the synthesized sample, and the inset shows the
3

N. Channa et al.
Colloids and Surfaces A: Physicochemical and Engineering Aspects 615 (2021) 126214
Fig. 1. A) XRD pattern of synthesized bulk
α
/β-Bi
2
O
3
, compared to commercial powders. B) UV–vis DRS analysis of the synthesized
α
2 3
/β-Bi O , and the inset shows
the Tauc plot obtained from Kubelka-Munk method.
corresponding Tauc plot. The broad spectrum in the region from 400 to
could attain better colloidal stability in a suspension [29]. In addition,
the zeta potential was analyzed at different pH; the obtained values were
plotted and given in Fig. S1. In strong acidic conditions i.e. at pH = 2.0,
4
50 nm is attributed to
α
-Bi
2
O
3
, while the additional plateau i.e. from
[17]. In principle,
4
70 to 550 nm displayed the contribution of β-Bi
2 3
O
these absorptions are associated with the composite electronic transition
from the valence band to the conduction band [17]. The estimated
the net negative charge of the
α
/β-Bi
2 3
O was stabilized to zero due to
+
abundant availability of H ions. On the contrary, at basic pH ( = 10.0),
bandgap energy of
α
/β-Bi
2
O
3
was around 2.78 eV (shown in the inset
the negative ionic surface characteristic was increased due to the
ꢀ
Fig. 1B); the presence of the heterojunction of β-phase influenced the
slight reduction in the bandgap. Overall, considering the band gap value
and the corresponding optical transition in the visible region, these data
confirm that the nanomaterial can be fruitfully activated by visible light.
abundance of OH ions on the
α
/β-Bi
2
O
3
surface, reaching a value of
about ꢀ 47 mV.
Moreover, the recorded BET specific surface area of the
α
2 3
/β-Bi O
2
composite was in the range of 7.6 m /g, which suggests that the ob-
tained composite is not porous and will allow minimum adsorption on
the surface or within the macropores (shown in FESEM images, Fig. 2).
3
.1.3. Morphology
Fig. 2 shows the FESEM images of
α
/β-Bi
2
O
3
at different magnifi-
materials with
layered interconnected and flowery microstructure. similar
morphology of bismuth oxides is also reported in various studies [27,
cation scales, presenting the typical morphology of Bi
2
O
3
3.2. Photocatalytic removal of Indigo Carmine (IC)
A
Fig. 3A shows the absorbance spectrum of the IC dye at different
2
3
8]. The observed thickness of the interconnected layers was around
irradiation times in the presence of the
α
/β-Bi
2 3
O composite under
0ꢀ 45 nm. Interestingly, the tiny voids and spaces between the inter-
visible light, while Fig. 3B shows the relative concentration and kinetic
profile of IC dye. The obtained kinetics revealed that IC’s removal fol-
lows a 1st order linear degradation kinetics and a calculated kinetic
connected layers revealed some macroporosity of the composite mate-
rial, facilitating interaction with contaminants for improved
photocatalytic performance.
ꢀ 2
-1
apparent rate (Kapp) of 3.67 × 10 min . In our earlier work [17,30],
the IC dye’s degradation mechanism was explained in detail; in brief, the
oxidation species attacked the IC dye and dissociated into
amine-sulfo-benzoic acid and isatin sulfonic acid and partially miner-
alized to carboxylic groups and phenol derivatives [30–32].
3
.1.4. Surface characteristics
At neutral pH, the observed zeta potential of the
α
2 3
/β-Bi O composite
was around ꢀ 35 mV, suggesting that the negative net charge of the
scattering sites around the composite surface could facilitate the inter-
action with positively charged ions/molecules and, at the same time,
Fig. 2. FESEM images of
α
/β-Bi
2
O
3
at different magnification scales.
4

N. Channa et al.
Colloids and Surfaces A: Physicochemical and Engineering Aspects 615 (2021) 126214
Fig. 3. A) Absorbance spectra of the IC dye removal using
α
/β-Bi
2
O
3
under LED lamp; B) relative concentration decrease at different treatment time (considering the
optical density at 610 nm; each point is the average of two replicates); inset: kinetic curve plot and calculated kinetic apparent constant (Kapp).
3
.3. Photocatalytic bacterial inactivation on solid and liquid media
dye, the main focus was to evaluate the photocatalytic inactivation of
bacteria using the composite. Therefore, two pathogen in-
/β-Bi
dicators (commonly found in drinking water) i.e. E. coli and S. aureus
α
2 3
O
Once the composite was proved to be effective towards an organic
Fig. 4. A) Photocatalytic inhibition of E. coli and S. aureus on solid media in the presence of
α
/β-Bi
2 3
O under visible light irradiation; B) Bacterial density (CFU/mL)
reduction of E. coli (EC, left) and S. aureus (SA, right) liquid suspensions incubated under visible light with and without (control) the presence of
control” (bacterial suspension incubated with the nanomaterial in the dark), was also included.
α
/β-Bi
2
3
O . A “dark
5

N. Channa et al.
Colloids and Surfaces A: Physicochemical and Engineering Aspects 615 (2021) 126214
were selected. The observed inhibition halo for both bacterial strains is
shown in Fig. 4A. Compared to the biotic control (without the presence
the permeability of the membrane to PI staining [11].
Compared to the obtained results in Fig. 4B, the fluorescence images
in Fig. 5 confirmed a similar trend of higher inactivation of E. coli cells
than the S. aureus. For instance, by comparing the values obtained after
60 min of treatments, the higher inactivation of E. coli result significant
if compared to that of S. aureus (p value = 0.0467). The estimated
proportions of live and dead cells of both strains i.e. after prolonged
photocatalytic exposure of 240 min, are given in Table 1, which again
revealed an increased proportion of dead cells over live in the case of
E. coli. Anyway, for both strains, the time required for the complete
sanitation of water can be compatible with standard WW treatments.
of
α
/β-Bi
2
O
3
), the petri dishes with the presence
α
/β-Bi
2 3
O showed
growth inhibition of both E. coli and S. aureus under the LED light
irradiation, showing an inhibition halo for E. coli and S. aureus of around
1
1 mm and 10 mm, respectively. These results revealed the potential of
/β-Bi towards inhibition of both bacterial strains.
Due to the well-known limitations (e.g. mass transfer limitations,
α
2 3
O
absorption, and complexation to organic compounds) that affect metal
bioavailability during tests on solid media [33], the effectiveness of the
α
/β-Bi
2 3
O composite was evaluated in liquid cultures, too. The obtained
results, obtained by assessing the number of CFU/mL (shown in Fig. 4B),
confirmed the bacterial cell density reduction for both strains after
3
.5. Photocatalytic degradation of mixed pollutants and pathogens in an
2
40 min of photocatalytic exposure. Indeed, the initial bacterial density
6
artificial WW
(
1 × 10 CFU/mL for both strains) was significantly reduced (about
9.99 % reduction) for both E. coli and S. aureus, respectively. However,
9
To investigate the photocatalytic response towards IC and bacteria’s
combined presence, mixed WW of IC and E. coli/S. aureus were tested
with and without the presence of . The obtained results of this
the reduction was found higher in the case of E. coli. A “dark control” in
which the bacterial suspension was mixed with the nanomaterial but
incubated in the dark, was also included for each strain. The cell den-
sities recorded during time for these dark controls were comparable to
those obtained by the controls cultured under the visible light, but in the
2 3
α/β-Bi O
artificial WW for both bacterial strains are shown in Fig. 6. As compared
to the previous results of IC removal alone, the removal kinetics of IC
ꢀ
2
-1
was reduced in the mixed WW i.e. from 3.67 × 10 min to
absence of the
α
/β-Bi
.4. Fluorescence microscopy of live/dead bacterial cells
To confirm if the has a biocidal effect on the bacterial
/β-Bi
2 3
O composite.
ꢀ 2
-1
0
.8 × 10 min , as estimated from the relative concentration decrease
in Fig. 6A. The decreased kinetics was probably due to the relative op-
tical density of bacterial suspension that hindered the irradiation pas-
3
sage and
α
/β-Bi
2 3
O activation, and besides, limited reactive species that
α
2 3
O
could have simultaneously targeted both the bacteria and IC dye
molecule and reduced the overall kinetics. Further, Fig. 6B shows the
control of both mixed WW solutions i.e. with and without the presence
strains, live/dead staining was adopted in combination with fluores-
cence microscopy. The fluorescent green-stained cells represent live
cells, whereas the red-stained represent dead ones. The acquired merged
live/dead fluorescence microscopy images of E. coli (top), and S. aureus
Table 1
(
bottom) are shown in Fig. 5, while the separate live and dead stained
Proportions of live and dead bacterial cells at different treatment times.
cells are shown in supplementary data Fig. S2. For both strains, in the
beginning, plenty of live green-stained cells could be observed, even if
the number of dead cells (red-stained) start to increase already after
E. coli
S. aureus
Time (min)
Live %
Dead %
Live %
Dead %
1
5 min for both strains. In contrast, at increasing photocatalytic expo-
15
30
60.24 ± 4.07
43.04 ± 3.32
19.12 ± 1.94
12.33 ± 0.18
0.33 ± 0.06
88.96 ± 0.76
39.76 ± 1.23
56.96 ± 1.40
80.88 ± 1.69
87.67 ± 5.19
99.67 ± 14.47
11.04 ± 0.25
90.56 ± 12.89
67.19 ± 9.21
35.47 ± 1.28
28.31 ± 1.05
1.90 ± 0.02
9.44 ± 1.74
32.81 ± 5.27
64.53 ± 4.19
71.69 ± 3.19
98.10 ± 2.42
22.88 ± 0.63
sure, the bacterial cells in the presence of resulted in the cell
α/β-Bi
2 3
O
6
1
2
0
membrane’s rupture, as indicated by the increasing number of red
stained cells during the treatment. The increased appearance of red-
20
40
stained cells at longer exposures revealed that
α
2 3
/β-Bi O , along with
Control*
77.12 ± 2.35
growth inhibition, could kill both bacterial strains. Potentially, during
the photocatalytic reaction, the nanomaterial produces ROS that could
attack the bacterial cell and leads to bacterial cell damage that increased
*
The control corresponds to a bacterial suspension irradiated with visible
light for 240 min, without the photocatalyst’s presence.
Fig. 5. Merged live/dead fluorescence microscopy images of E. coli and S. aureus at different treatment times: 15 min, 30 min, 60 min, 120 min, 240 min, and images
of controls (without ) after 240 min.
/β-Bi
α
2 3
O
6

N. Channa et al.
Colloids and Surfaces A: Physicochemical and Engineering Aspects 615 (2021) 126214
Fig. 6. A) Relative concentration decrease of IC alone and in mixed wastewater (WW) with E. coli/S. aureus with and without the presence of
α
/β-Bi
2 3
O ; B) Bacterial
density reduction of both E. coli and S. aureus in mixed WW with IC with and without the presence of
SA is referred to a mixed WW of IC and S. aureus.
α
2 3
/β-Bi O (Mix-EC referred to a mixed WW of IC and E. coli, Mix-
of
α
2
/β-Bi O
3
, the bacterial growth of both bacteria remained unaffected
advantage in the case of WW plants.
due to the presence of IC. Compared to the previous results (Fig. 4B),
almost a similar bacterial density reduction trend was observed for both
bacterial strains. The inactivation was more effective towards E. coli
than S. aureus. The control of all the mixed WW showed little change in
either removing IC (alone and in the mixed WW) or in the bacterial
density after prolonged exposure of irradiation under LED lights if the
composite was not added. The overall results revealed that the presence
of bacteria interfered with the photocatalytic degradation and removal
of the organic compound, as observed by reduced IC dye kinetics in the
mixed WWs. On the contrary, the inactivation of both bacterial strains
was unaffected in the presence of IC, as the observed density reduction
trend was similar compared to the bacterial suspension without the
presence of IC dye.
3.7. Photocatalytic mechanism
From the overall results, the encountered photocatalytic mechanism
of action of the
α
/β-Bi O powder can be explained. Previously, we re-
2
3
ported that ROS were generated on the
α
/β-Bi O surface due to resul-
2
3
ꢀ
+
tant reactions of the photogenerated electrons (e ) and holes (h ) with
•
¡
•
the dissolved O and OH radicals [17]. The cascade of photogenerated
2
ꢀ
+
e
and holes h shown in Fig. 7 i.e., photocatalytic induced reactions,
was facilitated due to heterojunction between
α
and β phases [17].
To further explore the photocatalytic activity and proof the oxida-
tive/reductive paths, we did some photocatalytic tests with and without
the addition of some reagents for the scavenging/quenching of holes and
+
ROS. Indeed, some researchers have used TEA for quenching h , BQ for
•
¡
2
3
.6. Stability and reuse of
To evaluate the
/β-Bi
α
/β-Bi
2
O
3
the reactive oxygen (O
) and IP for the OH- radicals [27,34,35], to
observe any significant change in the degradation kinetics due to their
presence in the dye solution. The addition of quenchers could influence
the photocatalysis process i.e. by reducing the dye degradation kinetic
rate because of the quenching of the generated reactive species or holes
around the photocatalyst surface. Therefore, the degradation of the IC
dye solution (10 ppm) was investigated under visible light, using
α
2 3
O
powder’s reuse and stability potential, it
was recovered, washed, dried, and reused for up to 3-cycles, for anti-
baterial tests on solid media and to observe any change in photocatalytic
response. The observed inhibition zone on inoculated petri dishes in the
presence of the recovered
α
/β-Bi
2
O
3
powder, shown in supplementary
/β-Bi for
data Fig. S3, revealed the stability and reusability of
α
2
O
3
α
/β-Bi
BQ, and IP.
Fig. 8 shows the C/Cₒ plots, and the kinetics curves along with the
2 3
O , with and without the presence of these quenchers i.e. TEA,
subsequent cycles of photocatalytic inactivation. This result highlight
that this nanomaterial can be used more than one time, which is an
Fig. 7. Mechanism of origination of reactive species at
α
2
/β-Bi O
3
surface, degradation of IC dye, and inactivation of E. coli and S. aureus bacterial cells.
7

N. Channa et al.
Colloids and Surfaces A: Physicochemical and Engineering Aspects 615 (2021) 126214
Fig. 8. A) Relative concentration of IC with and without quenchers; B) kinetic curves and Kapp
.
determined Kapp of IC dye degradation with and without the quenchers
in solution. It was observed that the degradation rate was drastically
reduced in the presence of each quencher compared to the results
inactivate both microorganisms through the cell wall/membrane’s
rupture. At the same time, E. coli was inactivated more rapidly than
S. aureus, which appeared to be more resistant to the ROS originated by
•
¡
without their addition, which revealed that both the O
icals were originated from the
2
and OH- rad-
the
α
/β-Bi
2
O
3
composite’s photocatalytic action.
α/β-Bi
2
O
3
surface and were responsible
for the removal of the IC dye. Moreover, this also supports that the
4. Conclusion
+
electrons are well separated from the h in generating the reactive
species quenched with quencher addition and decreasing degradation
The
thermal reduction of bismuth nitrate salt at 550 C. The obtained com-
posite with a balanced proportion of
-phase and the β-Bi O phase
contributed to the effective removal of IC dye and inactivation of Gram-
negative E. coli Gram-positive S. aureus. In the presence of
/β-Bi O , the
significant inhibition zone on the solid bacterial culture was observed,
and bacterial cell density reduction in liquid suspension was achieved.
The live/dead fluorescence microscopy analyses of the treated bacterial
suspension revealed an almost complete biocidal effect after the pro-
longed photocatalytic exposure up to 240 min. Further, the photo-
catalytic evaluation of the mixed WW of IC and E. coli/S. aureus revealed
that the removal kinetics of IC was affected and reduced due to inter-
ference of both bacteria, while the photocatalytic inactivation of both
strains remained unaffected even with the presence of IC. Finally, even if
a detailed photocatalytic investigation must be considered, the pre-
α/β-Bi O composite was obtained through facile solid-state
2
3
◦
kinetics. The ROS originated on the
α
/β-Bi
2 3
O surface, probably could
have degraded the organic dye, as well as the bacterial membrane/cell-
wall [17,36]. In the case of organic dyes, these reactions are responsible
for partial or complete degradation of the parent molecule i.e. with the
α
2
3
α
2
3
formation of intermediates and mineralization into CO
2
, H
2
O, NH
3
etc.
[
30,36]. Instead, in the case of bacteria, the ROS, and in particular the
•
•
¡
hydroxyl radicals ( OH), the superoxide (O
2
) and H O originated by
2 2
the photocatalytic reactions probably attacked the cell-wall/membrane
and their constituents, resulting in different kinds of damage, as shown
in the scheme given in Fig. 7. Indeed, it is widely known that these
molecules can cause the peroxidation of lipids and phospholipids, as
well as the oxidation of proteins present on the membranes [37]. These
reactions can disrupt the membrane itself or simply its lipid bilayer
organization, eventually leading to the efflux of cytosolic contents or cell
lysis. Indeed, ROS have potent antimicrobial activity against bacteria,
fungi and viruses, due to their quick action against various
sented data revealed that
α/β-Bi O was able to attack E. coli and
2
3
S. aureus and showed the potential for the combined treatment of
Gram-positive
and
Gram-negative
bacteria,
including
organic pollutants and microbial pathogens.
multidrug-resistant strains [38]. This mechanism of action has been
suggested for well-known metal such as silver [39], or other metal ox-
ides applied as antimicrobial [19]. Moreover, it is consistent with the
Author contributions
earlier reported results of the efficacy of β-Bi
2 3
O on E. coli and S. aureus
T.A.G. and A.C. conceived the presented idea, decided the methods
to be used and supervised the project, N.C. and T.A.G. carried out the
experiments, N.C., T.A.G. and A.C. analysed the data and prepared fig-
ures, tables and artworks, N.C., T.A.G. and A.C. prepared the first draft
of the manuscript, R.B.M., B.B. and A.T. obtained the funding. All au-
thors discussed the results and contributed to the final manuscript.
[
21]. These strains belong to different microbial groups, namely
Gram-negative and Gram-positive, and have different characteristics of
cell wall/membrane thickness, morphology, and chemical groups [40].
Therefore, both bacteria’s photocatalytic rupture could have occurred
2
differently, as reported for TiO [41]. The Gram-negative bacteria like
E. coli has an outer membrane rich of lipopolysaccharides and phos-
pholipids, a thin layer of peptidoglycan, and an inner phospholipidic
membrane. Whereas, Gram-positive bacteria, like S. aureus, have a thick
cell wall of peptidoglycans that covers the cytoplasmic membrane.
Furthermore, it is well-known that bacteria belonging to Staphylococci
groups can actively produce catalase, a heme protein enzyme that de-
composes hydrogen peroxide to water and oxygen, protecting the cell
from hydrogen peroxide-mediated leukocyte bactericidal mechanisms
Declaration of Competing Interest
The authors report no declarations of interest.
Acknowledgments
The authors acknowledge the technical support of Prof. Jennifer
Weidhaas from the University of Utah, USA, Prof. Francesca Bosco, and
Dr. Pravin Jagdale from Politecnico di Torino, Italy. This study was
financially supported by the United States Government and the Amer-
ican people through the United States Agency for International Devel-
opment (USAID) and USPCAS-W, MUET research project funding (grant
[
42]. Moreover, Staphylococci can usually produce exopolysaccharides
that could increase the resistance against the antibiotics and antimi-
crobial agents [43]. All of these considerations match with the obtained
results. The high proportion of dead (red-stained) cells for both the
treated strains suggested that the originated ROS could attack and
8

N. Channa et al.
Colloids and Surfaces A: Physicochemical and Engineering Aspects 615 (2021) 126214
number USPCASW-RGMC-Project-23). The contents are the authors’
sole responsibility and do not necessarily reflect the views of USAID or
the United States Government.
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