See discussions, stats, and author profiles for this publication at: https://www.researchgate.

net/publication/339031691

Dual Valorization of Olive Mill Wastewater by Bio-Nanosynthesis of

Magnesium Oxide and Yarrowia lipolytica Biomass Production

Article in Chemistry & Biodiversity · February 2020

DOI: 10.1002/cbdv.201900608

CITATIONS READS

38 1,008

5 authors, including:

Selma Hamimed Nejib Jebli

University of Jijel Ghent University: Faculty of Bioscinece Engineering / Carthage University: Faculty…
32 PUBLICATIONS 188 CITATIONS 15 PUBLICATIONS 71 CITATIONS

SEE PROFILE SEE PROFILE

Hanen Sellami Ahmed Landoulsi

Centre de Recherche et des Technologies des Eaux Institut Jacques Monod
49 PUBLICATIONS 811 CITATIONS 223 PUBLICATIONS 2,573 CITATIONS

SEE PROFILE SEE PROFILE

Some of the authors of this publication are also working on these related projects:

Water science New doctors search for jobs View project

The role of inflammatory pathways for prevention and treatment of cancer using dietary agents and traditional medicine View project

All content following this page was uploaded by Selma Hamimed on 01 October 2020.

The user has requested enhancement of the downloaded file.

 2018 2018
VOLUME 15 VOLUME 15
NO. 11 NO. 11

Cover Picture
Q. Wang, Y.-Q. Zhen, F. Gao, S. Huang, X.-L. Zhou,
Chem. Biodiversity 2018, 15, e1800386.
hyris Five New Diterpenoids from the Seeds of Euphorbia lathyris
DOI: 10.1002/cbdv.201900608 FULL PAPER

Dual Valorization of Olive Mill Wastewater by Bio-Nanosynthesis of

Magnesium Oxide and Yarrowia lipolytica Biomass Production
Selma Hamimed,*a Nejib Jebli,b Hanen Sellami,*c Ahmed Landoulsi,a and Abdelwaheb Chattia, c
a
Laboratory of Biochemistry and Molecular Biology, University of Carthage, Faculty of Sciences of Bizerte, CP
7021-Jarzouna, Tunisia, e-mail: selma.hamimed@yahoo.com
b
Laboratory of Hetero-Organic Compounds and Nanostructured Materials, University of Carthage, Faculty of
Sciences of Bizerte, CP 7021-Jarzouna, Tunisia
c
Laboratory of Wastewater Treatment, Water Research and Technology Center CERTE, Technopole Borj Cedria,
CP 8020-Soliman, Tunisia, e-mail: sellami_hanen@yahoo.fr

This research investigates an efficient dual valorization of olive mill wastewater in the biosynthesis of
magnesium oxide nanoparticles and in the depollution of the effluent by Yarrowia lipolytica growth evaluation.
After removal of polyphenols, the recovered biophenols were reacted with the magnesium precursor to provide
magnesium oxide nanoparticles. In order to confirm the biosynthesized magnesium oxide nanoparticles, several
analyses were undertaken. The Fourier transform infrared spectrum gives a broad absorption at 658 cm@1
confirming the presence of the magnesium oxide nanoparticles, while the UV/VIS absorption spectroscopy
reveals an intense transition with a maximum absorption at 300 nm. The X-ray diffraction and transmission
electron microscopy analyses show that nanoparticles are in pure cubic crystalline with spherical and hexagonal
shapes (average size is 19.4 nm). The zeta potential analysis illustrates a negative potential proving a good
stability of the biosynthesized nanoparticles. Nanoparticles were assigned for their in vitro antibacterial activity
against Escherichia coli, Enterobacter aerogenes, Salmonella typhimurium, Staphylococcus cohnii, and Bacillus
niacini. The evaluation of the growth of Yarrowia lipolytica on the recovered olive mill wastewater after removal
of polyphenols yielded 3.2 g/L of the Yarrowia biomass in 72 h without nutriment additions, providing an
important decrease of chemical oxygen demand (73 %).

Keywords: polyphenols, olive mill wastewater, magnesium oxide nanoparticles, Yarrowia lipolytica, antibacterial
activity.

Previous studies have also investigated the valoriza-

Introduction
Olive mill wastewater (OMW) is an agro-industrial
waste obtained from olive oil production. OMW is
typically harmful to the environment due to its high
chemical oxygen demand (COD) (80–200 g/L) and to
the presence of several phenol-type molecules (0.5–
24 g/L).[1,2] Due to its rich composition with phenolic
alcohols, phenolic acid, flavonoids, secoiridoids, and
lignans, several processes have been examined for the
treatment of OMW. Biological and physico-chemical
methods such as dilution, pyrolysis, coagulation-floc-
culation, electro-coagulation, filtration (micro, ultra
and nano), and many others process (aerobic or
anaerobic bioreactors, composting) were used.[3 –6]
tion of OMW by extraction and separation of their
compounds for further use in advanced applications
and/or to reduce its pollution charge. Moreover, the
recovery of phenolic compounds from OMW by liquid-
liquid solvent extraction has received a particular
attention. Ethyl acetate remains to be the preferred
solvent for such extraction.[7] The removal of phenol
compounds reduces the pollution charge of the
OMW.[8] In addition, biophenols of OMW have at-
tracted considerable attention as an economic and
natural source for several applications including food
products,[9] and biological activities such as
antioxidant,[10] antimicrobial,[11] anti-inflammatory,[12]
and cardioactive activities.[13]

Chem. Biodiversity 2020, 17, e1900608 © 2020 Wiley-VHCA AG, Zurich, Switzerland
 Chem. Biodiversity 2020, 17, e1900608
In the previous works, the application of OMW
biophenols in the domain of nanotechnology has
been rarely studied. Keyrouz et al.[14] proved that the
phenolic compounds of seaweed could act as reduc-
ing agents. By using rice husk waste, Liu et al.[15]
succeeded in the synthesis of silver nanoparticles and
confirmed the role of phenolic acids as bio-reducing
agents.
Nowadays, the exploitation of green and economic
sources in the synthesis of metal oxide nanoparticles is
of interest. Particularly, magnesium oxide nanopar-
ticles (MgO NPs) attract scientific community interest
due to their wide use in several domains. MgO NPs are
known as a safe material and are able to withstand
harsh process conditions.[16] Many physico-chemical
methods were developed for the synthesis of MgO
NPs including thermal decomposition,[17] sol-gel,[18]
chemical precipitation/aqueous wet chemical,[19]
surfactant,[20] flame spray pyrolysis,[21] hydrothermal/
solvothermal,[22] chemical gas phase deposition,[23]
laser ablation,[24] hot-melt extrusion,[25] and micro-
wave-assisted method, etc.[26] In addition, most of
these pathways suffer from the persistence of some
toxic chemical reagents adsorbed on the surface of
MgO NPs, which may cause harmful effects in medical
applications.[27]
In this work, it was found interesting to utilize the
biosynthesis method for the preparation of magnesi-
um oxide nanoparticles by using the polyphenols
recovered from olive mill wastewater. Many analyses
were utilized to identify the biosynthesized MgO
nanoparticles including X-ray diffraction (XRD), UV/VIS
spectroscopy, Fourier Transform Infrared (FT-IR) spec-
trum, Transmission Electron Microscopy (TEM), and
zeta potential. The phenol-free OMW was also treated
using Y. lipolytica in order to reduce the pollution of
OMW and to survey the biomass production of this
strain avoiding external nutriment additions.
Results and Discussion
Physicochemical Characterization of Olive Mill
Wastewater before and after Phenols Extraction
Table 1 shows the physicochemical characteristics of
OMW before and after phenols extraction. The
obtained results revealed a decrease on COD, total
solid (TS), total solid suspended (TSS), lipids and
phenols parameters, accompanied with a slight in-
crease of pH after phenols extraction.
www.cb.wiley.com (2 of 12) e1900608
Table 1. Chemical characterization of the olive mill wastewater.
Parameters OMW EOMW
pH[a] 4.76 � 0.2 5.12 � 0.1
Total solid [g L@1][a] 151.11 � 5.23 118.76 � 5.79
Total solid suspended [g L@1][a] 21.2 � 2.60 17.03 � 2.29
Phenols [gallic acid] [g L@1][a] 11.67 � 0.4 6.38 � 0.38
COD [g L@1][a] 189.70 � 0.2 96.80 � 0.1
Lipids [%][a] 9.0 � 0.01 –
[a]
Values are the averages of results obtained in triplicate � SD;
OMW: olive mill wastewater; EOMW: extracted olive mill
wastewater; @: absence.
Evaluation of Yarrowia lipolytica Growth on Olive Mill
Wastewater after Polyphenols Extraction
Yarrowia lipolytica has been used as a good candidate
in the treatment of olive mill wastewater, although the
concentration of polyphenols compounds in the
medium could limit its growth.[28] In this study, the
evaluation of Y. lipolytica growth on olive mill waste-
waters (crude and residual after partial removal of
polyphenols ‘EOMW’) is presented in Figure 1. An
important growth of Y. lipolytica after the partial
removal of phenols from OMW was noticed, which
could explain the efficiency of this biological treat-
ment, performed in the absence of nutriment addi-
tions. It should be also mentioned that a decrease of
the COD value within olive mill wastewater for about
73 % after 72 h of incubation was observed (Figure 2).
With these results, it is clear that the growth of Y.
lipolytica varies depending on the polyphenols con-
tent. When compared with some previous works on
the growth of Y. lipolytica using diluted OMW, nutri-
ment additions,[29] jar fermenter and bioreactor,[30] the
Figure 1. Effect of the olive mill wastewater (OMW) and
extracted olive mill wastewater (EOMW) on biomass concen-
tration (mg/mL) of Y. lipolytica. The results are expressed as
means � SD.
© 2020 Wiley-VHCA AG, Zurich, Switzerland
 Chem. Biodiversity 2020, 17, e1900608

the results reported by Dobrucka.[32] The band gap

energy was calculated by using the following
equation.[33]

Eg ¼ 1240=wavelength

The calculated band gap energy of the MgO NPs is

4.13 eV, similar to the value reported in previous
works.[34]

Fourier Transforms Infrared (FT-IR) Spectroscopy of MgO

NPs
Figure 2. COD (%) of the olive mill wastewater after phenol
extraction and treatment using Y. lipolytica. The results are
In order to gain more information (the functional
expressed as means � SD. groups and the types of the existing bends) on the
structure of the OMW polyphenols and the prepared
MgO NPs, a vibrational study using infrared absorption
was undertaken.
present treatment on the growth potential on EOMW The FT-IR spectrums of polyphenols extract and the
in shaker-flasks within 72 h seems to be more effective MgO NPs nanoparticles are shown in Figure 4. The
either in time duration or without any nutriment attribution of the most characteristic vibrational
addition. Further investigations on this treatment with modes is given in Table 2. The assignments of the
more details are ongoing. observed bends are comparative to the data reported
for similar compounds.[31,35]
In the high-frequency region, the OMW extract
Biosynthesis of MgO NPs by Using the Recovered
spectra shows a strong absorption band at 3343 cm@1,
Polyphenols
which is assigned to the OH stretching vibration of
UV/VIS Spectroscopy of MgO NPs. UV/VIS spectroscopy polyphenols. The bands observed at 2976 and
was used as a preliminary analysis to confirm the 2931 cm@1 are acquired to asymmetric and symmetric
biosynthesis of metal oxide, to determine the surface CH and CH2 stretching vibration of polyphenolic
plasmon resonance band (SPR), and to identify the compounds. The aromatic C=C stretching vibration of
band gap energy of metal oxide nanoparticles.[31] As aromatic moieties appears at 1654 cm@1. The absorp-
depicted in Figure 3, a clear SPR is observed with a
maximum absorbance at 300 nm, in agreement with
Table 2. FT-IR major peaks analysis.
Vibrational Frequency Assignments
[cm@1]
Polyphenols extract
3343 νasym OH
2976, 2931 νasym C@H
νsym C@H
1654 νasym C=C stretching
νsym C=C stretching
1382 νasym C@H bending
νsym C@H bending
1088, 1048 ν O@H in-plane-w vibration
881 ν aromatic C@H out of plane
bending
MgO NPs
3399, 1637 νstr O@H
1560, 1416 νasym O@H bending
νsym O@H bending
Figure 3. UV/VIS spectrum of MgO NPs nanoparticles synthe- 1023 ν O@H in plane bending
sized using OMW polyphenols extract. 658 νstr Mg@O Nps

www.cb.wiley.com (3 of 12) e1900608 © 2020 Wiley-VHCA AG, Zurich, Switzerland

 Chem. Biodiversity 2020, 17, e1900608

Figure 4. FT-IR spectrums of polyphenols extract and MgO NPs.

tion band at 1383 cm@1 corresponds to the C@O

stretching vibration. The O@H in-plane-w vibration is
observed at 1088 cm@1. The band at 881 cm@1 owes to
the aromatic C@H out of plane bending vibration.
In the MgO NPs spectra, the broad absorption
peaks at 3399 cm@1 and 1637 cm@1 are respectively
due to the stretching and the bending vibration of
O@H. The peak at 658 cm@1 is assigned to the Mg@O
stretching vibration in Mg(OH)2.[26] The absorption
peaks at 1416 and 1560 cm@1 are assigned to the
bending vibration of OH on the surface of MgO NPs.
The comparison of both recorded FT-IR spectrums Figure 5. X-ray diffraction analysis of MgO NPs.
(OMW extract and MgO NPs) shows the disappearance
of absorption bands corresponding to the phenols
and their derivatives and the appearance of new cubic (FCC) MgO that related to the angles 36.86°,
bands in favor of MgO NPs formation, mainly the 42.82°, 62.16°, 74.51°, and 78.44° associated with the
broad band at 658 cm@1, which is associated with the planes (111), (200), (220), (311) and (222), respectively.
special vibration of magnesium oxide These results are well matched with the standard data
nanoparticles.[36,37] These results revealed that phe- of JCPDS No. 71 –1176.[38] The peak at the range of
nolic compounds interact with metal as bio-reducing 40.49° corresponds to (101) of the hexagonal Mg(OH)2
and capping agents. crystalline structure (JCPDS No. 35 –821).[39] Thus, the
obtained XRD pattern indicates clearly a successful
synthesis of pure biogenic MgO NPs. The average
XRD Pattern of MgO NPs
crystal size calculated by using Scherrer’s formula
The obtained white powder was analyzed by X-ray applied on the major peak (42.82°) is around 19.4 nm.
diffraction to confirm the formation, the structure and
the size of MgO NPs, synthesized by using biophenols
of olive mill wastewater (Figure 5). The diffraction
indicates the presence of crystalline face-centered

www.cb.wiley.com (4 of 12) e1900608 © 2020 Wiley-VHCA AG, Zurich, Switzerland

 Chem. Biodiversity 2020, 17, e1900608
Zeta Potential Analysis of MgO NPs
The zeta potential is an important indicator of the
surface charge of nanoparticles in colloidal dispersion.
The zeta potential analysis of MgO NPs suspended in
Milli-Q water shows a negative potential value around
@12.5 mV, which indicates the stability of MgO NPs
(Figure 6). These negative potential could be owing to
the presence of oxygen atoms on the surface of
MgO.[40]
Morphological and Elemental Analysis of MgO NPs
The structural characterization, the morphology, and
the size distribution of the biosynthesized MgO NPs
were performed by transmission electron microscopy
(TEM) (Figure 7A– 7D). The morphology of MgO NPs is
Figure 6. Zeta potential analysis of MgO NPs.
Figure 7. (A, B, D) TEM images of the biosynthesized magnesium nanoparticles, (C) TEM of face-centered cubic MgO nanoparticles,
(E) EDX spectrum, (F) particle size distribution of biosynthesized MgO NPs.
www.cb.wiley.com (5 of 12) e1900608
predominantly spherical and agglomerated because of
the high surface energy, the interactions and the Van
der Waals forces between the MgO NPs.[41] The cubic
shape of MgO shown in Figure 7C confirms the XRD
spectrum results (Figure 5). A close look in Figure 7B
revealed also the presence of hexagonal shape with a
size of 13.8 nm. The EDX analysis results (Figure 7E),
confirms the characteristic signals of Mg and O atoms,
which support the coexistence of MgO NPs. The
synthesized biogenic MgO NPs were obtained in
varied size range of 20 –46 nm (Figure 7F). Previous
chemical methods of MgO nanoparticles reported a
size range of 16 –50 nm, which is in good agreement
with those given in this work.[42,43]
Proposed Scenario for the Biosynthesis of MgO NPs
It is known that the main biophenols compounds
found in OMW are phenolic acids, secoiridoids and
flavonoids.[2,44] These bioactive compounds contain
one or more aromatic rings substituted by the hydroxy
groups. A possible scenario of the magnesium reduc-
tion by using the phenolic groups of OMW (hydrox-
ytyrosol, tyrosol, and verbascoside) and the synthesis
of MgO NPs are presented in Scheme 1.
Herein, it could be hypothesized that the reaction
involves the adsorption of magnesium on the surface
© 2020 Wiley-VHCA AG, Zurich, Switzerland
 Chem. Biodiversity 2020, 17, e1900608

Scheme 1. Proposed scenario for the formation of MgO NPs using biophenols extracted from OMW.

of polyphenols. The presence of the hydroxy func-
tional group plays a double role as stabilizing and
reducing agents in this proposed mechanism. The
mode of binding of the hydroxy groups with magne-
sium is responsible for obtaining the hexagonal and
cubic forms. It is important to mention here that the
combinations of cubic and hexagonal were supported
by the TEM analysis. In addition, the difference, in
these coordination modes, could be due to steric
reasons.

www.cb.wiley.com (6 of 12) e1900608 © 2020 Wiley-VHCA AG, Zurich, Switzerland

 Chem. Biodiversity 2020, 17, e1900608
Antibacterial Activity of MgO NPs
The increase of drug resistance of bacteria has become
a major public health problem. Recently, several
studies investigated the use of nanoparticles as
alternatives of antibiotics.[45] Herein, the antibacterial
activities of biogenic magnesium oxide nanoparticles
(MgO NPs) against Enterobacter aerogenes, Salmonella
typhimurium, Escherichia coli, Bacillus niacini, and
Staphylococcus cohnii are showed in Figure 8. The
effect of MgO NPs is illustrated by the development of
inhibition zone (Figure 8a). The measured inhibition
zone of MgO NPs revealed a significant increase of
inhibition zone compared to ciprofloxacin (cip), as a
standard antibiotic. Interestingly, MgO NPs have
shown a greater significant zone of inhibition against
Enterobacter aerogenes (29.0 � 1.0 mm) followed by
Bacillus niacini (24.33 � 1.15 mm), Staphylococcus coh-
nii (20.3 � 1.52 mm), Salmonella typhimurium (18.33 �
0.57 mm), and Escherichia coli (17.66 � 0.5 mm) at
1.4 mg/ml (p < 0.01) (Figure 8b). These results revealed
that the sensitivity of microorganisms towards MgO
nanoparticles synthesized using biophenols extracted
from OMW is more effective than free phenolic
compounds derived from the OMW.[46]
Generally, metal oxide nanoparticles have demon-
strated wide-spectrum antibacterial properties against
both Gram-positive and Gram-negative bacteria. The
Ag-TiO2 nanoparticles/nanotubes demonstrated an
antibacterial activity against Bacillus subtilis and Escher-
ichia coli.[47] As shown in previous study, the green
synthesized ZnO NPs exhibited more enhanced anti-
bacterial activity towards various bacteria when com-
pared to chemical synthesized ZnO NPs.[48] Many
studies have also described the antibacterial activity of
MgO nanoparticles. Suresh et al.[49] reported that the
chemical synthesized MgO NPs exerted an antibacte-
rial activity against S. aureus and E. coli bacteria.
Pugazhendhi et al.[36] and El-Sayyad et al.[50] have also
found that the green synthesized MgO nanoparticles
has a powerful antibacterial activity against several
human pathogens: A. baumannii, E. cloacae, E. coli, E.
faecalis, S. typhimurium, S. aureus, and S. epidermidis. A
previous study revealed that the biogenic MgO nano-
particles synthesized by using aqueous leaf extracts
showed a potent antibacterial activity, which is more
effective in comparison to the titanium dioxide and
the silver nanoparticles.[51]
The antibacterial efficiency of magnesium oxide
nanoparticles may be due to the stability, the smaller
size and the shape of MgO nanoparticles.[52] Besides,
the MgO NPs affect the bacterial cells by the
www.cb.wiley.com (7 of 12) e1900608
production of reactive oxygen species (ROS).[53] To
conclude, nanomaterials are found to be highly
promising as alternatives to antibiotics and potentially
offering a useful benefit in pharmaceutical
applications.[54]
Conclusions
The current study developed an efficient and econom-
ic method for the valorization and the treatment of
OMW at the same time. The polyphenols extracted
from OMW was valorized through the biosynthesis of
MgO nanoparticles. The particle size of the obtained
MgO NPs varies from 20 to 46 nm, with spherical and
hexagonal shapes appearance. Moreover, the biogenic
MgO NPs exhibit an efficient antibacterial activity
against Enterobacter aerogenes, Bacillus niacini, Staph-
ylococcus cohnii, Escherichia coli and Salmonella typhi-
murium, which makes it a good candidate for further
promising pharmaceutical applications.
On the other hand, the residual OMW after partial
removal of polyphenols was treated using Yarrowia
lipolytica. In comparison to the crude OMW, a potent
stimulation on the growth of Y. lipolytica was revealed
in EOMW, leading to an important decrease of COD
(73 %). In view of the above results, OMW could be
interesting in the synthesis of new active material and
for further treatment considerations.
Experimental Section
Chemicals Materials
Current investigations are focused on the synthesis of
the MgO NPs. OMW was collected, in November 2018,
from three-phase extraction mill located in Bizerte
(Tunisia). The samples were stored at @20 °C and
defrosted before use.
Magnesium acetate (Mg(AcO)2 · 4H2O), ethyl acetate
and hexane were obtained from Biochem-Chemo-
pharma (France).
Extraction of Polyphenols from Olive Mill Wastewater
The extraction of phenolic compounds was carried out
according to the method described by De Marco
et al.[55] with some adjustments. OMW was acidified to
pH 2 (6 M HCl), then, removed the oil by using hexane.
The delipidated OMW was sonicated for 20 min in
ultrasonic bath, and then, treated five times with a
© 2020 Wiley-VHCA AG, Zurich, Switzerland
 Chem. Biodiversity 2020, 17, e1900608

Figure 8. Antimicrobial activity of MgO NPs against microorganisms. (a) Antibacterial image of MgO NPs. 1: ciprofloxacin, 2: 0.4 mg/
ml, 3: 0.8 mg/ml, 4: 1.4 mg/ml. (b) Bar diagram of inhibition zone of MgO NPs. The results are expressed as means � SD of triplicate
measurements. Significant differences are indicated by different letters (b and c) vs. positive control (p < 0.05).

www.cb.wiley.com (8 of 12) e1900608 © 2020 Wiley-VHCA AG, Zurich, Switzerland

 Chem. Biodiversity 2020, 17, e1900608
mixture of ethanol/ethyl acetate (1 : 2) in order to
extract the polyphenols. The recovered polyphenols
were evaporated to dryness under vacuum by using a
rotary evaporator (Büchi R-200). The extract was stored
at 4 °C.
Chemical Characterization of Olive Mill Wastewater
The crude olive mill wastewater (OMW) and the
residual olive mill wastewater after partial removal of
polyphenols (EOMW) were characterized for pH, total
solids (TS), and total suspended solids (TSS). Chemical
oxygen demand (COD) was determined by AFNOR.[56]
Lipids content was determined gravimetrically after
hexane extraction.[57] Phenolic compounds concentra-
tion was identified according to the Folin@Ciocalteu
method measured at 750 nm and expressed as gallic
acid equivalent.[58]
Microorganism, Growth Media and Analytical Method
Y. lipolytica strains (CLIB40) were obtained from CIRM-
Levures (France). The microorganism was grown in
Sabouraud Dextrose broth (Accumix) at 29 °C for 24 h.
The OMW and EOMW were filtered and centrifuged at
8000 rpm for 10 min. The pH of both olive mill
wastewaters (OMW and EOMW) was adjusted to 6.40
by using NaOH (6 M), inoculated with Y. lipolytica
(15 %) in an orbital shaker (Zhicheng, zhwy-103B,
China) with an agitation of 120 rpm at 29 °C.
The biomass was harvested by centrifugation at
9000 rpm for 15 min, washed with distilled water and
determined gravimetrically from dry weight at 105 °C.
Biosynthesis of Magnesium Oxide Nanoparticles
The synthesis starts by dissolving Mg(AcO)2 · 4H2O in
double distilled water to form a solution (0.01M). A
mixture of 5 ml of phenolic extract and ethanolic
solution (1 : 10) from OMW was added drop by drop
into the Mg(AcO)2 solution and then, heated at 80 °C
with continuous stirring for 2 h until the appearance
of brown color. The colloidal solution was cooled
down to room temperature, and then, dried in an
oven at 70 °C. Finally, the obtained product was
calcined in a muffle furnace (Vecstar VCTF5) at 500 °C
for 3 h. The powdered sample was grinded and stored
at 4 °C.
www.cb.wiley.com (9 of 12) e1900608
Characterization of MgO NPs
The biosynthesis of MgO NPs was monitored by
measuring the absorbance at a wavelength ranging
from 200 to 800 nm using UV/VIS spectrophotometer
(Cary-Varian).
An X-ray diffraction (XRD) analysis of dry nano-
particles powder was carried out by X’Pert Pro MPD
PANalytical device.
The characterization of phenolic extract and MgO
NPs using Fourier transform infrared spectroscopy (FT-
IR) was recorded on Nicolet IR200 FT-IR spectrometer
in the spectral range from 400 to 4000 cm@1.
The shape, size and elemental composition of MgO
NPs were characterized by using FEI Tecnai instrument
G20 transmission electron microscopy operating at
200 kV equipped with energy dispersive X-ray spectro-
scopy (EDX). Samples were prepared from white
powder of MgO NPs, which is dispersed in ethanol and
sonicated for 30 min, then, placed in formvar-coated
TEM grid.
The zeta potential (ZP) measurement of MgO NPs
in water was analyzed by Malvern Nano-Zetasizer ZS
(Malvern, UK).
In Vitro Antibacterial Activity of MgO NPs
The antibacterial activity of MgO NPs was performed
by using agar well diffusion. The MgO NPs powder
was sterilized in an oven at 120 °C for 20 min. The
bacterial strains used in the current study are Escher-
ichia coli (JW1772), Enterobacter aerogenes (AF395913),
Salmonella typhimurium (ATCC14028), Bacillus niacini
(AB021194), and Staphylococcus cohnii (AY395015).
Each strain was grown in nutrient broth, at 30 °C for
Enterobacter aerogenes and 37 °C for the other strains.
Approximately 100 μl of bacterial suspension (log
phase) were spread on agar Petri dishes. Then, the
sterilized MgO NPs with various concentrations 0.4,
0.8, and 1.4 mg/ml were added into each well and
incubated at the desired temperature for 24 h. Cipro-
floxacin antibiotic (10 μg/ml) was used as a positive
control. The antibacterial activity was estimated by
measuring the inhibition zone. All experiments were
performed in triplicates.
© 2020 Wiley-VHCA AG, Zurich, Switzerland
 Chem. Biodiversity 2020, 17, e1900608
Statistical Analysis
The statistical significance of differences between
mean values and comparison among groups was
performed by using the one-way ANOVA test followed
by Tuckey’s test with SPSS software version 25. Values
of p < 0.05 was considered statistically significant.
Acknowledgements
The authors are grateful to the Tunisian Ministry of
Higher Education and Scientific Research for funding.
We would like to thank Prof. Rakia Chouari (Plant
Toxicology and Environmental Microbiology Labora-
tory, Faculty of Sciences of Bizerte, Tunisia) for her
generous providing Enterobacter aerogenes
(AF395913.1) and Staphylococcus cohnii (AY395015.1).
Author Contributions Statement
Selma Hamimed and Nejib Jebli were responsible for
the analysis, data interpretation, and manuscript
writing. Hanen Sellami and Ahmed Landoulsi corrected
the manuscript. Abdelwaheb Chatti designed, cor-
rected, and approved the manuscript.
References
[1] D. Degirmenbasi, S. Takac, ‘Use of Olive Mill Wastewater as
a Growth Medium for Superoxide Dismutase and Catalase
Production’, Clean Soil Air Water 2018, 46, 1 –7.
[2] S. Dermeche, M. Nadour, C. Larroche, F. Moulti-Mati, P.
Michaud, ‘Olive mill wastes: Biochemical characterizations
and valorization strategies’, Process Biochem. 2013, 48,
1532 –1552.
[3] N. Rahmanian, S. M. Jafari, C. M. Galanakis, ‘Recovery and
Removal of Phenolic Compounds from Olive Mill Waste-
water – Review’, J. Am. Oil Chem. Soc. 2014, 91, 1 –18.
[4] L. G. C. Villegas, N. Mashhadi, M. Chen, D. Mukherjee, K. E.
Taylor, N. Biswas, ‘A short review of techniques for phenol
removal from wastewater’, Curr. Pollut. Rep. 2016, 2, 157 –
167.
[5] D. Sarris, M. Galiotou-Panayotou, A. A. Koutinas, M. Komai-
tis, S. Papanikolaou, ‘Citric acid, biomass and cellular lipid
production by Yarrowia lipolytica strains cultivated on olive
mill wastewater-based media’, J. Chem. Technol. Biotechnol.
2011, 86, 1439 –1448.
[6] S. Hachicha, J. Cegarra, F. Sellami, R. Hachicha, N. Drira, K.
Medhioub, E. Ammar, ‘Elimination of polyphenols toxicity
from olive mill wastewater sludge by its cocomposting
with sesame bark’, J. Hazard. Mater. 2009, 161, 1131 –1139.
[7] S. Khoufi, F. Aloui, S. Sayadi, ‘Extraction of antioxidants
from olive mill wastewater and electro-coagulation of
www.cb.wiley.com (10 of 12) e1900608
exhausted fraction to reduce its toxicity on anaerobic
digestion’, J. Hazard. Mater. 2008, 151, 531–539.
[8] N. Caporaso, D. Formisano, A. Genovese, ‘Use of phenolic
compounds from olive mill wastewater as valuable ingre-
dients for functional foods’, Crit. Rev. Food Sci. Nutr. 2018,
58, 2829 –2841.
[9] K. Gerasopoulos, D. Stagos, S. Kokkas, K. Petrotos, D.
Kantas, P. Goulas, D. Kouretas, ‘Feed supplemented with
byproducts from olive oil mill wastewater processing
increases antioxidant capacity in broiler chickens’, Food
Chem. Toxicol. 2015, 82, 42 –49.
[10] J. He, M. Alister-Briggs, T. de Lyster, G. P. Jones, ‘Stability
and antioxidant potential of purified olive mill wastewater
extracts’, Food Chem. 2012, 131, 1312 –1321.
[11] G. Bisignano, A. Tomaino, R. Lo Cascio, G. Crisafi, N. Uccella,
A. Saija, ‘On the in-vitro antimicrobial activity of oleuropein
and hydroxytyrosol’, J. Pharm. Pharmacol. 1999, 51, 971 –
974.
[12] M. J. Liu, J. X. Li, H. Z. Guo, K. M. Lee, L. Qin, K. M. Chan,
‘The effects of verbascoside on plasma lipid peroxidation
level and erythrocyte membrane fluidity during immobili-
zation in rabbits: a time course study’, Life Sci. 2003, 73,
883 –892.
[13] M. Pennacchio, E. Alexander, Y. M. Syah, E. L. Ghisalberti,
‘The effect of verbascoside on cyclic 3’,5’-adenosine mono-
phosphate levels in isolated rat heart’, Eur. J. Pharmacol.
1996, 305, 169–171.
[14] R. Keyrouz, M. L. Abasq, C. Le Bourvellec, N. S. Blanc, L.
Audibert, E. Argall, D. Hauchard, ‘Total phenolic contents,
radical scavenging and cyclic voltammetry of seaweeds
from Brittany’, Food Chem. 2011, 126, 831–836.
[15] Y. S. Liu, Y. C. Chang, H. H. Chen, ‘Silver nanoparticle
biosynthesis by using phenolic acids in rice husk extract as
reducing agents and dispersants’, J. Food Drug Anal. 2018,
26, 649 –656.
[16] P. K. Stoimenov, R. L. Klinger, G. L. Marchin, K. J. Klabunde,
‘Metal oxide nanoparticles as bactericidal agents’, Langmuir
2002, 18, 6679 –6686.
[17] F. Mohandes, F. Davar, M. Salavati-Niasari, ‘Magnesium
oxide nanocrystals via thermal decomposition of magne-
sium oxalate’, J. Phys. Chem. Solids 2010, 71, 1623 –1628.
[18] P. Ouraipryvan, T. Sreethawong, S. Chavadej, ‘Synthesis of
crystalline MgO nanoparticle with mesoporous-assembled
structure via a surfactant-modified sol-gel process’, Mater.
Lett. 2009, 63, 1862 –1865.
[19] W. Wang, X. L. Qiao, J. G. Chen, H. J. Li, ‘Facile synthesis of
magnesium oxide nanoplates via chemical precipitation’,
Mater. Lett. 2007, 61, 3218 –3220.
[20] F. Khairallah, A. Glisenti, ‘Synthesis, characterization and
reactivity study of nanoscale magnesium oxide’, J. Mol.
Catal. A 2007, 274, 137–147.
[21] T. Boningari, S. N. R. Inturi, V. I. Manousiouthakis, P. G.
Smirniotis, ‘Facile synthesis of flame spray pyrolysis-derived
magnesium oxide nanoparticles for CO2 sorption: Effect of
precursors, morphology, and structural properties’, Ind.
Eng. Chem. Res. 2018, 57, 9054 –9061.
[22] Y. Ding, G. T. Zhang, H. Wu, B. Hai, L. B. Wang, Y. T. Qian,
‘Nanoscale magnesium hydroxide and magnesium oxide
powders: Control over size, shape, and structure via
hydrothermal synthesis’, Chem. Mater. 2001, 13, 435 –440.
© 2020 Wiley-VHCA AG, Zurich, Switzerland
 Chem. Biodiversity 2020, 17, e1900608
[23] N. Pradeep, C. Venkatachalaiah, U. Venkatraman, C.
anthosh, A. Bhatnagar, S. K. Jeong, A. N. Grace, ‘Magnesium
oxide nanocubes deposited on an overhead projector
sheet: synthesis and resistivity-based hydrogen sensing
capability’, Microchim. Acta 2017, 184, 3349 –3355.
[24] T. X. Phuoc, B. H. Howard, D. V. Martello, Y. Soong, M. K.
Chyu, ‘Synthesis of Mg(OH)2, MgO, and Mg nanoparticles
using laser ablation of magnesium in water and solvents’,
Opt. Lasers Eng. 2008, 46, 829–834.
[25] S. M. Alshehri, R. V. Tiwari, B. B. Alsulays, E. A. Ashour, A. S.
Alshetaili, B. Almutairy, J. B. Park, J. Morott, B. Sandhu, S.
Majumdar, M. A. Repka, ‘Investigation of the combined
effect of MgO and PEG on the release profile of mefenamic
acid prepared via hot-melt extrusion techniques’, Pharm.
Dev. Technol. 2017, 22, 740– 753.
[26] K. Karthik, S. Dhanuskodi, C. Gobinath, S. Prabukumar, S.
Sivaramakrishnan, ‘Fabrication of MgO nanostructures and
its efficient photocatalytic, antibacterial and anticancer
performance’, J. Photochem. Photobiol. B 2019, 190, 8–20.
[27] H. Zhang, J. Hu, J. Xie, S. Wang, Y. Cao, ‘A solid-state
chemical method for synthesizing MgO nanoparticles with
superior adsorption properties’, RSC Adv. 2019, 9, 2011 –
2017.
[28] D. Sarris, M. Galiotou-Panayotou, A. A. Koutinas, M. Komai-
tis, S. Papanikolaou, ‘Citric acid, biomass and cellular lipid
production by Yarrowia lipolytica strains cultivated on olive
mill wastewater-based media’, J. Chem. Technol. Biotechnol.
2011, 86, 1439 –1448.
[29] D. Sarris, S. N. Stoforos, A. Malloucho, I. K. Kookos, G. A. A.
Koutinas, S. Papanikolaou, ‘Production of added-value
metabolites by Yarrowia lipolytica growing in olive mill
wastewater-based media under aseptic and non-aseptic
conditions’, Eng. Life Sci. 2017, 17, 695–709.
[30] C. Scioli, L. Vollaro, ‘The use of Yarrowia lipolytica to reduce
pollution in olive mill wastewaters’, War. Res. 1997, 31,
2520 –2524.
[31] D. Philip, ‘Synthesis and spectroscopic characterization of
gold nanoparticles’, Spectroschim. Acta A 2008, 71, 80 –85.
[32] R. Dobrucka, ‘Synthesis of MgO nanoparticles using
Artemisia abrotanum herba extract and their antioxidant
and photocatalytic properties’, Iran. J. Sci. Technol. 2018,
42, 547 –555.
[33] A. S. Bhadwa, R. M. Tripathi, R. K. Gupta, N. Kumar, R. P.
Singh, A. Shrivastav, ‘Biogenic synthesis and photocatalytic
activity of CdS nanoparticles’, RSC Adv. 2014, 4, 9484 –
9490.
[34] K. R. Nemade, S. A. Waghuley, ‘Synthesis of MgO nano-
particles by solvent mixed spray pyrolysis technique for
optical investigation’, Int. J. Met. 2014, 1– 4.
[35] D. A. Vanden Bout, ‘Metal nanoparticles: synthesis, charac-
terization, and applications’, J. Am. Chem. Soc. 2002, 124,
7874 –7875.
[36] A. Pugazhendhi, R. Prabhu, K. Muruganantham, R. Shanmu-
ganathan, S. Natarajan, ‘Anticancer, antimicrobial and
photocatalytic activities of green synthesized magnesium
oxide nanoparticles (MgO NPs) using aqueous extract of
Sargassum wightii’, J. Photochem. Photobiol. B 2019, 190,
86 –97.
[37] S. Kaur, J. Singh, R. Rawat, S. Kumar, H. Kaur, K. V. Rao, M.
Rawat, ‘A smart LPG sensor based on chemo-bio synthe-
www.cb.wiley.com (11 of 12) e1900608
sized MgO nanostructure’, J. Mater. Sci. Mater. Electron.
2018, 29, 11679 –11687.
[38] Y. S. Hii, J. Jeevanandam, Y. S. Chan, ‘Plant mediated green
synthesis and nanoencapsulation of MgO nanoparticle
from Calotropis gigantea: Characterisation and kinetic
release studies’, Inorg. Nano-Met. Chem. 2019, 1–12.
[39] I. Haas, A. Gedanken, ‘Synthesis of metallic magnesium
nanoparticles by sonoelectrochemistry’, Chem. Commun.
2008, 15, 1795 –1797.
[40] J. Jeevanandam, Y. S. Chan, M. K. Danquah, ‘Biosynthesis
and characterization of MgO nanoparticles from plant
extracts via induced molecular nucleation’, New J. Chem.
2017, 41, 2800 –2814.
[41] G. Sharma, R. Soni, N. D. Jasuja, ‘Phytoassisted synthesis of
magnesium oxide nanoparticles with Swertia chirayaita’, J.
Taibah Univ. Sci. 2017, 11, 471–477.
[42] S. Suresh, ‘Investigations on synthesis, structural and
electrical properties of MgO nanoparticles by sol-gel
method’, J. Ovonic Res. 2014, 10, 205– 210.
[43] M. R. Bindhu, M. Umadevi, M. K. Micheal, M. V. Arasu, N. A.
Al-Dhabi, ‘Structural, morphological and optical properties
of MgO nanoparticles for antibacterial applications’, Mater.
Lett. 2016, 166, 19 –22.
[44] L. Bravo, ‘Polyphenols: Chemistry, dietary sources, metabo-
lism, and nutritional significance’, Nutr. Rev. 1998, 56, 317 –
333.
[45] L. L. Wang, C. Hu, L. Q. Shao, ‘The antimicrobial activity of
nanoparticles: present situation and prospects for the
future’, Int. J. Nanomed. 2017, 12, 1227 –1249.
[46] I. Leouifoudi, H. Harnafi, A. Zyad, ‘Olive mill waste extracts:
polyphenols content, antioxidant, and antimicrobial activ-
ities’, Adv. Pharm. Sci. 2015, 1–11.
[47] D. Guin, S. V. Manorama, J. N. L. Latha, S. Singh, ‘Photo-
reduction of silver on bare and colloidal TiO2 nano-
particles/nanotubes: Synthesis, characterization, and tested
for antibacterial outcome’, J. Phys. Chem. C 2007, 111,
13393 –13397.
[48] S. Gunalan, R. Sivaraj, V. Rajendran, ‘Green synthesized ZnO
nanoparticles against bacterial and fungal pathogens’,
Prog. Nat. Sci. 2012, 22, 695–702.
[49] J. Suresh, R. G. R. Gandhi, S. Selvam, M. Sundrarajan,
‘Synthesis of magnesium oxide nanoparticles by wet
chemical method and its antibacterial activity’, Adv. Mater.
Res. 2013, 678, 297– 300.
[50] G. S. El-Sayyad, F. M. Mosallam, A. I. El-Batal, ‘One-pot green
synthesis of magnesium oxide nanoparticles using Penicil-
lium chrysogenum melanin pigment and gamma rays with
antimicrobial activity against multidrug-resistant microbes’,
Adv. Powder Technol. 2018, 29, 2616 –2625.
[51] L. Umaralikhan, M. J. M. Jaffar, ‘Green synthesis of MgO
nanoparticles and it antibacterial activity’, Iran. J. Sci.
Technol. 2018, 42, 477–485.
[52] N. P. A. Movahedi, ‘Fabrication, characterization and anti-
bacterial properties of MgO nanoparticles in zeolite matrix’,
Iran. J. Med. Microbiol. 2018, 12, 116–124.
[53] N. Y. T. Nguyen, N. Grelling, C. L. Wetteland, R. Rosario,
H. N. Liu, ‘Antimicrobial activities and mechanisms of
magnesium oxide nanoparticles (nMgO) against pathogen-
ic bacteria, yeasts, and biofilms’, Sci. Rep. 2018, 8, 1–23.
[54] N. Beyth, Y. Houri-Haddad, A. Domb, W. Khan, R. Hazan,
‘Alternative antimicrobial approach: nano-antimicrobial
© 2020 Wiley-VHCA AG, Zurich, Switzerland
 Chem. Biodiversity 2020, 17, e1900608
materials’, Evid.-Based Complement. Altern. Med. 2015, 1 –
16.
[55] E. De Marco, M. Savarese, A. Paduano, R. Sacchi, ‘Character-
ization and fractionation of phenolic compounds extracted
from olive oil mill wastewaters’, Food Chem. 2007, 104,
858 –867.
[56] AFNOR, ‘Determination de la demande chimique en oxy-
gène (DCO)’, NF T90-101, Association Française de Normal-
isation, Cedex, France, 2001.
[57] A. D’Annibale, G. G. Sermanni, F. Federici, M. Petruccioli,
‘Olive-mill wastewaters: a promising substrate for microbial
www.cb.wiley.com (12 of 12) e1900608
View publication stats
lipase production’, Bioresour. Technol. 2006, 97, 1828–
1833.
[58] J. Tabart, C. Kevers, A. Sipel, J. Pincemail, J. O. Defraigne, J.
Dommes, ‘Optimisation of extraction of phenolics and
antioxidants from black currant leaves and buds and of
stability during storage’, Food Chem. 2007, 105, 1268 –75.
Received October 28, 2019
Accepted February 3, 2020
© 2020 Wiley-VHCA AG, Zurich, Switzerland