Cedrus atlantica essential oil: Antimicrobial activity and effect on the physicochemical properties of cedar wood Surface

Abstract

This study aimed to investigate the chemical composition and antimicrobial effects of Cedrus atlantica essential oil, against two bacteria and six fungi that cause the degradation of cedar wood. The effects of C. atlantica essential oils, applied for different treatment times, on the physicochemical properties of cedar wood were also explored. Gas chromatography (GC)/mass spectrometry (MS)analysis of the studied essential oil showed that cedranone and iso-cedranol were the major components of C. atlantica essential oil. The minimum inhibitory concentrations (MICs) and minimum bactericidal/fungicidal concentrations were determined, using broth microdilution assays, and the physicochemical properties of cedar wood were determined using the contact angle measurement. The results demonstrated antibacterial activity against the two bacteria tested, with MICs ranging from 1% to 2%, and antifungal activity against all fungi tested, with MICs ranging from 0.5% to 1%. The cedar wood maintained its hydrophobic character, as assessed quantitatively, after treatment with C. atlantica essential oil, increasing its electron donor character after 15 min and 1 h of treatment.

Full text

Moroccan Journal of Biology
Number 16 (2019)
http://www.fst.ac.ma/mjb
e-ISSN: 2351-8456 - p-ISSN: 1114-8756
CONTACT S. Ibnsouda Koraichi  saad.ibnsouda@usmba.ac.ma  Laboratory of
Microbial Biotechnology, University Sidi Mohamed Ben Abdellah, Faculty of Science and
Technology, Fez, Morocco
Cedrus atlantica essential oil: Antimicrobial activity and effect on the
physicochemical properties of cedar wood Surface
Fadoua Bennouna1,3, Mohammed Lachkar3, Soumya El Abed1,2, Saad Ibnsouda Koraichi1,2
1Laboratory of Microbial Biotechnology, University Sidi Mohamed Ben Abdellah. Faculty of Science and
Technology, Fez, Morocco
2Regional University Centre of Interface, University Sidi Mohamed Ben Abdellah, Morocco
3Engineering Laboratory of Organometallic and Molecular Materials, University Sidi Mohamed Ben Abdellah.
Faculty of Science, Fez, Morocco
Abstract
This study aimed to investigate the chemical composition and antimicrobial effects of Cedrus
atlantica essential oil, against two bacteria and six fungi that cause the degradation of cedar
wood. The effects of C. atlantica essential oils, applied for different treatment times, on the
physicochemical properties of cedar wood were also explored. Gas chromatography
(GC)/mass spectrometry (MS)analysis of the studied essential oil showed that cedranone and
iso-cedranol were the major components of C. atlantica essential oil. The minimum inhibitory
concentrations (MICs) and minimum bactericidal/fungicidal concentrations were determined,
using broth microdilution assays, and the physicochemical properties of cedar wood were
determined using the contact angle measurement. The results demonstrated antibacterial
activity against the two bacteria tested, with MICs ranging from 1% to 2%, and antifungal
activity against all fungi tested, with MICs ranging from 0.5% to 1%. The cedar wood
maintained its hydrophobic character, as assessed quantitatively, after treatment with C.
atlantica essential oil, increasing its electron donor character after 15 min and 1 h of
treatment.
Keywords: Cedrus atlantica, antimicrobial activity, cedar wood, contact angle, physicochemical properties.
Introduction
Native to the Rif and Atlas
Mountains of North Africa, especially
Morocco and Algeria (Renau-Morata et al.,
2005; Maya et al., 2017), Cedrus atlantica
Manetti a large conifer, found at altitudes
ranging from 1,500 to 2,600 m. C.
atlantica is the most important timber
resource in Morocco, occupying a surface
area of 132,000 ha and representing 2.3%
of the national forest (Renau-Morata et al.,
2005). Essential oils derived from C.
atlantica are used in various products, as
drugs and perfumes (Adams, 1991). C.
atlantica essential oils have been already
studied and shown to possess larvicidal (Ez
Zoubiet al., 2017), antiviral (Loizzo et al.,
2008), antifungal, and antibacterial
activities (Hammer et al., 1999; Chebli et
al., 2003; Satrani, 2006; Derwich et al.,
2010; Rhafouri et al., 2014; Zrira et al.,
2016). However, the study of its antifungal
and antibacterial activities, especially
against bacteria and fungi isolated from
cedar wood, and the use of C. atlantica
essential oils for wood protection have
never been reported in the literature, to our
knowledge.
Biological organisms, such as
bacteria and fungi adhere to different
materials, including cedar wood, which
was often used as a raw material during the
building of historical monuments in
imperial cities, such as the old medina of
Fez. The growth of these latter is
associated with aesthetic degradation
(Dickinson, 1972; Chedgy et al., 2007;
Gobakken & Vestøl, 2012),
biodeterioration, and the reduction of wood

36
F. Bennouna et al. / Moroccan J. Biol. 16 (2019): 35-45
durability (Blanchette, 2000; Sterflinger et
al., 2013), and have been associated with
health risks due to the release of fungal
mycotoxins (Görs et al., 2007). Adhesion
is an important step in the biofilm
formation process and is governed by van
der Waals forces, electrostatic properties,
and acid-base interactions, which depend
on the hydrophobicity and electron donor–
electron acceptor properties of the material
and the microbial surface.
Recently, to protect the wood,
environmental concerns have required the
use of non-biocidal solutions, instead of
traditional methods, such as toxic
chemicals, that can provide decay
resistance but are associated with
environmental effects. Thus, the
identification of natural resources, such as
essential oils, that are able to prevent
microbial and fungal adhesion, by
modifying the physicochemical properties
of the cedar wood surface, combined with
antimicrobial activities against the
microorganisms that can degrade wood, is
our priority.
Thus, the purpose of this study was,
first, to investigate the chemical
composition of C. atlantica essential oils;
second, to examine their antimicrobial
activities against bacteria and fungi
associated with the deterioration of
historical wood; and finally, to evaluate the
effects of essential oils and treatment times
on the cedar wood physicochemical
properties.
Materials and methods
Plant materials and essential oil
extraction
The C. atlantica specimens were
collected in the region of Azrou, located in
the Middle Atlas (Morocco). The essential
oil extraction was performed by the
hydrodistillation of 200 g of sawdust, with
a Clevenger-type apparatus. The obtained
oil was dried with anhydrous sodium
sulfate and stored in sealed glass vials, at
4°C.
Essential oil analysis
The chemical composition of the
tested oil was determined, using gas
chromatography-mass spectrometry (GC-
MS). The analysis was performed using a
Trace GC Ultra gas chromatograph,
equipped with anapolar capillary TR-5
column (60 m  0.32 mm ID, 0.25-µm film
thickness), coupled to a mass detector (MS
Quadrupole). The analysis of C. atlantica
essential oil was performed by employing
the following GC conditions: initial
temperature of 40°C for 2 min, increasing
by 5°C per min until 280°C, followed by a
10-min hold. The injector was maintained
at a temperature of 220°C. Helium was the
carrier gas, at 1.2 ml/min; the sample
(1 µL) was injected in the split ratio mode
(10:1). The detector temperature was
300°C. The ionization energy was 70 eV.
Compounds were identified by comparing
their retention index (RI) and mass spectra
with those of components identified in the
literature and the National Institute of
Standards and Technology (NIST) Library.
RIs were calculated using a homologous
series of n-alkanes, C8-C18 (Sigma-Aldrich,
St Louis, MO).
Antibacterial activity test
The antibacterial activity of C.
atlantica essential oil was tested against
two Gram-positive bacteria: Bacillus
safensis and B. subtilis, which are known
for their abilities to deteriorate cedar wood.
They were isolated by Sadiki et al. (2017-
a), from decaying cedar wood found in an
old wooden house, located in the old
medina of Fez. The bacterial strains were
subcultured in Luria-Bertani (LB) agar and
incubated overnight, at 37°C. Then, the
bacterial inoculum was prepared, at a final
concentration of 2106colony-forming
units (CFU)/ml, using a physiologic saline
solution.
The minimum inhibitory
concentration (MIC) of the essential oil
was determined using the broth

37
F. Bennouna et al. / Moroccan J. Biol. 16 (2019): 35-45
microdilution assay (Bouhdid et al., 2009;
Tianet al., 2014), with some modifications.
The MICis defined as the lowest
concentration of an antibacterial agent that
inhibits bacterial growth (Lancini et al.,
1993). To determine the MIC, 50 µL of
Mueller Hinton Broth (MHB),
supplemented with bacteriological agar
(0.15% w/v), was deposited from the
second to the twelfth well. C. atlantica
essential oil was dissolved in Mueller
Hinton Broth (MHB), containing 0.15% of
agar and diluted until the 11th well, so that
the final concentration was ranged between
8-0.00781% (v/v). Finally, 50 µL of
bacterial suspension, prepared at a
concentration of 2106 CFU/ml, was added
to each well.
The twelfth well was considered
the growth control, containing only the
bacterial suspension and Mueller Hinton
Broth, supplemented with agar (0.15 %
w/v). The incubation was performed at
37°C, for 20 h. Then, 5 µL of resazurin
was added to each well, followed by
further incubation for 2 h, to determine the
MIC of C. atlantica essential oil (Bouhdid
et al., 2009). The minimum inhibitory
concentration of the essential oil
corresponded with the lowest
concentration that prevented the reduction
of the blue resazurin dye into pink
resorufin.
A 5-µL volume from each negative
well was deposited on an LB plate and
incubated for 24 h, at 37°C, to determine
the minimum bactericidal concentration
(MBC), which is defined as the lowest
essential oil concentration that results in
negative subcultures.
Antifungal activity test
Thielavia hyalocarpa, Aspergillus
niger, and four fungi of the genus
Penicillium: P. commune (PDLd” and
PDLd10), P. crustosum, and P. expansum
were selected for their abilities to rot cedar
wood. They were isolated by Zyani et al.
(2009) and El Abed et al. (2010), and
identified in our laboratory. The fungal
strains were subcultured in Malt-Extract
(ME) agar medium, at 25°C for 10 days.
Then, the fungal spores were harvested by
scraping the culture surface, using a sterile
physiologic saline solution containing 1%
dimethyl sulfoxide (DMSO). The spore
suspensions were concentrated to a final
concentration of 2104 spores/ml, by
centrifugation at 7,000 rpm, for 15 min at
4°C (CLSI document M38-A2. 2008).
The determination of the MIC was
performed as indicated in the antibacterial,
test using the broth microdilution assay,
except that Malt Extract Broth (MEB),
supplemented with bacteriological agar
(0.15% w/v) was used instead of MHB.
The 96-well plate was incubated at 25°C,
for 48 h. Then, 5 µL from negative wells
was deposited on ME plates and incubated
at 28°C for 72 h, to determine the
minimum fungicidal concentration (MFC).
Wood preparation
The investigated substrate in this
study was cedar wood (C. atlantica),
which was obtained from a woodworking
shop in Fez City, Morocco. The roughness
of the wood samples (30 mm ×10 mm ×4
mm) was set in a range from 0.8 to 1µm,
using a rugosimeter. Then, the samples
were cleaned with distilled water, oven-
dried, and autoclaved at 121°C for 20 min.
Wood treatment
A 20-μL volume of the tested
essential oil was deposited on the cedar
wood surface, at room temperature
(252°C) (Sadiki et al., 2014). The
samples were analyzed with contact angle
measurements, after 15 min and after 1h, to
evaluate the effects of the essential oil
treatment time on the cedar wood
physicochemical properties. Experiments
were conducted in duplicate.
Contact angle measurements and
surface energy components
The Lifshitz-van der Waals, acid-
base, and surface free energy values of

38
F. Bennouna et al. / Moroccan J. Biol. 16 (2019): 35-45
untreated and treated cedar woods were
calculated from contact angle
measurements, which were performed by
the sessile drop method, using a
goniometer (GBX Instruments) (De Meijer
et al., 2000). Three contact angle
measurements were made on each wood
sample, using three liquids (two of which
must be polar), with well-known surface
energy components (Table1) (Van Oss et
al., 1988). Once the contact angles have
been measured, the Lifshitz-van der Waals
and acid-base surface tension components
can be obtained, using the following three
equations (Van Oss, 1993):
Table 1. Surface tension properties of pure liquids used to measure
contact angles (Van Oss, 2003).
Liquid
γLW (mJ/m2)
γ+ (mJ/m2)
γ- (mJ/m2)
Water (H2O)
21.8
25.5
25.5
Formamide (CH3NO)
39
2.3
39.6
Diiodomethane (CH2I2)
50.5
0
0
The Lewis acid-base component is expressed as
follows:
The wood sample hydrophobicity was evaluated
by the approach described by Van Oss et al. (1988),
through contact angle measurements. In this approach,
the degree of hydrophobicity for a given material is
expressed as the free energy
of the interaction between
two entities of this material
when immersed in water
(w): ΔGiwi. Therefore, the
material is considered to be
hydrophilic when the
interaction between the two
entities is lower than the
interaction of each entity
with water (ΔGiwi> 0);
otherwise, the material is
considered to be
hydrophobic ∆Giwi< 0.
ΔGiwi is calculated as
follows:
Results and discussion
Chemical composition of Cedrus
atlantica essential oil
The analysis of C. atlantica
essential oil resulted in twenty-two
components, constituting 100% of the total
composition (Table 2). The sesquiterpenes
represented the major constituents
(80.26%), among which, 47.17% were
oxygenated sesquiterpenes, and 33.09%
were hydrocarbon sesquiterpenes. The
sesquiterpene fraction was principally
constituted by γ-himachalane, β-
himachalane, γ-calamenene, δ-cadinene,
iso-cedranol, cedranone, cedrol, and
caryophyllene oxide. Cedranoneand iso-
cedranol were identified as the primary
components in the sesquiterpenes fraction,
with percentages of 19.35% and 13.78%,
respectively. The monoterpenes
represented 15.08% of the total identified
volatiles, of which 10.76% were
oxygenated monoterpenes, and 4.32%
were hydrocarbon monoterpenes,
represented by a single compound
(sabinene). The chemical composition of
C. atlantica essential oil has been the
subject of some investigations in Morocco,
especially the study by Derwich et al.
(2010), who reported -pinene as the
major component of C. atlantica leaf oil
(14.85%), followed by himachalane
(10.14%), -himachalane (9.89%), and -
himachalane (7.62%). The percentages of

39
F. Bennouna et al. / Moroccan J. Biol. 16 (2019): 35-45
Table 2. Chemical composition of C. atlantica
essential oil.
N°
Compounds
RI
% Area
1
Sabinene
969
4.32
2
Rose oxide
1127
1.15
3
α-terpineol
1142
2.62
4
Borneol
1163
1.32
5
p-cymen-8-ol
1183
2.42
6
Trans-carveol
1217
1.45
7
Bornylacetate
1285
1.8
8
Tetradecane
1398
3.93
9
Epi-Cedrane
1441
0.58
10
γ-himachalene
1476
4.05
11
β-himachalene
1499
7.23
12
γ-Cadinene
1513
0.48
13
γ-Calamenene
1520
7.77
14
δ-Cadinene
1524
7.34
15
γ-Dehydro-ar-Himachalene
1526
1.81
16
NI
-
0.73
17
α-calacorene
1542
3.83
18
Oxido-Himachalene
1574
0.87
19
Caryophyllene oxide
1591
8.73
20
Cedrol
1611
4.44
21
Cedranone
1620
19.35
22
Iso-cedranol
1661
13.78
Hydrocarbon monoterpenes
4.32
Oxygenated monoterpenes
10.76
Hydrocarbon sesquiterpenes
33.09
Oxygenated sesquiterpenes
47.17
Others
4.66
Total identified compounds
100
Notes: RI: Retention index; NI: Not Identified.
-himachalane and -himachalane
identified in their study were relatively
similar to our percentages. Rhafouri et al.
(2014) have shown that-pinene, manool,
and bornyl acetate represent the primary
constituents of cedar wingless seeds, with
percentages of 46.16%, 25.47%, and
10.18%, respectively. Zrira & Ghanmi
(2016) reported the chemical composition
of C. atlantica sawdust-derived essential
oil [-(E)-atlantone (19.3%), -
himachalane (15.1 %), 8-cedren-13-ol,
(13.1%), -himachalane (5.1%),
cedroxyde (4.6%), and deodarone (4.6 %)],
and recently, Ez Zoubi et al. (2017) have
reported the presence of -himachalane
(35.34%), -himachalane (13.62%), -
himachalane (12.6%), cedrol (10.32%),
iso-cedranol (5.52%), and -pinene
(5.5%), in the aerial parts of C. atlantica.
The same components were found in
Cedrus libani, with different percentages
[himachalol (22.50%), -himachalane
(21.90%), and -himachalane (10.50%)]
(Loizzo et al., 2008). Other studies have
shown the chemical composition of
oleoresin on the cones of Cedrus libani,
which is grown in Turkey [-pinene
(24.78%), abieta-7,13-diene (16.67%),
abieta-8,11,13-triene (6.85%), manool
(5.83%), terpinen-4-ol (3.74%), -
terpineol (3.42%), p-cymene (2.89%), and
limonene (2.69%)] (Necmettin et al.,
2005). The quantitative differences
observed among the chemical
compositions of the various C. atlantica
essential oils, and the absence of some
major constituents in our C. atlantica
essential oil, such as-(E)-atlantone (Zrira
& Ghanmi 2016), -himachalane (Ez
Zoubiet al., 2017), himachalol (Loizzo et
al., 2008), and -pinene (Necmettin et al.,
2005; Derwich et al., 2010; Rhafouriet al.,
2014), could be explained by differences in
geographical factors and climatic
conditions, that are specific to each region
(Mansouri et al., 2010), differences in the
parts of the plants being extracted, and
differences in the harvest time (Marcum &
Hanson, 2006; Muñoz-Bertomeu et al.,
2007).
Antibacterial activity
Table 3 shows MIC and MBC
values obtained in the antibacterial test for
cedar wood essential oil. The results
showed that the essential oil possessed
good antibacterial activity against the
studied bacterial strains studied, as the
MIC values ranged between 1% and 2%.
B. safensis and B. subtilis were both
found to be susceptible to C. atlantica
essential oil, with MIC values of 2% and
1%, respectively. The essential oil
exhibited abacteriostatic effect against B.
subtilis (MBC/MIC>4), and a bactericidal
effect against B. safensis (MBC/MIC=4)
(CLSI document M07-A9. 2012).
Few studies examining the effects
of C. atlantica essential oil have been

40
F. Bennouna et al. / Moroccan J. Biol. 16 (2019): 35-45
Table 3. The minimum inhibitory concentrations and the
minimum bactericidal/fungicidal concentrations of cedar wood
essential oil.
Strains
MIC
%(v/v)
MBC-MFC
%(v/v)
MBC/MIC
MFC/MIC
Bacteria
B. safensis
2
8
4
B. subtilis
1
8
8
Fungi
P. commune
(PDLd”)
1
˃8
-
P. commune
(PDLd10)
1
˃8
-
P. expansum
0.5
8
16
P. crustosum
0.5
˃8
-
T. hyalocarpa
0.5
8
16
A. niger
1
˃8
-
published. According to these reports, C. atlantica
essential oils have demonstrated effective antibacterial
activity, with MIC values of 0.4 µl/ml against
Escherichia coli and Bacillus cereusand0.2 µl/ml
against B. Subtilis (Zrira & Ghanmi 2016). Derwich et
al. (2010) revealed a low to moderate antibacterial
activity for C. atlantica leaf oil against a range of
bacteria tested, with MIC values between 0.25 mg/ml
and 1.62 mg/ml (MIC=0.98mg/ml for Pseudomonas
aeroginosa, and MIC=1.31 mg/ml for Enterococcus
faecalis). Satrani (2006) also concluded that C.
atlantica essential oil has antimicrobial activity against
Escherichia coli, B. subtilis, Micrococcus luteus, and
Staphylococcus aureus. A similar study demonstrated
that the essential oils derived from Atlas Cedar winged
and wingless seeds were able to inhibit the growth of
Escherichia coli, at a concentration of 1/100 v/v
(Rhafouri et al., 2014).
The antibacterial activity of the
hydromethanolic extract of C. atlantica cones and its
purified compounds were also investigated. Maya et al.
(2017) revealed the interesting antimicrobial activity of
hydromethanolic extract against a large panel of
bacterial strains. Indeed, among the purified
compounds, dehydroabietic acid was the most active,
with MIC values of 15.1 and 31.2 µg/ml against
Enterococcus faecalis and Staphylococcus aureus,
respectively.
The antibacterial activity of our essential oil
can be attributed to its chemical composition,
especially the presence of terpene alcohols (iso-
cedranol, cedrol, trans-carveol, p-cymen-8-ol, borneol,
and-terpineol),which represent 26.03% of the oil
(Satrani, 2016).Other studies have shown that the
essential oils that possess the strongest antibacterial
properties are rich in phenolic compounds (Baydar et
al., 2004; Rota et al., 2008).
Thus, the presence of phenolic
compounds, especially
hydroxyl groups, play an
important role in antimicrobial
activity (Zinoviadou et al.,
2009).
Antifungal activity
C. atlantica essential
oil showed antifungal activity,
as reflected by the obtained
MIC values. The MICs
determined for all fungal
strains tested in this study
oscillated between 0.5% and
1% (v/v) (Table 3). The
essential oil tested has
fungistatic activity against
almost all fungal strains
studied. Thus, similar
susceptibility levels were
identified for P.
commune (PDLd” and
PDLd10) and A. niger, with
MIC values of 1% (v/v). P.
expansum, P. crustosum, and
T. hyalocarpa showed similar
levels of susceptibility with
MIC values of 0.5% (v/v).
The lipophilicity of
essential oils enables their
penetration into the membrane
structures of the fungi, causing
membrane expansion,
increased membrane
permeability and fluidity, the
disruption of membrane-
embedded proteins, and
changes in the ion transport
process in fungi (Burt, 2004;
Oonmetta-aree et al., 2006;
Khanet al., 2010; Fadli et al.,
2012). Decreased lipids, which
are major components of the
cell membrane, suggest a
reduction in membrane
stability and the increased
permeability of water-soluble
materials (Helal et al., 2007).

41
F. Bennouna et al. / Moroccan J. Biol. 16 (2019): 35-45
Terpenes, which are the primary
constituents of essential oils, reportedly
disrupt or penetrate the lipid structures of
cells, by saturating the cell membrane
(Prashar et al., 2003). Some studies have
shown that the treatment of fungi with
essential oils decreased the lipid contents,
affected the cell membrane structure, and
inhibited fungal growth (Helal et al., 2006;
Helal et al., 2007; Tao et al., 2014).
The fungi toxicity and antifungal
activities of C. atlantica essential oil could
be attributed to fungal membrane
disruption, due to the accumulation of the
essential oil compounds on the cytoplasmic
membrane.
Effects of Cedrus atlantica essential oil
on the physicochemical properties of
cedar wood
According to Vogler (1998), and
the approach of Van Oss et al. (1988,
1989), the untreated cedar wood surface
was qualitatively and quantitatively
hydrophobic, with values of θW=
81.50.73° and ∆Giwi=−64.38mJ/m2.
These results are consistent with those
obtained by Meijer et al. (2000), who also
reported the qualitative hydrophobic
character of cedar wood, with θW=692°.
Table 4 shows that untreated cedar wood
has an electron donor character, γ−, more
than an electron acceptor character, γ+.
The cedar wood essential oil had
remarkable effects on cedar wood surface
hydrophobicity and the electron
donor/electron acceptor properties after
treatment. The results showed that the
degree of hydrophobicity did not change
much, quantitatively, even after 15 min of
treatment, with ∆Giwi=−41.46mJ/m2. In
addition, the electron donor character,
using C. atlantica essential oil was on
average 2.5-fold higher than that of
untreated wood.
Similar results were reported in our
recent study, focused on the treatment of
the cedar wood surface, using Rosmarinus
officinalis essential oil (Bennouna et al.,
2018). After 15 min of treatment, the
physicochemical properties of the wood
surface were modified, maintaining its
hydrophobic character, quantitatively, with
an increase in the electron donor character
(∆Giwi=−26.49mJ/m2; γ− =16.290.58
mJ/m2). Similar results were reported by
Barkai et al. (2015, 2016), after treatment
with -ionone (∆Giwi=−7.52 mJ/m2; γ−
=27.520.41mJ/m2) and carvone (∆Giwi=
−5.31 mJ/m2; γ−=29.110.43mJ/m2).
However, unlike our results, several
studies have shown that untreated cedar
wood samples can become hydrophilic
following treatments with Mentha
pulegium and Cananga odorata essential
oils (Bennouna et al., 2018), essential oil
components (Carvacrol and 1.8-cineol)
(Barkai et al., 2015, 2016), Myrtus
communis, and Thymus vulgaris extracts
(Sadiki et al., 2014, 2015, 2017-b).
The effects of essential oil
treatment time were also evaluated in this
study. Contact angle measurements and
surface energy components were
calculated after 1 h of essential oil
treatment. The results showed that the
cedar wood always retained its
hydrophobic character, quantitatively, with
∆Giwi =−11.62 mJ/m2. The electron
donor/electron acceptor properties were
also affected. Treatment for 1 h resulted in
a 6-fold increase compared with that of the
untreated wood, and a 2.5-fold increase
compared with the 15-min treatment. The
values of the electron acceptor character
were almost negligible for both untreated
and treated cedar wood.
The modification of surface
properties that were noted in this study can
be attributed to the chemical composition
of C. atlantica essential oil. The
maintenance of the hydrophobic character
of cedar wood can be explained by the low
percentage of terpene alcohols (26.03%),
which have a hydrophilic character, due to
the presence of hydroxyl groups, compared
with the percentages of other hydrophobic
compounds in essential oils (73.97%).

42
F. Bennouna et al. / Moroccan J. Biol. 16 (2019): 35-45
Table 4. Contact angle measurements, surface energy parameters (Lifshitz–van der Waals (γLW), electron donor
(γ–) and electron acceptor (γ+)) of untreated and treated cedar wood.
Contact angles(°)
Surface energy: components
and parameters (mJ/m2)
Untreated wood
81.50
±0.73
54.50
±0.57
21.9
±0.2
47.1
0.44
3.74
-64.38
HEC-15 min
62.60
±0.03
30.30
±0.56
15.9
±0.9
48.76
±0.22
0.85
±0.03
10.19
±0.20
-41.46
HEC-1h
50.60
±0.33
33.60
±0.15
10.20
±0.61
49.89
±0.09
0.07
±0.01
25.46
±0.41
-11.62
Conclusion
The analysis of C. atlantica
essential oil revealed cedranone (19.35%)
and iso-cedranol (13.78%) to be the major
components, followed by caryophyllene
oxide (8.73%), γ-calamenene (7.77%), -
cadinene (7.34%), -himachalane (7.23%),
cedrol (4.44%), sabinene (4.32%), and-
himachalane (4.05%). All of the bacterial
and fungal strains that were isolated from
decaying cedar wood and were tested in
this study were found to be susceptible to
C. atlantica essential oil. The
physicochemical properties of cedar wood
surfaces were found to change after
treatment with C. atlantica essential oil,
although the wood retained its hydrophobic
character, quantitatively, after both 15 min
and 1h of treatment. An increase in the
electron donor/electron acceptor properties
was noticed, and after 1 h of treatment,
they were 2.5-fold than that of the 15-min
treatment. Therefore, the C. atlantica
essential oil, as a natural product, can be
used as an alternative to synthetic chemical
products, to produce an anti-adhesive and
antimicrobial cedar wood surface and
prevent biofilm development.
Acknowledgements
The authors would like to
acknowledge the support and technical
assistance of Interface Regional University
Center (University Sidi Mohamed Ben
Abdellah, Fez), and National Center for
Scientific and Technical Research
(CNRST-Rabat).
References
Adams RP (1991) Cedar wood oil-
Analyses and properties. In Essential oils
and Waxes, Springer-Verlag Berlin
Heidelberg, Germany, pp. 159–173.
Barkai H, El Abed S, Sadiki M,Balouiri M,
Maataoui H, Ibnsouda Koraichi S(2016)
Evaluation of hydrophobic-hydrophilic
properties and anti-adhesive potential of
the treated cedar wood by two essential oil
components against bioadhesion of
Penicillium expansum spores. Journal of
Applied Sciences 16: 372–379.
Barkai H, Sadiki M, El Abed S, El harchli
H, Boutahari S, Ibnsouda Koraichi, S
(2015) The effect of carvacrol and carvone
treatments on the cedar wood surface
physico-chemical properties. International
Journal of Scientific & Engineering
Research 6: 767–771.
Baydar H, Saǧdiç O, Özkan G, Karadoǧan
T (2004) Antibacterial activity and
composition of essential oils from
Origanum, Thymbra and Satureja species
with commercial importance in Turkey.
Food Control 15: 169–172.
Bennouna F, Ibnsouda Koraichi S, Barkai
H, Lekbach Y, El Abed S, Lachkar M
(2018) Essential oil effect on the

43
F. Bennouna et al. / Moroccan J. Biol. 16 (2019): 35-45
physicochemical characteristics of
different wood species. Asian Journal of
Scientific Research 11: 568–573.
Blanchette RA (2000) A review of
microbial deterioration found in
archaeological wood from different
environments. International
Biodeterioration & Biodegradation 46:
189–204.
Bouhdid S, Abrini J, Zhiri A, Espuny MJ,
Manresa A (2009) Investigation of
functional and morphological changes in
Pseudomonas aeruginosa and
Staphylococcus aureus cells induced by
Origanum compactum essential oil.
Journal of Applied Microbiology 106:
1558–1568.
Burt S (2004) Essential oils: their
antibacterial properties and potential
applications in foods-a review.
International Journal of Food
Microbiology 94: 223–253.
Chebli B, Achouri M, Idrissi Hassani M,
Hmamouch M (2003) Antifungal activity
of essential oils from several medicinal
plants against four postharvest citrus
pathogens. Phytopathologia Mediterranea
42: 251–256.
Chedgy RJ, Morris PI, Lim YW, Breuil C
(2007) Black stain of western red cedar
(Thuja Plicata Donn) by Aureobasidium
pullulans: The role of weathering. Wood &
Fiber Science 39: 472–481.
Clinical and Laboratory Standards Institute
(2008) Reference Method for Broth
Dilution Antifungal Susceptibility Testing
of Filamentous Fungi; Approved
Standard—Second Edition, CLSI
document M38-A2, 950 West Valley
Road, Suite 2500, Wayne, PA 19087 USA.
Clinical and Laboratory Standards Institute
(2012). Methods for Dilution
Antimicrobial Susceptibility Tests for
Bacteria That Grow Aerobically ;
Approved Standard—Ninth Edition, CLSI
document M07-A9, 950 West Valley
Road, Suite 2500, Wayne, PA 19087 USA.
De Meijer M, Haemers S, Cobben W,
Militz H (2000) Surface energy
determinations of wood: Comparison of
methods and wood species. Langmuir 16:
9352–9359.
Derwich E, Benziane Z, Boukir A (2010)
Chemical Composition and In Vitro
Antibacterial Activity of the Essential Oil
of Cedrus atlantica. International Journal
of Agriculture & Biology 12: 381–385.
Dickinson DJ (1972) Disfigurement of
decorative timbers by blue stain fungi.
International pest control 14: 21–25.
El Abed S, Hamadi F, Latrache H, Iraqui
Houssaini M, Ibnsouda Koraichi S (2010)
Adhesion of Aspergillus niger and
Penicillium expansum spores on Fez cedar
wood substrata. Annals of Microbiology
60: 377–382.
Ez Zoubi Y, El-akhal F, Farah A,
Taghzouti K, El Ouali Lalam A (2017)
Chemical composition and larvicidal
activity of Moroccan Atlas Cedar (Cedrus
atlantica Manetti) against Culex pipiens
(Diptera : Culicidae). Journal of Applied
Pharmaceutical Science 7: 030–034.
Fadli M, Saad A, Sayadi S, Chevalier J,
Mezrioui NE, Pags JM, Hassani L (2012)
Antibacterial activity of Thymus
maroccanus and Thymus broussonetii
essential oils against nosocomial infection-
Bacteria and their synergistic potential
with antibiotics. Phytomedicine 19: 464–
471.
Gobakken LR, Vestøl GI (2012) Surface
mould and blue stain fungi on coated
Norway spruce cladding. International
Biodeterioration & Biodegradation 75:
181–186.
Görs S, Schumann R, Häubner N, Karsten
U (2007) Fungal and algal biomass in
biofilms on artificial surfaces quantified by
ergosterol and chlorophyll a as biomarkers.
International Biodeterioration &
Biodegradation 60: 50–59.
Hammer KA, Carson CF, Riley TV (1999)
Antimicrobial activity of essential oils and

44
F. Bennouna et al. / Moroccan J. Biol. 16 (2019): 35-45
other plant extracts. Journal of Applied
Microbiology 86: 985–990.
Helal GA, Sarhan MM, Abu Shahla ANK,
Abou El-Khair EK (2006) Effects of
Cymbopogon citratus L. essential oil on
the growth, lipid content and
morphogenesis of Aspergillus niger ML2-
strain. Journal of Basic Microbiology 46:
456–469.
Helal GA, Sarhan MM, Abu Shahla ANK,
Abou El-Khair EK (2007) Effects of
Cymbopogon citratus L. essential oil on
the growth, morphogenesis and aflatoxin
production ofAspergillus flavus ML2-
strain. Journal of Basic Microbiology 47:
5–15.
Khan A, Ahmad A, Akhtar F, Yousuf S,
Xess I, Khan LA, Manzoor N (2010)
Ocimum sanctum essential oil and its
active principles exert their antifungal
activity by disrupting ergosterol
biosynthesis and membrane integrity.
Research in Microbiology 161: 816–823.
Lancini GC, Parenti F, Gallo GG (1993)
Antibiotics : a multidisciplinary approach.
Second edition, Springer Science &
Business Media, Salerno, Italy.
Loizzo MR, Saab A, Tundis R, Statti GA,
Lampronti I, Menichini F, Gambari R,
Cinatl J, Doerr H W (2008) Phytochemical
analysis and in vitro evaluation of the
biological activity against herpes simplex
virus type 1 (HSV-1) of Cedrus libani A.
Rich. Phytomedicine 15: 79–83.
Mansouri N, Satrani B, Ghanmi M, El
Ghadraoui L, Aafi A, Farah A (2010)
Valorisation des huiles essentielles de
Juniperus thurifera et de Juniperus
oxycedrus du Maroc. Phytothérapie 8:
166–170.
Marcum DB, Hanson BR (2006) Effect of
irrigation and harvest timing on
peppermint oil yield in California.
Agricultural Water Management 82: 118–
128.
Maya BM, Abedini A, Gangloff SC,
Kabouche A, Kabouche Z, Voutquenne-
Nazabadioko L (2017) A new δ-
tocotrienolic acid derivative and other
constituents from the cones of Cedrus
atlantica and their in vitro antimicrobial
activity. Phytochemistry Letters 20: 252–
258.
Muñoz-Bertomeu J, Arrillaga I, Segura J
(2007) Essential oil variation within and
among natural populations of Lavandula
latifolia and its relation to their ecological
areas. Biochemical Systematics & Ecology
35: 479–488.
Necmettin Y, Hakki Mehmet A, Siegfried
N, Hubert K, Tuncay Efe F(2005)
Chemical composition of the essential oils
from oleoresin on cones of Cedrus libani.
Asian Journal of Chemistry 19: 2300–
2306.
Oonmetta-aree J, Suzuki T, Gasaluck P,
Eumkeb G (2006) Antimicrobial properties
and action of galangal (Alpinia galanga
Linn.) on Staphylococcus aureus. LWT -
Food Science & Technology 39: 1214–
1220.
Prashar A, Hili P, Veness RG, Evans CS
(2003) Antimicrobial action of palmarosa
oil (Cymbopogon martinii) on
Saccharomyces cerevisiae. Phytochemistry
63: 569–575.
Renau-Morata B, Nebauer SG, Sales E,
Allainguillaume J, Caligari P, Segura J
(2005) Genetic diversity and structure of
natural and managed populations of Cedrus
atlantica (Pinaceae) assessed using random
amplified polymorphic DNA. American
Journal of Botany 92: 875–884.
Rhafouri R, Strani B, Zair T, Ghanmi M,
Aafi A, El Omari M, Bentayeb A (2014)
Chemical composition, antibacterial and
antifungal activities of the Cedrus atlantica
(Endl.) Manettiex Carrière seeds essential
oil. Mediterranean Journal of Chemistry 3:
1034–1043.
Rota MC, Herrera A, Martínez RM,
Sotomayor JA, Jordán M J (2008)
Antimicrobial activity and chemical
composition of Thymus vulgaris, Thymus
zygis and Thymus hyemalis essential oils.
Food Control 19: 681–687.

45
F. Bennouna et al. / Moroccan J. Biol. 16 (2019): 35-45
Sadiki M, Barkai H, Ibnsouda Koraichi S,
Elabed S (2014) The effect of the Thymus
vulgaris extracts on the physicochemical
characteristics of cedar wood using angle
contact measurement. Journal of Adhesion
Science & Technology 28: 1925–1934.
Sadiki M, El Abed S, Balouiri M, Barkai
H, El Bergadi FZ, El Farricha O, Ibnsouda
Koraichi S (2017-a) Combined effect of
essential oils against bacteria associated
with deterioration of historical wood.
Journal of Materials & Environmental
Science 8: 594–602.
Sadiki M, El Abed S, Barkai H, Balouiri
M, Nasri A, Ibnsouda Koraichi, S (2017-b)
The modification of cedar wood surface
properties for the prevention of fungal
adhesion. International Journal of
Adhesion & Adhesives 75: 40–46.
Sadiki M, El Abed S, Barkai H, Laachari
F, Ibnsouda Koraichi S (2015) The impact
of Thymus vulgaris extractives on cedar
wood surface energy: Theoretical and
experimental of Penicillium spores
adhesion. Industrial Crops & Products 77:
1020–1027.
Satrani B (2006) Composition chimique et
activité antimicrobienne des huiles
essentielles extraites par hydrodistillation
fractionnée du bois de Cedrus atlantica
Manetti. Acta Botanica Gallica 153: 97–
104.
Sterflinger K, Piñar G (2013) Microbial
deterioration of cultural heritage and works
of art - Tilting at windmills? Applied
Microbiology & Biotechnology 97: 9637–
9646.
Tao N, Jia L, Zhou H (2014) Anti-fungal
activity of Citrus reticulata Blanco
essential oil against Penicillium italicum
and Penicillium digitatum. Food Chemistry
153: 265–271.
Tian J, Chen Y, Ma B, He J, Tong J, Wang
Y (2014) Drosera peltata Smith var. lunata
(Buch.-Ham.) C. B. Clarke as a feasible
source of plumbagin: Phytochemical
analysis and antifungal activity
assay.World Journal of Microbiology &
Biotechnology 30: 737–745.
Van Oss CJ (1993) Acid-base interfacial
interactions in aqueous media. Colloids
and Surfaces A: Physicochemical &
Engineering Aspects 78: 1–49.
Van Oss CJ (2003) Long-range and short-
range mechanisms of hydrophobic
attraction and hydrophilic repulsion in
specific and aspecific interactions. Journal
of Molecular Recognition 16: 177–190.
Van Oss CJ, Chaudhury MK, Good RJ
(1988) Interfacial Lifshitz-van der Waals
and polar interactions in macroscopic
systems. Chemical Reviews 88: 927–941.
Van Oss CJ, Ju L, Chaudhury MK, Good
RJ (1989) Estimation of the polar
parameters of the surface tension of liquids
by contact angle measurements on gels.
Journal of Colloid & Interface Science
128: 313–319.
Vogler EA (1998) Structure and reactivity
of water at biomaterial surfaces. Advances
in colloid & interface science 74: 69–117.
Zinoviadou KG, Koutsoumanis KP,
Biliaderis CG (2009) Physico-chemical
properties of whey protein isolate films
containing oregano oil and their
antimicrobial action against spoilage flora
of fresh beef. Meat Science 82: 338–345.
Zrira S, Ghanmi M (2016) Chemical
Composition and Antibacterial Activity of
the Essential of Cedrus atlantica (Cedar
wood oil). Journal of essential oil-bearing
plants 19: 1267–1272.
Zyani M, Mortabit D, Mostakim M, Iraqui,
M, Haggoud A, Ettayebi M, Ibnsouda
Koraichi S (2009) Cellulolytic potential of
fungi in wood degradation from an old
house at the Medina of Fez. Annals of
Microbiology 59: 699–704.