Chemical- vs sonochemical-assisted synthesis of ZnO nanoparticles from a new zinc complex for improvement of carotene biosynthesis from Rhodotorula toruloides MH023518

Article abstract of DOI:10.1002/aoc.6086

The complex [ZnL₂] {HL = 2-(((3,4-dimethylphenyl)imino)methyl)phenol} was introduced by two routes (chemical vs. sonochemical) and acted as a source of different size zinc-oxide (ZnO) nanoparticles (NPs) (nonsonicated ZnO NPs of Brunauer–Emmett–Teller (BET) surface area of 19.208 m² g^?1, total pore volume of 0.03493 cc g^?1, and particle diameter of 20.49–114.89 nm vs. sonicated ZnO NPs of BET surface area of 38.383 m² g^?1, total pore volume of 0.08723 cc g^?1 and particle diameter of 15.09–55.31 nm). The NPs generate cell stress that leads to the formation of reactive oxygen species (ROS). Consequently, carotene biosynthesis can be enhanced to overcome the ROS. Rhodotorula toruloides MH023518, genetically identified with 18S rRNA, produced carotenoids (124 ± 4.5 mg L^?1) and lipids (0.897 ± 0.01 mg L^?1) under no NPs' stress, but these values increased to total carotenoids of 264 ± 0.6 and 297.78 ± 0.8 mg L^?1 and total lipids of 0.97 ± 0.007 and 1.096 ± 0.015 mg L^?1 under stress conditions of 50 ppm of the nonsonicated and sonicated ZnO NPs, respectively. These results may represent potential industrial utilization of the as-formed ZnO NPs (particularly the sonicated sample) as carotene and lipid stimulator. Additionally in this article, the soluble proteins, total antioxidants, and catalase and superoxide dismutase specific activities of the yeast in the presence of various concentrations of the as-formed ZnO NPs were determined.

Full text of DOI:10.1002/aoc.6086

Received: 7 July 2020
DOI: 10.1002/aoc.6086
Revised: 21 September 2020
Accepted: 14 October 2020
F U L L P A P E R
Chemical- vs sonochemical-assisted synthesis of ZnO
nanoparticles from a new zinc complex for improvement of
carotene biosynthesis from Rhodotorula toruloides
MH023518
1
2
Ahmed B.M. Ibrahim
| Ghada Abd-Elmonsef Mahmoud
1
Department of Chemistry, Faculty of
Science, Assiut University, Assiut, 71516,
Egypt
The complex [ZnL ] {HL = 2-(((3,4-dimethylphenyl)imino)methyl)phenol} was
2
introduced by two routes (chemical vs. sonochemical) and acted as a source of
different size zinc-oxide (ZnO) nanoparticles (NPs) (nonsonicated ZnO NPs of
2
Department of Botany and Microbiology,
Faculty of Science, Assiut University,
Assiut, 71516, Egypt
2
−1
Brunauer–Emmett–Teller (BET) surface area of 19.208 m g , total pore
−1
volume of 0.03493 cc g , and particle diameter of 20.49–114.89 nm
Correspondence
2
−1
vs. sonicated ZnO NPs of BET surface area of 38.383 m g , total pore volume
of 0.08723 cc g− and particle diameter of 15.09–55.31 nm). The NPs generate
cell stress that leads to the formation of reactive oxygen species (ROS). Conse-
quently, carotene biosynthesis can be enhanced to overcome the ROS.
Rhodotorula toruloides MH023518, genetically identified with 18S rRNA,
Ahmed B.M. Ibrahim, PhD, Department
of Chemistry, Faculty of Science, Assiut
University, Assiut 71516, Egypt.
Email: aibrahim@aun.edu.eg
1
−1
−1
produced carotenoids (124 ± 4.5 mg L ) and lipids (0.897 ± 0.01 mg L )
under no NPs' stress, but these values increased to total carotenoids of
64 ± 0.6 and 297.78 ± 0.8 mg L− and total lipids of 0.97 ± 0.007 and
1
2
1
.096 ± 0.015 mg L− under stress conditions of 50 ppm of the nonsonicated
1
and sonicated ZnO NPs, respectively. These results may represent potential
industrial utilization of the as-formed ZnO NPs (particularly the sonicated
sample) as carotene and lipid stimulator. Additionally in this article, the
soluble proteins, total antioxidants, and catalase and superoxide dismutase
specific activities of the yeast in the presence of various concentrations of the
as-formed ZnO NPs were determined.
K E Y W O R D S
carotenoids, nano-structures, Rhodotorula, ultrasonic, zinc oxide
1
| INTRODUCTION
catalytic, antimicrobial, and ultraviolet filtering proper-
[
6,7]
ties.
ZnO—catalogued as a nontoxic material by Food
[8]
Nanomaterials—thanks to their high surface area and
unique characteristics—are convenient for utilization in
[3]
and Drug Administration (FDA) —is biocompatible to
[
9]
human cells surpassing other chemotherapeutic agents
in targeting cancer cells and is used in manufacturing of
various cosmetics including moisturizers, make-ups, face
[1]
[2]
medicine as antiviral,
antibacterial,
antifungal,
[4]
[5]
insecticidal, and antitumor agents. The zinc-oxide
nanoparticles (ZnO NPs) are highly applicable nano-
material that possesses chemical stability as well as
[
10]
powders, hand creams, and lotions.
Moreover, ZnO is
a
common photo-catalyst for degradation of
Appl Organomet Chem. 2020;e6086.
wileyonlinelibrary.com/journal/aoc
© 2020 John Wiley & Sons, Ltd.
1 of 13
https://doi.org/10.1002/aoc.6086

2
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IBRAHIM AND MAHMOUD
[
11]
environmental pollutants
and finds usefulness in gas
used in the complex synthesis, and the ultrasound syn-
thesis of the complex was carried out by the help of an
ultrasonic generator (Sonics Vibracell, SONICA-2200,
USA) utilizing frequency of 20 kHz and power output of
750 W. CHN data were elucidated on the element
Analyzer (elementar Analysensysteme GmbH—vario EL
III). Fourier-transform infrared spectroscopy (FT-IR)
analyses were obtained on a Nicolet iS10 spectrophotom-
eter. [ZnL2] solution electrical conductivity in
dimethylformamide (DMF) was determined by a conduc-
tivity meter (Jenway 4320), and the thermal data (ther-
mogravimetric analysis [TGA] and differential thermal
sensor, biosensor, optical device, drug delivery, and other
[12–14]
applications.
Cell exposure to NPs generates reactive oxygen spe-
cies (ROS) that whose amount depends on the NPs' con-
and excessive ROS disrupt the cells causing
DNA damage, lipid peroxidation, inactivation of proteins,
and enzymes and membrane perturbation.
ever, the cells usually scavenge the invading ROS by
enzymatic and nonenzymatic mechanisms to overcome
this situation.
Red yeasts are capable of producing valuable metabo-
lites such as lipids, proteins and carotenoids.
[15]
centration
[16,17]
How-
[18]
[
19]
analysis [DTA]) of 5.53 mg of [ZnL ] were measured by a
2
Rhodosporidium toruloides (R. glutinis, anamorphic form)
carotenoid acts physiologically as an antioxidant contrib-
uting in the yeast nonenzymatic defense system under
thermal analyzer (Shimadzu DTG 60-H) under regular
airflow and heating rate of 40 ml min− and 10 C per
minute, respectively. The packing phase of sonicated and
nonsonicated ZnO NPs was determined by a Philips PW
1
ꢀ
[20,21]
elevated oxidative stress situations.
Carotene
composition—which accumulates in the lipid drop-
1710 X-ray diffractometer utilizing CuK radiation,
α
[22]
ꢀ
ꢀ
lets —may change and some pigments with optimal
antioxidation properties, for example, β-carotene, may
40 mA and 40 kV over 2θ = 20 –90 with step size of
ꢀ
0.06 . Transmission electron microscopy (TEM) analyses
[23,24]
result in high amounts.
for detection of morphology and size of [ZnL ] and the
2
As coordination compounds are usually nontoxic and
of flexible structures, several zinc coordination com-
pounds are suitable precursors for preparation of ZnO
as-prepared ZnO NPs were carried out by a JEOL JEM-
100CX II transmission electron microscope, whereas the
ImageJ software was used for estimation of the size of the
compounds. The ZnO samples' textural characteristics
including their surface areas and pore volumes were
investigated on a NOVA 3000 high-speed gas sorption
analyzer (version 6.10) after degassing the NPs for 3 h at
[25–29]
NPs.
However, sonolysis is known to cause cavita-
tion during synthesis of various products, as it affords for-
mation, collapse, and growth of bubbles that result in
introduction of high pressure and temperature spots
[30–34]
ꢀ
affecting the size of the products.
we managed to prepare a new zinc complex of the ligand
-(((3,4-dimethylphenyl)imino)methyl)phenol by two
In this research,
200 C in vacuum.
2
routes (chemical vs sonochemical) that both afforded
different size ZnO NPs upon calcination. Subsequently,
we studied the NPs' size effect on the biosynthesis of
carotene by Rhodotorula toruloides MH023518. Being
influenced by the NPs' stress and carotene biosynthesis,
herein is also investigated the yeast defense mechanism
against ROS by determining the superoxide dismutase
2.2 | Synthesis of [ZnL2]
The zinc complex was prepared following two different
routes as follow:
Route (1): To a suspension of HL (1.125 g, 5 mmol)
and triethylamine (0.7 ml, 5 mmol) in ethanol (50 ml),
ZnCl (335 mg, 2.5 mmol) was added. The yellow product
2
(
SOD) and catalase (CAT) specific activities, total lipids,
was stirred for an hour before filtering, washing with eth-
anol and diethyl ether and drying over calcium chloride.
Route (2): To a suspension of HL (1.125 g, 5 mmol)
and triethylamine (0.7 ml, 5 mmol) in ethanol (50 ml),
[35]
and total antioxidants (TA).
Generally speaking, this
research could be helpful in industrial stimulation of
carotene by stress of ZnO NPs when the appropriate
concentration and size of the NPs are used.
ZnCl (335 mg, 2.5 mmol) was added. The reaction mix-
2
ture was then transferred for ultrasonication for 15 min,
whereas the reaction temperature was maintained below
ꢀ
2
2
| EXPERIMENTAL
40 C by the help of an ice bath. The yellow product was
then centrifuged, washed with ethanol and diethyl ether,
and dried over calcium chloride.
.1 | Materials and physical
measurements
[ZnL ] (Yield = [nonsonicated: 1.126 g, 89%; soni-
2
cated: 1.075 g, 85%]): Anal. Calcd. for ZnC H N O : C,
30
28 2 2
[36]
The ligand HL was prepared as described
analytical grade salicylaldehyde, 3,4-dimethylaniline and
absolute ethanol were used. ZnCl (Sigma Aldrich) was
where
70.11; H, 5.49; N, 5.45%. Found (nonsonicated): C,
70.47; H, 5.28; N, 5.64%. Found (sonicated): C, 71.03; H,
5.87; N, 6.11%. FT-IR for nonsonicated/sonicated [ZnL2]
2

IBRAHIM AND MAHMOUD
3 of 13
−
1
(
cm ): 1608/1604 ν(C N), 581/578 ν(Zn O), 483/482
2.5 | Growth conditions
−
1
2
−1
ν(Zn N). Molar conductance: 3.12 and 7.58 Ω cm mol
DMF) for the nonsonicated and sonicated complex.
(
Yeast inoculums were prepared by scraping 24 h old yeast
culture grown on YPD plats into 250 ml Erlenmeyer flasks
containing 50 ml of YPD broth medium. Flasks were incu-
ꢀ
2
.3 | Synthesis of ZnO NPs
bated for 18 h at 28 ± 1 C until reaching a final con-
6
[43]
centration of approximately 2 × 10 cells per milliliter.
Carotenoid was produced on modified glucose asparagine
The nonsonicated and sonicated ZnO NPs were prepared
by calcination of the nonsonicated and sonicated [ZnL ]
1000 mg) in a preheated oven at 600 C for 4 h, before
leaving the decomposition products to cool naturally.
−1
yeast medium (GAY) containing (g L ): glucose, 30.0;
yeast extract, 1.5; KH PO , 1.5; L-asparagine, 0.12 and
2
ꢀ
(
2
4
[
21,44]
MgSO .7H O, 0.5 at initial pH of 5.
Sterilized chloram-
4
2
−
1
phenicol (250 mg ml ) with a membrane of 0.22 mm pore
size was added.
[45]
After sterilization, the flasks were
2
.4 | Rhodotorula isolation and
inoculated with 1 ml of the yeast culture and incubated at
28 ± 1 C on a rotary shaker (150 rpm) for 72 h. Biomass
ꢀ
identification
was collected by centrifugation at 4000 g rpm for 15 min at
room temperature and washed thoroughly with distilled
water for removal of any medium residual.
R. toruloides MH023518 was isolated from Egyptian faba
bean roots as carotene target yeast using dilution plate
method on yeast extract peptone dextrose (YPD) medium
(
2
dextrose 20, yeast extract 10, peptone 20 and agar
−
1
ꢀ
[37,38]
0 g L ) and stored at 4 C.
The red yeast was iden-
2.6 | Effect of sonicated and nonsonicated
ZnO NPs on R. toruloides growth and
metabolites
tified first according to its phenotypic characteristics
including colony characteristics, color, ascospore forma-
tion,
characteristics,
pseudohyphae,
and
the
physiological
[39]
and the identification was confirmed
Different concentrations of nonsonicated and sonicated
ZnO NPs (0, 10, 25, 50, 100, 150, and 200 ppm) were
suspended in double distilled water and vortexed for 30 s.
The NPs were added to sterile GAY medium prior to
inoculation with freshly prepared yeast culture and incu-
by genotypic characters. For molecular identification,
yeast culture was prepared in 250 ml Erlenmeyer baffle
flask containing 50 ml of the YPD medium incubated at
ꢀ
2
5 ± 1 C for 2 days. Molecular identification was per-
[40]
ꢀ
formed in SolGent, Daejeon, South Korea.
used for the polymerase chain reaction (PCR) were (1) 5 -
ATTGGAGGGCAAGTCTGGTG-3
CTTGGTCATTTAGAGGAAGTAA-3 bound to conserve
Primers
bation at 28 ± 1 C on a rotary shaker (150 rpm) for 72 h.
0
Biomass was collected by centrifugation at 4000 g rpm for
15 min at room temperature and washed thoroughly with
distilled water. Experiment was performed with three
replicates of each treatment.
0
0
and
(2)
5 -
0
[41]
regions of the yeast 18S rRNA gene.
Amplification
reactions were performed using PCR (ABI, 9700). The
PCR reaction mixture was prepared using SolGent EF-
[
42]
Taq
deoxynucleoside triphosphate (dNTP) (T) 0.5 μl, primer
F-10p) 1.0 μl, primer (R-10p) 1.0 μl, EF-Taq (2.5 U)
as follows: 10X EF-Taq buffer 2.5 μl, 10 mM
2.7 | Analytical methods
(
2.7.1 | Consuming sugars assay
0
.25 μl, template 1.0 μl and DW to 25 μl. One round of
amplification was denatured at 95 C for 15 min followed
by 30 cycles of denaturation at 95 C for 20 s, annealed at
0 C for 40 s, and extended at 72 C for 1 min with final
extension step at 72 C for 5 min. The PCR products were
then purified with the SolGent PCR Purification Kit-Ultra
SolGent, Daejeon, South Korea) prior to sequencing.
Finally, the purified PCR products were reconfirmed
using size marker) by electrophoreses on 1% agarose gel,
ꢀ
Total sugars were determined in medium supernatant
ꢀ
[46]
using anthrone method
in which the sample (0.1 ml)
ꢀ
ꢀ
5
reacted with 2.25 ml of freshly prepared anthrone regent
(100 mg anthrone + 50 ml of 98% H SO ) in a boiling
ꢀ
2
4
water bath for 10 min, before cooling and the sugar con-
tent was estimated at 620 nm according to the glucose
(
−
1
standard graph (0.1–10 g L ).
(
and the obtained sequence was further analyzed using
BLAST from the National Center of Biotechnology Infor-
mation (NCBI) website (http://www.ncbi.nlm.nih.gov)
and deposited in GenBank database for accession
numbers.
2.7.2 | Total lipids extraction and assay
[
47]
Total lipids were extracted as reported : Yeast cells
were suspended in chloroform-methanol mixture (1:1),

4
of 13
IBRAHIM AND MAHMOUD
left in darkness overnight and treated with sulfuric
acid (98%) to transform all extracted fats into fatty
acids. Total lipids were determined using sulfo-
phospho-vanillin method at 490 nm according to the
vegetable oil standard graph (1–10 mg per 10 ml)
in chloroform.
2.7.5 | Statistical analysis
All biological tests were conducted in triplicate and statis-
tically analyzed via analysis of variance (ANOVA) (one-
way) using the SPSS 10.0 software (SPSS, Chicago, IL,
USA). Significant variations and means were compared
with Duncan's multiple tests (DMRT) with 5% level.
2
.7.3 | Total carotenoids extraction and
assay
3 | RESULTS AND DISCUSSION
3.1 | Characterization of [ZnL2]
Carotenoids were extracted with petroleum ether-acetone
(1:1), after disintegrating the yeast cells using liquid
[
36]
nitrogen until complete cell breakage and the extraction
process was repeated until colorless cell residue was
obtained. The residue was centrifuged at 4000 g for
HL was prepared as described.
[ZnL ] was prepared
2
via a conventional, and an ultrasound-assisted route
where in both cases triethylamine was added to facilitate
the ligand hydroxyl group deprotonation. Indeed,
sonolysis is known of its ability for introducing disaggre-
gation and reducing the size of the synthesized materials
1
5 min, washed thoroughly with distilled water and dried
ꢀ
at 40 C overnight until constant dry weight. Carotenoid
was quantified in the pigmented layer at 450 nm
according to the standard graph of β-carotene (2 to
via introducing high pressure and temperature spots dur-
−1
[44,48]
[30–34,54–57]
1
2 μg ml ).
ing synthesis in the reaction medium.
Addi-
tionally, it was confirmed that chemical and
sonochemical synthetic routes for the complex formation
resulted not only in coordination compounds of different
sizes but also in obtaining derived NPs of different
2
.7.4 | Antioxidants assay
[
30,58]
Fresh yeast cells were disintegrated using mechanical
disruption with liquid nitrogen until complete cell
breakage and extracted with 100 mM potassium phos-
phate buffer (pH 7.8) containing polyvinylpyrrolidone
sizes.
The tetra-coordinate zinc complexes especially
those with salicylaldimine ligands are usually tetrahedral
3
(SP hybridization), and the involvement of sal-
icylaldimine ligands in coordination was reported to be
(
PVP, 0.1 g) and EDTA (0.1 mM). The homogenate
via the imine nitrogen and the hydroxyl oxygen atoms in
ꢀ
[59–61]
was centrifuged at 18,000 rpm for 10 min at 4 C and
the supernatants were collected and used for the assay
of SOD and CAT activities, TA, and soluble proteins
a monobasic manner.
The analytics only showed
negligible differences between the nonsonicated and son-
icated complex regarding the CHN composition, FT-IR
data and DMF solution electrical conductivity clearing
[49]
(
SP).
of epinephrine (adrenochrome) as described
modifications as the reaction medium contained
0 mM of sodium carbonate buffer (pH 10.2), 0.1 mM
of ethylenediaminetetraacetic acid (EDTA), 100 μl of
SOD (EC 1.15.1.1) was assayed by autoxidation
[50]
with
the same formula of [ZnL ] for the complex in both cases
2
(Figure 1). The IR spectra of HL and [ZnL ] (non-
2
5
sonicated and sonicated) were studied. The mostly impor-
−
1
tant characteristic bands of HL exist at 3426 cm
−
1
−1
protein extract and 100 μl of epinephrine (5.5 mg ml ).
Autoxidation of epinephrine was spectrophotometri-
cally determined at 480 nm for 1 min, and the activity
was reported as specific activity units per milligram
protein. Catalase (CAT; 1.11.1.6) was assayed following
υ(OH) and 1615 cm ν(C N). In the complex, the
Zn(II) coordination sites are linked with two nitrogen
[
51]
the consumption of H O for 1 min at 240 nm.
TA
2
2
of the yeast methanol extract were evaluated by the
phosphomolybdenum method using the ascorbic acid
[52]
standard curve at 695 nm
and SP in the yeast
extract were determined by Folin reagent according to
the bovine serum albumin (BSA) calibration curve at
[53]
7
50 nm.
All colorimetric measurements were
performed using a T60 ultraviolet-visible (UV–Vis)
spectrophotometer with a 2 nm fixed slit.
2
FIGURE 1 Structure of [ZnL ]

IBRAHIM AND MAHMOUD
5 of 13
and two oxygen atoms. Hence, the υ(OH) vibration dis-
appeared, whereas the ν(C N) band shifted to a lower
wavenumber of 1608/1604 cm for the nonsonicated/
Thermal analysis (TGA and DTA) of the complex was
performed and proved no significant weight loss up to
200 C corresponding to no solvent molecules involved in
−
1
ꢀ
sonicated [ZnL ] indicating the formation of Zn(II)
the structure of the complex. Additionally, the complex
thermogram (Figure 2) showed formation of final decom-
2
[61]
azomethine (N) bonds.
Besides at the wavenumbers of
−
1
5
81/578 and 483/482 cm in spectra of [ZnL ], two
position
product
(ZnO;
Calcd.
=
15.606%,
2
ꢀ
bands corresponding to ν(Zn O) and ν(Zn N) vibrations
have appeared.
Found = 15.371%) at around 580 C; therefore, we heated
ꢀ
the complex at 600 C to obtain the ZnO NPs.
FIGURE 2 Thermogravimetric
analysis (TGA) curve of [ZnL ]
2
FIGURE 3 The powder X-ray
diffraction (PXRD) pattern of the
nonsonicated (red) and sonicated (black)
zinc-oxide nanoparticles (ZnO NPs)

6
of 13
IBRAHIM AND MAHMOUD
FIGURE 4 Transmission electron microscopy (TEM) images of nonsonicated [ZnL
2
] (bar = 500 nm, top left), sonicated [ZnL
2
]
(
bar = 100 nm, top right), nonsonicated zinc-oxide (ZnO) (bar = 100 nm, bottom left) and sonicated ZnO (bar = 100 nm, bottom right);
insets are particle size distribution histograms
[
64]
3.2 | Characterization of nonsonicated
indexed in the ICDD file 04-016-6648
clearing highest
and sonicated ZnO NPs
purity for the NPs: Both patterns displayed peaks attrib-
uted to the (100), (002), (101), (102), (110), (103), (200),
(112), (201), (004), and (202) planes of hexagonal struc-
Preparation of the sonicated and nonsonicated ZnO NPs
[
64]
was based on heating the sonicated and nonsonicated
ture (space group: P6 mc),
whereas the diffraction
3
ꢀ
[
ZnL ] in air at 600 C for 4 h. Sonochemical-assisted
peaks obtained from the nonsonicated ZnO NPs were
intense and sharper than the peaks obtained from the
2
techniques for synthesis are considered simple, fast, and
ecofriendly that usually help in preparation of small sized
sonicated ZnO NPs which is indicative for the size reduc-
[58]
[45]
NPs.
Interestingly, sonolysis as-derived end products
tion in the sonicated sample.
Indeed, the crystallite
[
62,63]
are usually of excellent purity and high surface area.
size was determined from the PXRD pattern by
[
65]
The powder X-ray diffraction (PXRD) patterns of the
nonsonicated and sonicated ZnO NPs (Figure 3) are not
significantly different and all determined peaks were
employing Debye–Scherrer equation ; the obtained
crystallite size was of 9–30 for the nonsonicated sample
(25 nm corresponding to the predominant 101 peak), and

IBRAHIM AND MAHMOUD
7 of 13
TABLE 1 Surface areas and pore volumes and diameters for
nonsonicated and sonicated ZnO NPs
revealed almost spherical and oval morphology for all
investigated particles. TEM proved no uniformity in size
of the nonsonicated [ZnL ] particles as the complex parti-
2
Nonsonicated
ZnO NPs
Sonicated
ZnO NPs
cles fall in a wide range of 57.52–146.03 nm (aver-
age = 93.47 nm), however ultrasonication resulted in
much smaller particles of 18.74–38.83 nm (aver-
Characteristics
BET surface area
19.208
18.231
0.03493
7.242
38.383
38.383
0.08723
9.090
2
−1
(
m g
)
age = 25.27 nm) of [ZnL ]. Regarding the ZnO particles,
2
External surface area
2
−1
TEM indicated the occurrence of 96.16% of the non-
sonicated ZnO in the “nano” range (1–100 nm) with larg-
est particle diameter of 114.89 nm, whereas the largest
particle diameter of the sonicated ZnO NPs was of
(
m g
)
Total pore volume
−
1
(
cc g )
Average pore
diameter (nm)
55.31 nm. TEM also revealed the smallest particle diame-
ter and average particle diameter of 15.09 and 34.52 nm
for the sonicated ZnO NPs and of 20.49 and 47.62 nm for
the nonsonicated ZnO NPs.
Abbreviations: BET, Brunauer–Emmett–Teller; NPs, nanoparticles; ZnO,
zinc-oxide.
Being that the material specific surface area and
mesoporosity improvement can significantly increase its
active sites which are responsible for its activity, the N2
sorption isotherms of the nonsonicated and sonicated
ZnO NPs were determined. It sounds that sonication of
the NPs' precursor appreciably influenced the as-formed
ZnO textural features: calcination of the conventionally
this crystallite size was reduced by 5–10 nm in the soni-
cated sample (19 nm corresponding to the predominant
1
01 peak).
Perfect analyses of the size and morphology of the as-
formed [ZnL ] complex and ZnO particles were accessi-
2
ble by TEM (Figure 4) that has proved the relationship
between the precursor and the NPs' size. The analysis
prepared [ZnL ] afforded NPs of Brunauer–Emmett–
2
FIGURE 5 Effect of nonsonicated (top left and bottom left) and sonicated (top right and bottom right) zinc-oxide nanoparticles (ZnO
NPs) on R. toruloides MH023518 dry mass (g L−1) and residual sugars (g L−1
)

8
of 13
IBRAHIM AND MAHMOUD
2
−1
Teller (BET) surface area of 19.208 m g , whereas the
sonicated complex after calcination afforded NPs of
3
of approximately 573 base pairs with 100% similarity to
R. toruloides (MN689692), 99% similarity to R. toruloides
KY104927 and 99% similarity to R. toruloides JQ963341.
Therefore, the yeast isolate was identified as R. toruloides
(MH023518) according to the NCBI website.
2
−1
8.383 m g which is almost twice the BET surface area
of the nonsonicated ZnO NPs (Table 1). Additionally,
Barrett, Joyner, and Halenda (BJH) distribution analysis
assigned mesoporous morphology for the ZnO NPs with
average pore larger for the sonicated ZnO sample.
3
.4 | Effect of ZnO NPs on sugar
consumption and R. toruloides growth
3
.3 | Yeast identification
In the presence of ZnO NPs, inverse relationship
between the yeast dry mass and residual sugar
(Figure 5) was observed. The ZnO NPs diminished the
The yeast was isolated from roots of Egyptian faba bean
on YPD medium and identified preliminary according to
its phenotypic characters as Rhodotorula sp. The colony
characters were red, smooth with entire margin, and the
microscopic characters were ovoid cell shape with multi-
lateral budding and absence of pseudohyphae, mycelia,
arthroconidia, ascospores, ballistoconidia, basidiocarps,
teliospores, chlamydospore, and endospores. Rhodotorula
sp. utilized different carbon sources, for example, glucose,
fructose, sucrose, galactose, and maltose, but could not
−
1
yeast growth from 2.2 ± 0.019 g L (zero ppm) to
−
1
1.67 ± 0.03 g L
(200 ppm, nonsonicated) and
1.63 ± 0.01 g L− (200 ppm, sonicated); however, the
residual sugar increased from 5.083 ± 0.1 g L (zero
ppm) to 12.76 ± 0.1 g L (200 ppm, nonsonicated)
and 12.88 ± 0.01 g L− (200 ppm, sonicated). In litera-
ture, good culture conditions are required for red yeast
1
−
1
−
1
1
growth, but cultivation stress conditions are important
[21,39]
[66]
grow on lactose.
gene sequences indicated that the isolate has a sequence
Molecular analysis of 18S rRNA
for specific metabolite production.
yielded biomass of 2–13 g L
R. toruloides
−
1
depending on the
FIGURE 6 Effect of nonsonicated (top left and bottom left) and sonicated (top right and bottom right) zinc-oxide nanoparticles (ZnO
NPs) on R. toruloides MH023518 total carotenoids (mg L−1) and total lipids (g L−1
)

IBRAHIM AND MAHMOUD
9 of 13
[
47,67]
264 ± 0.6 mg L−1 for nonsonicated ZnO NPs) was found
corresponding to 50 ppm of ZnO NPs, whereas total
carotenoids of 124 ± 4.5 mg L− were produced from the
control sample. After reaching the maximum, total carot-
enoids decreased as the concentration of ZnO NPs
increased reaching to 152 ± 2.7 and 192.2 ± 4.37 mg L
in the presence of 200 ppm of the nonsonicated and soni-
cated ZnO NPs, respectively. Carotenoids act as antioxi-
substrate type,
and the retardation in Rhodotorula
biomass production in this study may result from
hydrogen peroxide which can be generated under
stress conditions of nanomaterials.
NPs afford higher cell activities, and this has been
proved by studying the growth and physiological activi-
ties of R. toruloides.
1
[68,69]
The smaller
−
1
[70]
Small-sized NPs of zinc
exhibited enhanced antibacterial effect that was not
demonstrated in the presence of bulk Zn samples of
dants to protect yeast cells against photo-oxidation and
[71]
[72]
[75]
larger size,
besides ZnO NPs
especially those of
stress conditions.
Under no stress conditions,
−
1 [76]
size as small as of 5–10 nm demonstrated selective
size-dependent antimicrobial cell activities in compari-
son with larger-size ZnO samples.
R. glutinis yielded carotenoids of 0.5–185 mg L .
However, stress conditions including nanomaterials stim-
ulate the carotenoid production from the red yeast
[73,74]
[76]
for
detoxifying the active radicals and maintaining intracel-
lular stability state during the stress situation to face and
[
77]
3.5 | Effect of ZnO NPs on total
repair the damage.
Biologically, zinc is a significant
carotenoids and total lipids production by
R. toruloides
element involved in many cell enzymes and is also
important for the nucleic acid synthesis regulations with
[
78,79]
various metabolic pathways.
Besides, it was reported
In general, sonicated ZnO NPs produced more caroten-
oids in comparison with the nonsonicated ZnO NPs
significant decrease in carotene biosynthesis at concen-
[
35]
trations of ZnO NPs higher than the critical,
and this
(
(
Figure 6) and the highest carotenoids' concentration
explains our results as carotenoid concentration was
diminished at ZnO NPs' concentrations ≥ 50 ppm.
−1
297.78 ± 0.8 mg L
for sonicated ZnO NPs and
FIGURE 7 Effect of nonsonicated (top left and bottom left) and sonicated (top right and bottom right) zinc-oxide nanoparticles (ZnO
NPs) on R. toruloides MH023518 soluble proteins (mg g−1 fresh yeast) and total antioxidants (mg g−1 protein)

10 of 13
IBRAHIM AND MAHMOUD
In harmony with carotene production results, total
studied and—in general—the sonicated sample indicated
lipids followed the same trend (Figure 6): increasing ZnO
NPs concentration to 50 ppm resulted in increment of
total lipids to 0.97 ± 0.007 mg L− for the nonsonicated
higher stress activity than the nonsonicated one. SP
−
1
ranged from 4.9
±
0.016 mg
g
(0 ppm) to
1
−1
6.19 ± 0.02 mg g (nonsonicated ZnO, 200 ppm) and
6.47 ± 0.07 mg g (sonicated ZnO, 200 ppm) fresh yeast,
−1
−1
ZnO NPs and 1.096 ± 0.015 mg L for the sonicated
ZnO NPs in comparison to total lipids of
whereas ZnO NPs (100 ppm) resulted in the highest SP
−1
−1
0
.897 ± 0.01 mg L for the control sample. However, the
content of 7.02 ± 0.038 mg g (nonsonicated ZnO) and
7.47 ± 0.016 mg g− (sonicated ZnO) fresh yeast. TA
1
total lipids decreased down to 0.63 ± 0.004 and
−
1
−1
0
.77 ± 0.001 mg L in the presence of 200 ppm of the
ranged from 3.62 ± 0.045 mg g
(0 ppm) to
−1
nonsonicated and sonicated ZnO NPs, respectively.
Research documented degradation of cellular macromol-
ecules (proteins, DNA, carbohydrates, and lipids) by ROS
generated under NPs' stress as ROS react with unsatu-
rated fatty acids forming lipid radicals or toxic lipid
4.46 ± 0.11 mg g (nonsonicated ZnO, 50 ppm) and
4.03 ± 0.04 mg g− (sonicated ZnO, 50 ppm) protein,
whereas the TA decreased to 3.36 ± 0.02 mg g (non-
sonicated ZnO, 200 ppm) and 3.81 ± 0.04 mg g (soni-
cated ZnO, 200 ppm) protein (Figure 7). The results
indicated alternation in the cellular proteins to adapt the
1
−
1
−1
[67,80]
hydroperoxides.
[
81]
cell to the stress factors
and the considered toxicity
mechanism of ZnO NPs is generation of ROS (e.g., H O )
2
2
3
.6 | Effect of ZnO NPs on the
that could disrupt the electron transfer reactions in the
inner membrane of mitochondria.
[
82,83]
R. toruloides antioxidant system
CAT and SOD specific activities (Figure 8) have
increased as ZnO NPs' concentration increased (CAT and
SOD [control sample] = 0.02 and 0.67 ± 0.04, CAT and
The relationships between the as-prepared ZnO NPs con-
centration and SP, TA, and CAT and SOD activities were
FIGURE 8 Effect of nonsonicated (top left and bottom left) and sonicated (top right and bottom right) zinc-oxide nanoparticles (ZnO
NPs) on R. toruloides MH023518 catalase (CAT) (specific activity) and superoxide dismutase (SOD) (specific activity)

IBRAHIM AND MAHMOUD
11 of 13
SOD [nonsonicated ZnO, 200 ppm] = 0.075 ± 0.0013 and
methodology;
project
administration;
resources;
4
2
.89 ± 0.04 and CAT and SOD [sonicated ZnO,
00 ppm] = 0.118 ± 0.006 and 5.76 ± 0.29). This is with
software; supervision; validation; visualization. Ghada
Abd-Elmonsef Mahmoud: Conceptualization; data
curation; formal analysis; funding acquisition;
investigation; methodology; project administration;
the exception that the highest CAT and SOD activities for
nonsonicated sample (0.094 ± 0.0018 and 5.23 ± 0.138,
respectively) were recorded in the presence of ZnO NPs
resources;
software;
supervision;
validation;
(150 ppm). The optimal NPs uptake in microbial cells
visualization.
results in maximum specific activities of SOD and
[84]
CAT
and concentrations higher than the optimal of
ORCID
the NPs result in diminishing the cellular SOD, CAT, and
[17,35]
Ahmed B.M. Ibrahim https://orcid.org/0000-0003-4748-
135
Ghada Abd-Elmonsef Mahmoud https://orcid.org/
000-0002-4760-9871
carotenoid contents.
SOD functions to dispropor-
6
tionate two superoxide anions into H O and O , whereas
2
2
2
[18,76]
CAT breakdowns H O to O and H O.
When ZnO
2
2
2
2
0
NPs interact with the microbial cell surface, oxidative
stress ultimately dependent on the microbial cell mem-
brane polarity is induced resulting in cell growth
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[
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How to cite this article: Ibrahim ABM,
Mahmoud GA-E. Chemical- vs sonochemical-
assisted synthesis of ZnO nanoparticles from a new
zinc complex for improvement of carotene
biosynthesis from Rhodotorula toruloides
MH023518. Appl Organomet Chem. 2020;e6086.
https://doi.org/10.1002/aoc.6086
[
3
74, 1.