Toxics 2032738 C
Toxics 2032738 C
Toxics 2032738 C
Nur Zaida Zahari 1,2*, Pavitra A/P Murugayah1, Fera Nony Cleophas1,2, Sahibin Abd Rahim1 and Ahmad Norazhar 4
Mohd Yatim1 5
1 Environmental Science Programme, Faculty of Science & Natural Resources, Universiti Malay- 6
sia Sabah, UMS Road, 88400 Kota Kinabalu, Sabah Malaysia. 7
2 Small Islands Research Center, Universiti Malaysia Sabah, UMS Road, 88400, Kota Kinabalu, 8
Sabah, Malaysia 9
* Correspondence: zaidazahari@ums.edu.my ; Tel.: +60138988754; (N.Z.Z) 10
11
Abstract: Heavy metal pollution in the environment is a major concern for humans as it is non- 12
biodegradable and can bring a lot of effects on the environment, humans as well as plants. At 13
present, a solution to this problem is suggested in terms of a new innovative eco-friendly technology 14
known as phytoremediation. The use of bast fiber plants collected from the bast surrounding are 15
known as non-edible crops with a short life cycle. This plant composed of cellulose and hemicellu- 16
lose with a mixture of lignin or pectinis makes it a significant crop for many industrial sectors. Due 17
to its low maintenance requirements with minimum economic investment, bast fiber plants have 18
Citation: Zahari, N. Z..; Murugayah, been widely used in phytoremediation. Nevertheless, these plants have the ability to extract metals 19
P.; Cleophas, F.N.; Rahim, S.A.; from the soil through their deep roots, combined with their commercial prospects, making them an 20
Yatim, A.N.M. Phytoremediation: ideal candidate as a profit-yielding crop for phytoremediation purposes. Therefore, a 21
A Novel Approach of Bast Fiber
comprehensive review is needed for a better understanding of the morphology and 22
Plants (Hemp, Kenaf, Jute and Flax)
phytoremediation mechanism of four commonly bast fiber plants which are hemp (Cannabis sativa), 23
for Heavy Metals Decontamination
kenaf (Hibiscus cannabinus), jute (Corchorus olitorius) and flax (Linum usitatissimum). This review ar- 24
in Soil – Review. Toxics 2022, 10, x.
ticle summarizes existing research on the phytoremediation potential of these plants grown in dif- 25
https://doi.org/10.3390/xxxxx
ferent toxic pollutants such as Lead (Pb), Cadmium (Cd), and Zinc (Zn). This work also discusses 26
Academic Editor: Firstname Last- several aids including natural and chemical amendments to improve phytoremediation. The role of 27
name
these amendments in the bioavailability of contaminants, their uptake, translocation, and bioaccu- 28
Received: date mulation, as well as their effect on plant growth and development, has been highlighted in this 29
Accepted: date paper. This paper helps in identifying, comparing, and addressing the recent achievements of bast 30
Published: date fiber plants for the phytoremediation of heavy metals in contaminated soil. 31
1. Introduction 34
Heavy metal contamination in soil has a negative impact on the environment espe- 64
cially on soil quality and plant growth. Once the plant is saturated with heavy metal, the 65
plant dies due to the interruption in photosynthesis and protein synthesis. Elimination of 66
heavy metals is difficult as it is irreversible, and remediation needs to be done. Remedia- 67
tion can be divided into in-situ and ex-situ remediation. In-situ remediation is a process 68
of remediation that doesn’t require transport of contaminated soil to off-site treatment 69
facilities. Ex-situ remediation, on the other hand, is the remediation technique that re- 70
quires excavation of contaminated soil to an off-site treatment facility [3]. This process 71
requires additional costs. However, the treatments are controlled and accelerated and pro- 72
vide better results in a shorter time. Examples of in-situ remediation are surface capping, 73
encapsulation, electro-kinetics, soil flushing, immobilization, phytoremediation, and bio- 74
remediation. Examples of ex-situ remediation techniques are landfilling, soil washing, so- 75
lidification, and vitrification [3]. 76
toxicity of the contaminants. Third, the roots, the rate of growth, the plant biomass, the 93
adaptation to the climate, and the tolerance to salty soil [5]. Among all these aspects, the 94
main production of high biomass in parts of plants will affect more on the phytoremedia- 95
tion capacity [6]. 96
It is also crucial to understand that edible plants are not appropriate for phytoreme- 97
diation because they may affect the health of humans or animals once it is consumed[7]. 98
Therefore, fiber crops are said to be the best fit for phytoremediation. This is because fiber 99
plants involve a cycle of planting and harvesting, which will help to reduce the heavy 100
metal contamination in the soil over time, and the harvested fiber is used to manufacture 101
biomaterials such as paper and textiles. In this case, it does not enter the food chain and 102
affects the environment negatively, such as harming human or animal health. Apart from 103
that, different plants have different methods for the removal and accumulation of heavy 104
metals. For example, some plants can stabilize or decrease the mobility of the pollutants 105
in the soil through accumulation by roots through root hairs to stop contaminants' run- 106
off, bulk erosion, and air-borne transport [8]. Other plants may be involved in the process 107
of the plant uptake and release to the atmosphere through transpiration which is known 108
as phytovolatilization. Many phytoremediation processes are possible through better re- 109
lationships in between plants, microbes, soil, and contaminants. These different processes 110
of phytoremediation perform different management options for a better end product to 111
the environment [6]. 112
This paper discusses the potential of four commonly used bast fiber plants namely 113
Cannabis sativa (Hemp), Hibiscus cannabinus (Kenaf), Corchorus olitorius (Jute) and Linum 114
usitatissimum (Flax) for phytoremediation of selective heavy metals cadmium (Cd), lead 115
(Pb) and zinc (Zn) from contaminated soil. The main goal of this paper is to provide ref- 116
erences for suitable bast fiber plants for heavy metal treatment . In addition, this review 117
summarises these plants' ability to accumulate heavy metal elements and reveals their 118
potential for use as phyotoaccumulators or phytostabilizers via their uptake mechanisms.. 119
This emerging technology can be improved with natural and chemical amendments that 120
make heavy metals bioavailable and soluble. 121
122
123
Phytovolatilization – vo- 124
latilization of metals into 125
Phytoextraction – uptake of metals
the atmosphere 126
from plant root to the above part tis-
sues 127
128
129
130
131
132
133
134
Rhizodegradation – degradation of
organic pollutants in the root zone 135
Phytostabilization – reduction of bi-
oavailability of metals in root 136
137
138
139
140
141
Figure 1. Mechanisms involved in the phytoremediation process. 142
Toxics 2022, 10, x FOR PEER REVIEW 4 of 19
2.1 Morphology and Characteristics of Bast Fiber Plants (Hemp, Kenaf, Jute and Flax) 144
Bast fibre is a natural fibre derived from the bast environment of certain dicotyle- 145
donous angiosperm plant stems. It is made up of cellulose and hemicellulose combined 146
with a lignin or pectin mixture. In this paper, the potential of four different fiber plants 147
from various places in the uptake of heavy metals from contaminated soil was high- 148
lighted. The four fiber plants are Hemp (Cannabis sativa), Kenaf (Hibiscus cannabinus), Jute 149
(Corchorus olitorius), and Flax (Linum usitatissimum). 150
151
Hemp is a member of the Cannabaceae plant family, and the fibre derived from this 152
plant is one of the strongest forms of natural fibre [9]. It has the potential to be an envi- 153
ronmentally friendly and highly sustainable crop if it is well managed. On the other hand, 154
Kenaf and Jute comes from the same family of Malvacea. Kenaf is one of the non-wood 155
fibers that can be used for reinforcement and it is the world’s third traditional crop after 156
wood and bamboo which originated from Asia and Africa [10]. Jute fibers are totally bio- 157
degradable as it is partially wood [11]. Flax is a member of the Linaceae family of plants, 158
and due to its exceptional qualities, flax fibres are significant raw material for textiles [11]. 159
The Flax and Hemp do not have much difference because they are both cellulose fibers 160
except that Hemp has ten chromosomes (2n = 20) while Flax has 15 pairs of chromosomes 161
(2n = 30) [12] wheareas, Kenaf and Jute are woody-stemmed herbaceous dicotyledons 162
grown in the tropics and subtropics. 163
164
165
166
167
168
169
170
171
172
Table 2. Morphology and specifics characteristics of bast fiber plants (Hemp, Kenaf, Jute and Flax) 173
174
Fiber Plants Morphology 175
Roots Stems Leaves Flowers Seeds Reference 176
177
Hemp Root system is The stems are nor- The first true leaves are Male flowers and female Hemp seeds are [13] 178
(Cannabis sa- well developed mally hollow with di- single leaflets, later flowers available. Female achenes seeds. Seeds in 179
tiva) with depth of ameter ranging from leaves become palmately flowers are more com- ellipsoid shape, 2 to 7 180
about 1 to 1.5m 5 to 25 mm. The base compound. The second pact. mm long and 2 to 4 mm 181
and top stem has dif- leaf pair consist of three wide in diameter. Seeds 182
ferent diameters. Ma- leaflets per leaf, the third vary in colour from
183
ture plant reaches up leaf pair has five leaflets light brown to dark
184
to 5 m. per leaf, and so on, up to green
185
eleven leaflets per leaf.
186
187
188
189
Kenaf It has a prolific It mainly has un- Young leaves are simple It produces large showy, The seeds are normally [14] 190
(Hibiscus can- root system with a branched stems and and entire. Divided leaf light yellow, creamy col- brown with 6 mm long 191
nabinus) long taproot and grows up to 4.5 m tall can produce 3 to 10 entire oured flowers that are and 4 mm wide. The 192
extensive lateral young leaves prior to the bell-shaped and widely seeds of Kenaf are pro- 193
roots. first divided leaf open. The flowers duced by the fruits, 194
are solitary, short-stalked known as fruit capsules 195
and auxiliary which are 8 in 1.9 to 2.5 cm long and 196
to 13 cm in diameter with 1.3 and 1.9 cm in diame- 197
5 petals, 5 sepals, and ter with many seeded 198
numerous stamens. around 20 to 26 seeds.
199
200
201
202
203
204
Table 2. Specifics characteristics of bast fiber plants (Hemp, Kenaf, Jute and Flax) 205
206
Fiber Plants Morphology 207
Roots Stems Leaves Flowers Seeds Reference 208
[15] 209
Jute It has extensive The height range of The leaves are edible It consists of small pale- Seeds are greyish black, 210
(Corchorus lateral branching the jute plant is be- with a bitter taste. Leaves yellow flower, bracts angled. 211
olitorius) and deep tap root tween 2 and 4 m. The are usually 6-10 cm long lanceolate; 2 to 3 cm 212
system. stems are about 1 to 2 and 3.5-5 cm broad. wide, sepals 3 mm long, 213
cm in diameter with and petals are 5 mm 214
few branches. The long.
215
colour of the stem,
216
petiole and leaf var-
217
ies.
218
219
220
221
Flax It has short and It has one main stem, The leaves are normally The flowers parts are The seeds are flat, oval [16] 222
(Linum usitatis- branched tap root but two or more small and in lance normally in the units of and pointed at one end. 223
simum) which can extend branches (tillers) may shaped. five and they can range Normally the seeds are 224
to a depth, 1m develop from the base from a dark to a very covered mucilage giv- 225
with side when plant density is light blue, white or pale ing it a high shine 226
branches spread- low or high of soil ni- pink 227
ing to 30cm trogen levels. 228
229
230
231
232
233
2.2 Application of Bast Fiber Plants (Hemp, Kenaf, Jute and Flax) 234
Fiber plants are useful not only for phytoremediation but also in a variety of other 235
fields. Bast fibre of hemp plants is used in the automotive industry and textile industry 236
while the whole plant part is used for feedstock and biofuel. Hurds are used for paper 237
production and as a building material such as fiberglass. Hemp oil from the seeds is used 238
in shampoo, soaps, and bathing gels. Seeds are also applicable in the food industry as 239
hemp milk and are used as a salad dressing. Technical commercial products such as oil 240
paints, ink, and coatings are also produced by these plants [17]. However, the usage of the 241
plants is based on the quality of the hemp. On the other hand, jute is the second most 242
important fiber plant in the world, and it is also one of the cheapest-grown fiber plants in 243
the tropical region. It is traditionally used to manufacture packaging materials such as 244
sacking, ropes, twines, and carpet backing cloth. Moreover, diversified jute is also used in 245
the production of home textiles, composites, geotextiles, paper pulp, technical textiles, 246
chemical products, handicrafts, and fashion accessories. The woody central core is used 247
as a rural building material, for fences, fuel, and for charcoal-making. In the Philippines, 248
the leaves of jute are used to treat headaches [18]. 249
250
Kenaf also has its own uses and one of them is the paper production . Kenaf paper is 251
stronger and more resistant to yellowing compared the wood paper and it requires less 252
bleaching agent. Furthermore, Kenaf seeds produce edible oil, which is one of the best 253
cooking oils. Dried Kenaf leaves are consumed as a vegetable in some countries because 254
it contains 30% of crude protein. The fruit of Kenaf helps in lowering blood pressure and 255
the presence of vitamin C and antioxidants in Kenaf help in fighting some diseases. Kenaf 256
will be used in new applications such as medicines, textiles, natural fiber compounds and 257
environmental cleaning [19]. Flax is used for fruit, medications and textiles and has there- 258
fore been used for food processing. It has been of considerable significance for human 259
civilization and growth for more than 8000 years. At the time, flax was commonly used 260
for the manufacture of fabrics, and now, oil is the main production[20]. 261
262
263
Table 1. World countries ranking of producing fibre plants. 264
265
Types of fi- Hemp
ber plants Kenaf (Hibiscus Jute (Corchorus Flax (Linum
(Cannabis sa-
cannabinus) olitorius) usitatissimum)
Ranking tiva)
1 China India India Russia
2 Canada China Bangladesh Canada
United Syaye
3 Thailand China Kazakhstan
of America
4 France Brazil Uzbekistan China
5 Chile Vietnam Nepal United States
6 North Korea Cuba South Sudan India
7 Indonesia Zimbabwe
8 Pakistan Egypt
9 Pakistan Vietnam
10 Cambodia Bhutan
References [21] [22] [23] [24]
266
2.3 Case study on Phytoremediation of Heavy Metals Pb, Zn, and Ni by Bast Fiber Plants 267
In this study, Hemp (Cannabis sativa), Kenaf (Hibiscus cannabinus), Jute (Corchorus 268
olitorius), and Flax (Linum usitatissimum) were chosen to compare their potential for phy- 269
toremediation of Pb, Cd, and Zn in the soil. Hemp plants were harvested from agricultural 270
activities with acidic soil value. The concentrations of these metals were higher in the root 271
than in the leaves and shoots. Hemp can tolerate high concentrations of Zn and most of 272
the Zn absorbed is retained in the roots [25]. The uptakes of these heavy metals are signif- 273
icantly influenced by the pH of the soil. This statement is supported by the study caried 274
out by Gray et al., [26], where the results showed that increasing in pH will caused a sig- 275
nificant reduce in the concentration of cadmium in clover, lettuce, carrot and ryegrass. 276
277
Research conducted by Nizam et al., [27], highlighted that the concentration and up- 278
take of Pb by the shoot were significantly higher than the root in the Kenaf plant. Most of 279
the varieties grown in Pb contaminated soil accumulated more Pb in shoots than roots, 280
indicating that Pb was easily transported from root to shoot in Pb contaminated soil. This, 281
could be related to the Pb content and its relationship with other essential ions during 282
nutrient uptake. Other studied by Shehata et al.,[7], mentioned that Kenaf plants were ir- 283
rigated with wastewater and sulfur soil addiction with humic acid were used as foliar 284
spraying and it showed the significant highest accumulation of cadmium which 0.87 285
mg/kg in the roots and 0.36 mg/kg in the shoots. They noticed that humic acids are the 286
most active components in soil and compost as it improves the uptake and accumulation 287
of heavy metals in the tissues' plant [28]. Cecília et al., [29], studied the phytoremediation 288
of zinc and the results showed that Kenaf is able to take 233 mg/kg of zinc in the roots and 289
264 mg/kg in the shoots. 290
291
Furthermore, the studies about phytoremediation in untreated industrial 292
wastewater form textile factories by Ahmed & Slima [30], shows that there was very high 293
concentration of Cd in the roots with 261.83 mg/kg and 41.35 mg/kg in the shoots of the 294
Jute. In contrast the concentration of Pb in the roots was 367.83 mg/kg while in the shoots 295
was 370.43 mg/kg. This findings showed that the nutrients in the roots and shoots were 296
decreased significantly due to contamination stress. Lead (Pb) is a toxic heavy metal that 297
can inhibit plant growth, seedling development and root elongation [31]. They also state 298
that flax is a fibre plant that is suitable for growing in industrially polluted areas because 299
its root system removes significant amounts of heavy metals from the soil and can be used 300
as a potential crop for cleaning soil of heavy metals [32]. Hosman et al., [33], studied the 301
bioremediation potential of flax under different concentration of Pb, Cd and Zn. The aver- 302
age ability of the flax plant to remove heavy metals from soil was 49% for Cd, 68.6% for 303
Pb, and 71.76% for Zn. Following that, the highest accumulation of Cd was found in the 304
root, while the highest accumulation of Pb and Zn was found in the capsule. He also re- 305
ported that by increasing in metal concentration in the soil, there was a gradual increase 306
in metal uptake in flax plant. Several phytotoxicity effects was observed when these metals 307
are exceeded the endogenous level [34]. 308
309
310
311
312
313
314
315
316
317
318
319
Toxics 2022, 10, x FOR PEER REVIEW 3 of 19
Table 2. Heavy metal concentration in Bast Fiber Plants. Listed tissues represent those 320
with the highest concentration of metals in roots, leaves, and shoots. 321
322
protein with low molecular weight can facilitate the metal ion into the plant cells and 341
translocate them via the xylem. In phytoremediation technologies, the addition of nutri- 342
ents to plants may results in healthy plant growth with the development of flowers, 343
leaves, and branching of the root system thus can increase the level of uptake contaminant 344
in the study area. However, an excessive amount of nutrients given to the plants can result 345
in a significant reduction in plant growth. This symptom is known as nutrient toxicity. In 346
a nutrient-enriched environment, the bioavailable fraction of metals may be reduced be- 347
cause of the binding to the nutrient anions. The uptake of heavy metals in plants may also 348
be affected by competition since nutrient cations compete with the metal for uptake sites 349
[53]. Thus, the uptake of the metal under investigation decreases with increasing concen- 350
tration of nutrients. However, a generous availability of nutrients promotes plant growth, 351
which in turn creates an increasing number of uptake sites for metal in plants. This may 352
increase the uptake as well as the metal concentrations in plants. 353
354
Interactions between plants and microbes are crucial factors in determining the effi- 355
ciency of phytoremediation [54]. These interactions are implicated to play an essential role 356
in plant metal uptake. The beneficial microbes associated with plants directly improve the 357
efficiency of the phytoremediation process by altering metal accumulation in plant tissues 358
and indirectly by promoting shoot and root biomass production. Whiting et al., [55], re- 359
ported that the biomass and zinc concentration in shoots of Thlaspi caerulescens has been 360
increased with the presence of rhizospheric bacteria. These bacteria can promote plant 361
growth by inhabiting the plant roots [56] and are known as plant growth-promoting rhi- 362
zobacteria (PGPR)[57]. The generation of phytohormones, specialized enzymatic activity, 363
nitrogen fixation in the atmosphere, and pathogen-depressing chemicals such sidephores 364
and chelating compounds all contribute to the role of PGPR in promoting plant 365
growth[58]. Sidephores and chelating compounds have been shown to promote plant 366
growth even in the presence of heavy metals [59]. 1- aminocyclopropane- carboxylic acid 367
deaminase is another plant growth-promoting compound that has been studied in rela- 368
tion to heavy metals (ACC deaminase). ACC is an intermediate of ethylene produced by 369
stressed plants, and bacteria that produce ACC deaminase can reduce ethylene levels in 370
plants, promoting plant growth [60]. 371
372
In another study, Belimov et al., [61] discovered that bacteria containing ACC deam- 373
inase can improve plant growth in metals polluted conditions. Whereas, Braud et al., [62], 374
studied the phytoextraction of agricultural Cr and Pb with sidephore- producing bacteria, 375
and highlighted that the inoculated Maize plant with the bacteria enhanced the bioavail- 376
ability and uptake of Cr and Pb. Khan et al., [63], investigated the (Ni) accumulation of 377
mycorrhizal and non-mycorrhizal flax plants at various concentrations of Ni, i.e., 0, 250, 378
350, and 500 ppm. He reported that the accumulation of metals was higher in mycorrhizal 379
than the non-mycorrhizal plants. Additionally, mycorrhizal plants showed noticeably 380
greater growth and development than non-mycorrhizal plants. The production of 381
phytohormones by Arbuscular Mycorrhizal Fungi (AMF) can improve nutrient and water 382
uptake as well as improves metal bioavailability and aid in the phytoremediation process 383
[64]. Figure 3 showed the mechanism of plant-microbe association that support metal phy- 384
toremediation. 385
386
387
388
389
390
391
392
Toxics 2022, 10, x FOR PEER REVIEW 5 of 19
393
394
Improved plant 395
growth and yield
396
397
Production of glu-
398
tathione-phyto-
chelatin mediated 399
resistance
PGPR 400
Metal transformation
401
Phytoprotection into soluble forms
via bacteria metal 402
resistance Sidephores
403
production
404
Improved root Metals tolerance from EPS 405
length and root (extracellular polymeric sub-
area stances) formation 406
407
408
Figure 3: The mechanism of plant-microbe association that support metals phytoremedia- 409
tion[65]. 410
411
412
2.5 Molecular Mechanisms Involved in Microbial Resistance to Heavy Metals 413
Microorganisms have been involved in the mechanisms of adapting with heavy metals 414
either in water or soil [66]. Some metals, such as copper, nickel, and cobalt, are given to micro- 415
organisms as micronutrients for use in redox processes, to stabilise molecules through electro- 416
static interactions, to act as components of various enzymes, and to regulate osmatic pressure. 417
Otherwise, non-essential metals are recognized to have little nutritional value and may be toxic 418
to microorganisms. To overcome the toxicity value, there are six metal mechanism that exists 419
in the microorganism includes the exclusion of permeability barrier, intra, and extra-cellular 420
sequestration, active transport efflux pumps, enzymatic detoxification, and reduction in the 421
sensitivity of cellular targets to metal ions. 422
423
2.5.1 Metal Exclusion by Permeability Barrier 424
The metal exclusion by the permeability barrier involved the changes in the cell wall, 425
membrane, or envelope of microorganisms. This mechanism is an attempt by the organism to 426
protect metal-sensitive and essential cellular components. Previous research has shown that 427
bacteria form an extracellular polysaccharide coating that has the ability to bio-absorb heavy 428
metal ions and prevent them from interacting with vital cellular components [67]. These 429
bacteria's exopolysaccharide coating may provide sites for metal cation attachment [67]. For 430
example, there are several strains of bacteria that demonstrated the ability to bind metals 431
extracellularly such as Klebsiella aerogenes, Pseudomonas putida, and Arthrobacter viscosus. 432
According to Scott & Palmer, [67] a protective layer of exopolysaccharide improves the survival 433
of K. aerogenes strains in Cd (II) solutions. When compared to strains without their protective 434
layer, these strains show a 2-fold increase in Cd (II) accumulation. This protective layer appears 435
to help reduce toxicity by preventing metal ion uptake and keeping metal ions away from 436
sensitive cellular components. 437
438
2.5.2 Active Transport of the Metals Away from the Microorganisms 439
One of the largest categories of metal resistance systems is an active transport or efflux 440
system by microorganisms. These methods involved the cytoplasmic export of harmful metals. 441
Toxics 2022, 10, x FOR PEER REVIEW 6 of 19
3. It is suggested that Hemp, Kenaf, and Jute are suitable species for soil remediating of heavy 524
metals Pb and Zn. Therefore, these species can be successfully cultivated for phytoremedia- 525
tion purposes since their root system can remove significant amounts of heavy metals from 526
the soil. 527
528
529
Author Contributions: Conceptualization, N.Z.Z and P.M.; data curation, N.Z.Z and P.M.; 530
methodology, N.Z.Z, and F.N.C.; validation, S.A.R and N.Z.Z.; writing—original draft prepara- 531
tion, N.Z.Z and P.M.; writing—review and editing, N.Z.Z and A.N.M.Y.; supervision, N.Z.Z and 532
S.A.R; funding acquisition, N.Z.Z and F.N.C. All authors have read and agreed to the published 533
version of the manuscript. 534
535
Funding: This work was supported by the Project No. (SDN0063) the Centre for Research and 536
Innovation, Universiti Malaysia Sabah, Malaysia. 537
538
Institutional Review Board Statement: Not applicable. 539
540
Informed Consent Statement: Not applicable. 541
542
Data Availability Statement: Not applicable. 543
544
Acknowledgments: The authors express their sincere appreciation to the Researchers Support- 545
ing for this project. 546
547
Conflicts of Interest: The authors declare no conflicts of interest. 548
549
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