Archives of

1. Introduction
icrobial oils are called single-cell oils [1]
as oleaginous microorganisms produce
them. Owing to their ability and specific
characteristics, these microorganisms
have attracted many researchers in the
last few decades. Traditionally, microorganisms, including
bacteria, yeast, mold, and microalgae that are capable
of storing lipid greater than 20% of their dry weight, are
considered oleaginous microorganisms [2]. Microbial oil
production does not require land or other resources to produce
food and is not affected by weather or seasons [3].
Lipid storage occurs when microorganisms are grown in an
environment with excess carbon, which reduces the use of
other nutrients, especially nitrogen. Therefore, the ratio of
Carbon to Nitrogen (C/N) plays a vital role in stimulating
lipid storage [4, 5].
In recent years, attention has been focused on bacteria for
lipid production in biotechnology and industrial applications.
Bacterial lipids include Triacylglycerol (TAG-long
chain fatty acid) and Wax esters (WE-ester as long-chain
primary fatty acid and long-chain primary alcohol). They
are used to produce food additives, cosmetics, lubricants,
chemical oils, candles, and biofuels [6, 7].
Most bacterial species can synthesize Polyhydroxyalkanoates
(PHAs) as storage compounds [8, 9], while a
few genera of bacteria can store triacylglycerol and wax
ester [10]. The amount and structure of bacterial lipid compounds
depend on several factors, including the bacterium
itself, the carbon source structure, the time of cultivation,
and the amount of carbon and nitrogen present in the culture
medium [8, 11-13].
The triacylglycerol accumulation has been described by
Actinomycetes, including Mycobacterium, Streptomyces,
Acinetobacter, Nocardia, and Rhodococcus [10]. Among
the bacterial genera of triacylglycerol storage, Rhodococcus
is one of the most promising, as some species store
more than 20% of their biomass weight in triacylglycerol
form and are considered oleaginous bacteria. Members
of this genus are found in various natural environments,
from arid and tropical soils to cold ecosystems and marine
sediments [14-16]. Besides, Rhodococcus can produce and
store triacylglycerol in several types of substrates under
low nitrogen conditions. These conditions include specific
carbon sources such as sugar, organic acids, and hydrocarbons
[8, 17, 18], as well as complex carbon sources present
in the industrial wastes that show remarkable bacterial
versatility during substrate degradation [19, 20].
The genus Kocuria is a member of the Micrococcus
family of Actinobacteria. Members of the genus cells
are coccoid, Gram stain positive, non-encapsulated, immobilized,
chemoorganotrophic, highly aerobic, mesophilic,
and catalase-positive metabolisms. Polar lipids
included diphosphatidylglycerol, phosphatidylglycerol,
and phosphatidylinositol. They are present in one species.
The major fatty acids are C15:0 anteiso (above 50%),
C15 iso, and C16:1; each contains 10% fatty acid. The percentage
of Gas Chromatography (GC) is between 60%
and 75% of total DNA [21].
Numerous studies have focused on searching for and
finding inexpensive and alternative raw materials for
use as a substrate for microbial lipid production. These
materials can be found in agricultural, forestry, and food
waste [20, 22, 23]. Whey is a waste from the dairy industry,
which is produced in bulk worldwide (production
of 1 kg of cheese results in 9 kg of whey). The whey
consists mainly of lactose (5%-7%), with lower amounts
of glucose, cactus, and protein (0.8%-1.2%) and lipid
(3%-0.06%) [24]. Final disposal of whey is a significant
problem for the dairy industry because it produces a significant
amount of contamination in the environment,
and its cleaning is costly [25]. Bioconversion of whey
to valuable microbial oil is an attractive and effective
method that can significantly reduce the environmental
impacts created by the release of waste. It simultaneously
results in the low-cost production of high lipid used
biodiesel, biological lubricants, chemical oils, cosmetics,
and other quantities of biofuels [26-28].
The agricultural waste consists of cellulose, hemicellulose,
lignin, protein, and ash. Many agricultural wastes
are composed of lignocellulose, a complex carbohydrate
polymer of cellulose, hemicellulose, and lignin. The percentages
of cellulose, hemicellulose, lignin, and other
compounds in lignocellulose are in the ranges of 35%-
50%, 20%-35%, 15%-20%, and 15%-20%, respectively
[29]. One of the essential agricultural wastes is corn
stalks, which refer to the stems, leaves, and stalks that remain
on the field after harvest. Corn stalks are one of the
first biomass resources used to produce cellulose ethanol
in the United States. It has already been shown that corn
stalks containing lignin can be converted to lipids with
using Rhodococcus [30]. The lignocellulose biomass,
consisting of lignin, hemicellulose, and cellulose, is an
abundant, sustainable source for large-scale, low-cost
production [19, 31]. The bioconversion of lignocellulose
to bacterial lipid involves several steps: pretreatment
of lignocellulose biomass, hydrolysis of carbohydrate
structure to usable sugar, microbial lipid production,
product isolation, and purification [1, 32, 33].
M
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The primary purpose of this study is to evaluate the production
capacity of microbial oil from Kocuria Y205 isolate
to produce microbial lipid from cheap raw materials
available in industrial and agricultural compounds that
can be used in industrial applications for the first time.
2. Materials and Methods
Sample collection and culture medium
Soil samples were collected from agricultural fields
around the city of Qom (Iran) and stored at ambient temperature
until the experiment begins. Wheat stems were
prepared as lignocellulose compounds from agricultural
fields around Qom. Whey was produced as industrial
waste from the dairy industrial Qom.
To investigate lipid production in bacteria from MSM
synthetic medium containing glucose (40 g/L), we used
SO4 (NH4)2 (2 g/L), KH2PO4 (7 g/L), NaH2PO4 (2 g/L),
MgSO4.7H2O (1.5 g/L), and yeast extract (1 g/L) (MERCK-
Germany) [34, 35].
Isolation of bacteria
Nutrient Agar (NA) and Tryptic Soy Agar (TSA) medium
were used for bacterial isolation. Serial dilutions (10-
6 dilutions) of soil samples were performed aseptically.
Aliquots of 0.1 M suspension were made from pure 10-4,
10-5, and 10-6 dilutions, respectively. The cultures were
purified, and the media were incubated at 37°C. Purified
cultures were obtained from the isolated bacteria colonies,
and the isolates were obtained for subsequent steps [36].
Morphological and biochemical tests of isolates
Purified isolates were characterized by biochemical
analysis using catalase tests, oxidase tests, urea tests,
H2S, and anaerobic growth tests (according to Bergey’s
Manual of Systematic Bacteriology). Morphological
identification was performed with Gram staining, endospore
staining, and motion test [36].
Primary evaluation of lipid production
The lipid production of isolates was evaluated by Sudan
black staining. They were cultured in a TSA solid
medium with 3% w/w glycerol as an additional carbon
source; the plates were incubated for 7 days at 30˚C [37].
Preparation of seed cultures
After evaluation analysis by Sudan Black staining, the
selected isolated was cultured in TSA broth agar medium
for 24 hours in 250-mL flasks on a rotary shaker (150
rpm) at 30°C, and the growth was measured Optical
Density (OD) at 600 nm wavelength with a spectrophotometer
(CE-9500-England) [38].
Lipid accumulation experiments with the different
carbon source
For cultivating with whey, first liquid pass the Whatman
paper to remove suspended particles and then removed
the microorganisms using sodium gluconate pH
above 7.5, the solution was autoclaved, again it was
passed through Whatman paper and added 50 mL solution
to 100 mL flasks and then inoculate the prepared
bacterium and culture on a rotary shaker 150 rpm at
30°C for 24, 48, 72, and 96 hours [38].
For the preparation of cultivating with lignocellulose
compounds, the wheat stem crushed by 0.5 to 1 cm
and boiled in a volume of 200 g on 1000 ml flask for
20 minutes, then removed the suspended particles with
Whatman paper, the solution was autoclaved, Equal to
the base medium added 50 mL solution to 100 mL flasks
and then inoculate the prepared bacterium and culture on
a rotary shaker 150 rpm at 30°C for 24, 48, 72, and 96
hours [39].
To produce lipids with pure carbon sources, the isolates
were transferred to the MSM synthetic medium containing
nitrogen. About 50 mL of this medium was cultured
in 100-mL flasks on a rotary shaker at 150 rpm at 30°C
for 24, 48, 72, and 96 hours. Using the same medium,
glycerol with 3% (w/w) was used as a carbon source instead
of glucose [40].
Lipid extraction method
We used the standard method of Fluch et al. (1957) to
extract and calculate lipid weight [38]. To calculate the
dry weight, we first centrifuge 10 mL of broth medium
at 6000 rpm. Next, we extract the supernatant and then
wash the precipitate 3 times with distilled water and
dried at 80°C until constant weight (typically 24 h) [38].
Fourier Transform Infrared Spectroscopy (FTIR)
To confirm the presence of lipid in the samples, Fourier
Transform Infrared Spectroscopy (FTIR) device analysis
(Bruker-Tensor27-Germany) was performed according
to the standard method with range spectrum analysis of
the device from 400 cm-1 to 4000 cm-1 [37]. The experiment
was conducted for all carbon sources.
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Gas Chromatography (GC) analysis
Gas chromatography [38] device analysis (Agilent-
7890B-USA) was used to identify fatty acids. The
experiment was performed according to the standard
method and using an HP 5890 A GC equipped with an
INNOWAX capillary column (30 m, 0.53 mm, 1 mm)
and a flame ionization detector. The injection volume
was 0.5 mL, and hydrogen was used as carrier gas (13
mL/min). A temperature program was used to efficiently
separate the methyl esters (90°C for 5 min, temperature
increase of 6°C/min, 220°C for 10 min). For quantitative
analysis, tridecanoic acid was used as an internal standard
[41]. GC analysis was performed on lignocellulose
carbon source samples.
Transmission Electron Microscopy (TEM) analysis
Electron microscopy was used to observe the lipid
granules in the isolate. Cells were washed, suspended in
0.1 M potassium phosphate buffer (pH 7.5), and fixed
with glutaraldehyde for 24 hours. Then, the cells were
washed with a solution of sucrose 0.32 M in phosphate
buffer and embedded in low viscosity resin [42]. Electron
imaging (Zeiss-EM900- Germany) was performed
at Pasteur Institute of Iran.
Molecular identification based on 16S rRNA
Genomic DNA was extracted by boiling [43]. DNA
extracted by NanoDrop was checked for accuracy and
concentration [44]. Universal primers were used for the
amplification of the 16s rRNA gene fragment with a
length of 20 bp (total length 40 bp), 27F (Forward–5’-
AGAGTTTGATCCTGGCTCAG-3’), and 1492R
(Reverse-5’-GGGTTACCTTGTTACGACTT-3’). The
rRNA amplification reaction (30 μL) consists of master
mixes with DNA and primers. The PCR cycling conditions
were as follows: an initial denaturation at 94˚C for
5 min, followed by 30 cycles of denaturation for 1 min
at 94˚C, annealing for 1 min at 55˚C, extension for 2 min
at 72˚C, and a final extension at 72˚C for 5 min [45]. The
sequencing was performed at the Genetic Coding Institute
(Tehran, Iran). The phylogenetic tree drawing was
also plotted using the MAG7 software using the neighborhood
attachment model with the highest similarity
[46]. Sequence results were recorded on the NCBI site.
Statistical analysis
Significant differences between biological samples,
cultivated on different carbon sources and different accumulation
periods, were evaluated using a two-way
Analysis of Variances (ANOVA) in SPSS (version 25).
The significance level was set at less than 0.05.
3. Results
Isolation of bacteria
Seventeen bacteria were isolated from soil samples,
and biochemical and morphological tests were performed
on them. The results of these experiments can
be seen for the selected Kocuria Y205 isolate as follows,
Gram-Staining, Catalase, Urea, Oxidase are Positive,
and H2S, Anaerobic Growth, Endospore, Motion are
Negative Respectively.
The bacteria Cells are coccus-shaped, Colonies on
TSA agar are circular, smooth, and entire, orange in color.
The Substrate mycelium and aerial mycelia are not
observed, diffusible colored pigments are not produced
on any tested media.
Lipid accumulation
After bacterial culture on TSA medium and addition of
carbon source for 7 days, the isolates were tested using
Sudan black. Then, the isolate with the ability to store
lipid was selected to be evaluated. The microscopic examination
images can be seen in Figure 1.
Cultivation in different carbon sources
The performance of the Kocuria Y205 isolate at 4 different
carbon sources indicates higher lipid production
in the glycerol carbon source than glucose in pure carbon
sources and higher whey lipid production than lignocellulose
compounds in the waste carbon sources. The results
of which are visible in Table 1.
Comparing lipid production by an isolate from all
carbon source
Comparing lipid percentage in isolates indicates higher
production in whey than all carbon sources (Figure 2).
Quantitative analysis of lipid production by FTIR
spectrophotometry
FTIR analy sis was used in three different carbon
sources to prove lipid production, and all the analyzed
samples showed the presence of carbon and functional
groups (lipids) and confirmed the presence of aliphatic
carbon chains (lipids) in the samples. All carbon and hydrogen
bonding results were found to be the precursor of
the primary lipid structure and the methyl group, which
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is a hydro phobic functional group and was observed
from a methane molecule (CH4) by removal of hydrogen
and other lipid groups in the sample. These compounds
include carbonyl groups, alkenes, alkyls, and glycerol.
The above groups indicate unsaturated fatty acid in the
samples. This finding confirms the presence of triacylglycerol
in the composition obtained from lipolysis. The
interpreta tion is presented in Table 2 for each carbon
source, respectively, and microbial lipid graphs obtained
from FTIR analysis are shown in Figure 3.
GC analysis
The lipid samples obtained from isolate culture on lignocellulose
compounds for fatty acid identification were
tested with GC. The results of which based on the percentage
of carbon in the sample in Table 3 indicate the pres-
Figure 1. The results of the Kocuria Y205 ability to produce lipid, the white arrows in the image represent the lipid granules
stored in the isolate a) 7-day isolate culture and b) 7-day isolate culture with the addition of carbon source of glycerol
Table 1. Kocuria Y205 isolate performance in different carbon sources based on mg/mL
No.
Kocuria Y205
24 hours 48 hours 72 hours 96 hours
Carbon Source Performance in mg/mL
1 Whey
Dry weight 15.5 17.5 19.0 20.6
Amount of lipid 3.5 4.3 4.6 5.0
Lipid % 22.58 24.57 24.21 24.27
2 Lignocellulose compounds
Dry weight 12.0 17.0 24.0 30.0
Amount of lipid 1.0 2.6 3.6 3.9
Lipid % 8.3 15.29 15.0 13.0
3 Glucose
Dry weight 10.0 14.0 18.0 22.0
Amount of lipid 0.7 1.6 2.2 2.5
Lipid % 7.0 11.42 12.22 11.36
4 Glycerol
Dry weight 24.0 28.0 34.0 38.0
Amount of lipid 3.8 4.1 4.7 5.1
Lipid % 15.83 14.64 13.82 13.42
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ence of fatty acid in the lipid composition obtained from
isolate culture in lignocellulose compounds, which proves
the conversion of lignocellulose compounds into lipid
during the decrease of nitrogen by bacteria, the carbon
structure of the fatty acid produced and investigated, 14
and 15-hydroxy pentadecyl glycerol, the carbon structure
is 16 to 19-dihydroxyl glycerol, and 20 to 24 is orchols.
Transmission Electron Microscopy (TEM) analysis
Transmission Electron Microscopy (TEM) was used to
observe the lipid storage granules. The images are shown
in Figure 4. The storage granules of lipid produced in
these images are seen in bright color.
Molecular analysis
The 16s rRNA sequence results showed more than
96% similarity with Kocuria sp. strain JSM 1684076
(accession number: MG893104.1) is found in the Gene
Bank, demonstrating the close kinship of both strains.
The native Kocuria Y205 strain number MN818672.1 is
registered at NCBI. The result of drawing a phylogeny
tree is shown in Figure 5.
The evolutionary history is plotted using the neighborhood
attachment method with the most similarity
4. Discussion
This study aimed to investigate the potential of a native
and novel species of genus Kocuria isolate to produce
single-cell oil from low-carbon sources. This study
showed the ability of this strain to produce single-cell oil
from pure carbon sources of glucose and glycerol as well
as carbon sources of whey and lignocellulose compounds
under low nitrogen conditions. Also, the amount of lipid
produced in whey sources was higher than pure carbon
sources. FTIR analysis was used to quantify single-cell
oil production. The results of all samples showed the
presence of carbon and functional groups, which meant
the formation of lipids. In the sample, it was shown that
transmission electron imaging was also used to view the
storage granules, which is visible in the images of the
storage granules. Carbon sources are the essential factor
in determining the type of fatty acid produced in strains.
The difference in the produced fatty acid in the samples
is visible in the FTIR analysis of the samples of different
carbon sources. Bacterial compatibility with early life
environment and genetic variation and type of carbon affect
the diversity of fatty acids.
Genus Kocuria is part of the family of Actinomycetes,
and they are widely distributed in nature and can produce
many biologically active substances [47]. Li Tuo et
al. isolated Kocuria from the soil with the characteristics
of being aerobic, Gram-stain positive, coccus-shaped,
and approximately 1.1-1.8 μm in diameter, and pale yellow
in color. Substrate mycelia and aerial mycelia were
not observed; diffusible pigments were not produced on
any tested media. They grow well on ISP 2 agar, ISP 4
agar, TSA, NA, LB agar, MA, and R2A agar. Growth
occurs at 10˚C-37˚C (optimum 37˚C), pH 6.0-11.0 (pH
6.0-7.0) and with NaCl concentration of 0%-7% (w/w)
(0%). No growth at 45˚C and no growth at pH 5.0. Cells
reactions were positive for catalase, nitrate reduction and
hydrolysis of gelatin, H2S production, oxidase activity,
Rasouli A, et al. Single-cell Oil Production By Kocuria Y205. Arch Hyg Sci. 2021; 10(2):143-154.
Figure 2. Comparison chart of the lipid percentage of the isolate Kocuria Y205 in different carbon sources
24 48 72 96
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urease, and methyl red test [48]. Hansda et al. isolated
bacteria from dry tailing copper mines with the potential
of metal resistance. The isolated bacteria were cocci
shape, gram stain positive, pink and translucent in color,
catalase positive, and H2S production positive. Their
morphological, physiological, biochemical, and molecular
analysis characteristics showed that the isolated strain
belonged to genus Kocuria [49].
The ability of lipid production and accumulation by
Kocuria Y205 was compared with Rhodococcus, the
best known bacterial producer of single-cell oils [20].
To investigate microbial oil production, Ana Rito Castro
et al., two different strains of Rhodococcus opacus on
three carbon sources of glucose, acetate, and hexadegan
and yeast and peptone extracts as nitrogen limiters cultivated,
and both strains were able to store the highest
amount of lipid within 72 hours. Thin Layer Chromatography
(TLC) method was used to quantify the lipid production
in the samples, indicating the presence of oil in
the samples and qualitatively evaluating the oil produced
by GC analysis. It was used to show triacylglycerol production
in both strains that are closely related [50]. The
results of the Ana Rito study and the results of the present
study demonstrate the ability of the native Kocuria
Y205 isolate to store and produce triacylglycerol using
low-cost sources.
Marcia and Alvarez investigated the production of microbial
oil and biomass. Five strains of Rhodococcus
were cultured on whey, and the results showed that Rhodococcus
opacus produced more than 45% lipid and the
Table 2. Results of FTIR analysis with a different carbon source
No. Carbon Source Wave Number (cm-1) Functional Group
1
Whey
3383.5 = C – H stretch
2 2924.52 - 2853.17 -CH3 (Methyl groups)
3 1745.26 Carbonyl groups
4 1594.84 - 1421.28 - 1375.96 CH2 binding
5 1117.55 - 1073.19 - 1035.59 C – O – C stretching in esters
6
Lignocellulose compounds
3546. 45 - 3463.53 - 3415.31 = C – H stretch
7 2919.7 - 2848.35 -CH3 (Methyl groups)
8 1723.09 - 163.3 - 1614.13 Carbonyl groups
9 1447.31 - 1374. 03 CH2 binding
10 1057.76 C – O – C stretching in esters
11
Glucose
3450.23 - 3415.31 = C – H stretch
12 2934.78- 2839.78 -CH3 ( Methyl groups)
13 1723.09 - 1636.3- 1614.13 Carbonyl groups
14 1447.31 - 1374.03 CH2 binding
15 1667.76 C – O – C stretching in esters
16
Glycerol
3412.09 = C – H stretch
17 2950.13 – 2881.36 -CH3 (Methyl groups)
18 1717.3 , 1631.48 , 1631.48 Carbonyl groups
19 1406.82 CH2 binding
20 1226.5 , 1046.09 C – O – C stretching in esters
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remaining bacteria produced less than 5% lipid [41]. The
results of the Marcia and Alvarez study indicate that all
Rhodococcus bacteria are not capable of using whey for
lipid production, but in the present study, the native isolate
of Kocuria Y205 could produce the highest amount of
lipid production from the whey substrate, indicating bacterial
adaptation for lipid production from whey.
Herrero et al. used several Rhodococcus strains on
lignocellulose olive waste. They could produce lipid
with varying percentages, indicating the ability of these
bacteria to convert this type of carbon source into lipid.
However, examination of the produced lipid showed
the formation of triacylglycerol [51]. Due to the similar
structure of lignocellulose biomass, plant resources,
including cellulose, lignin, and hemicellulose, have different
percentages depending on the growing conditions
and type of plant. Comparison of the present study and
the study of olive plant waste showed the enzymatic
ability of newly isolate Kocuria Y205 to use lignocellulose
compounds for lipid production.
Rasouli et al. examined microbial oil production from
the genus Rhodococcus erythropolis, using MSM medium
and glycerol, glucose, wheat straw, and whey, for the
carbon sources, they could produce microbial oil under
the same conditions. The FTIR test proved the production
of oily groups and also used the GC test to examine
the structure of the fatty acid produced [52]. In many
studies, the genus Rhodococcus is a species suitable for
microbial oil production. In this study, we could produce
microbial oil for the first time with a new species using
the bacterial isolate of Kocuria Y205 with similar conditions
in the above research and prove the production of
carbon groups by FTIR test. Also, we tested the structure
of the fatty acid produced by the GC test.
Figure 3. FTIR analysis image for the isolates of Kocuria Y205 in different carbon sources
A: The whey source; B: Lignocellulose compounds; C: Glucose; and D: Glycerol.
Table 3. The fatty acid composition produced during culture on lignocellulose compounds
Bacterial
Isolate
The Total Amount
of Fatty Acids
The Relative Percentage of Fatty Acids (%, w/w)
C14 C15 C16 C17 C18 C19 C20 C21 C22 C23 C24
Kocuria Y
205 15.29% 2.30 1.87 0.63 4.27 2.66 3 3.84 7.05 7.86 0.18 21.45
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5. Conclusion
Microbial oil has many applications, including biodiesel
use as an environmentally friendly alternative
to organic hydrocarbon sources. Microbial oil also can
be used as dietary supplements due to the type of fatty
acid produced in it and used as chemical oils in the pharmaceutical
and cosmetic industries. The results of this
study, the first kind on the country, showed that the native
Iranian isolate Kocuria Y205 which belongs to the
family of Actinobacteria, Micrococcales, and Micrococcaceae,
can use pure carbon sources and bio-conversion
of industrial and agricultural waste as a cheap raw material
for lipid production and in particular, it is used as
a biodiesel. The results of this study will increase the
theoretical knowledge about this bacterium and the production
of microbial oil. The above bacterial isolate is
available in the Microorganisms Storage Complex of
Azad University of Qom with Kocuria Y205 (accession
number: MN818672.1) specifications.
Figure 5. The result of drawing a phylogenetic tree with Mega 7 softwar
Figure 4. Electron image of the Kocuria Y205 strain cultured on TSA medium with glycerol as an additional carbon source, a)
image with a magnification of 300 nm, and b) image with a magnification of 400 nm
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Ethical Considerations
Compliance with ethical guidelines
This article is a meta-analysis with no human or animal
sample.
Funding
This research did not receive any specific grant from
funding agencies in the public, commercial, or non-profit
sectors.
Authors' contributions
Conceptualization, methodology, investigation, resources,
project administration: Seyyed Soheil Aghaee
and Alireza Rasouli; Software, validation, formal analysis,
data curation, writing – original draft preparation,
writing, visualization, supervision, project administration,
and funding acquisition: Alireza Rasouli; Review
& editing: All authors.
Conflict of interest
The authors declared no conflict of interest regarding
the publication of the current article.
Acknowledgments
We would like to thank the staff of the Azad University
of Qom, and especially Dr. Ali Javadi, the Head of the
Microbiology Department, for providing the necessary
equipment to carry out the research. Mr. Zand Monfarad,
the Head of the Chemistry Laboratory at the Azad University,
and Ms. Mahyar Zeinivand, the PhD student of
Microbiology, for her contribution to the interpretation
of the GC and FTIR analysis.
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