FEMS Microbiology Letters, 367, 2020, fnaa144

doi: 10.1093/femsle/fnaa144
Advance Access Publication Date: 24 August 2020
Research Letter

R E S E A R C H L E T T E R – Biotechnology & Synthetic Biology

A novel sophorolipid-producing Candida keroseneae

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GBME-IAUF-2 as a potential agent in microbial
enhanced oil recovery (MEOR)
Zahra Ganji1 , Keivan Beheshti-Maal1, *,† , Ahmadreza Massah2 and
Zarrindokht Emami-Karvani1
1
Department of Microbiology, Faculty of Biological Sciences, Falavarjan Branch, Islamic Azad University,
Falavarjan, Isfahan, Iran and 2 Department of Chemistry, Faculty of Science, Shahreza Branch, Islamic Azad
University, Shahreza, Isfahan, Iran
∗
Corresponding author: Department of Microbiology, Faculty of Biological Sciences, Falavarjan Branch, Islamic Azad University, Falavarjan 84515/155,
Isfahan, Iran. Tel/Fax: +98(31)3742 0136; E-mail: beheshtimaal@iaufala.ac.ir
One sentence summary: This is the first report of sophorolipid production by Candida keroseneae that could be highly recommended for applications in
microbial enhanced oil recovery and food industries according to its high thermal, pH and saline stability.
Editor: Paul Christakopoulos
†
Keivan Beheshti-Maal, http://orcid.org/0000-0003-3226-4783

ABSTRACT
The biosurfactants have extensive applications in food and petroleum microbiology. The aims of this research were
isolation and characterization of thermo-tolerant biosurfactants from highly producing yeast strains. The Bushnell Hass
medium was used for screening the biosurfactant-producing yeasts. Biosurfactant presence was evaluated using oil
displacement assay and surface tension test. The best biosurfactant-producing strain was named Candida keroseneae
GBME-IAUF-2 and its 5.8s-rDNA sequence was deposited in GenBank, NCBI, under the accession number MT012957.1. The
thin layer chromatography and Fourier-transform infrared spectroscopy analysis confirmed that the extracted biosurfactant
was sophorolipid with a significant surface activity. The purified sophorolipid decreased the surface tension of water from
72 to 29.1 mN/m. Its maximum emulsification index, E24% , was recorded as 60% and preserved 92.06–97.25% of its original
activity at 110–120◦ C. It also preserved 89.11% and 84.73% of its original activity in pH of 9.3 and 10.5, respectively. It
preserved 96.66–100% of its original activity in saline extreme conditions. This is the first report of sophorolipid production
by the yeast C. keroseneae. According to the high thermal, pH and saline stability, the sophorolipid produced by C. keroseneae
GBME-IAUF-2 could be highly recommended for applications in microbial enhanced oil recovery as well as food industries
as an excellent emulsifying agent.

Keywords: biosurfactant; Candida keroseneae; emulsification index; oil displacement assay; sophorolipid; surface tension

INTRODUCTION of precious crude oil, 60–70%, is still trapped in petroleum

reservoirs (Das 2018). A popular chemical procedure, enhanced
Recently, the global demand for oil and its derivatives has
oil recovery (EOR), has been applied for extracting the remaining
increasingly developed because of the growth of industrial-
oil from reservoirs using surfactants or surface active agents.
ization and human urbanization. After usual primary and
These amphiphilic compounds decline the crude oil viscosity
secondary oil recovery procedures, a considerable amount
and result in the oil mobilization and recovery facilitation.

Received: 24 April 2020; Accepted: 20 August 2020


C FEMS 2020. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com

1
 2 FEMS Microbiology Letters, 2020, Vol. 367, No. 17
Biosurfactants as biological alternative could be considered
as an asset in comparison with chemical surfactants because
they are non-toxic, highly active in extreme pH, temperatures
and salinity levels, biodegradable, ecologically friendly and
could be produced using renewable waste resources (Thavasi,
Jayalakshmi and Banat 2011; Geetha, Banat and Joshi 2018;
Akbari et al. 2020). Biosurfactants are classified into five groups
of glycolipids, lipopeptides, phospholipids, fatty acids and poly-
meric biosurfactants (Mukherjee, Das and Sen 2006; Geetha,
Banat and Joshi 2018). Glycolipid biosurfactants have several
applications in biomedicine (as antiviral, antifungal, antibac-
terial and antiadhesive agents), food and agricultural industry,
pulp and textile, cosmetic and pharmaceutical industries as
well as oilfield industries [as microbial enhanced oil recovery
(MEOR)]. MEOR is defined as the use of microorganisms or their
bioproducts, such as biosurfactants, for increasing the recovery
of remaining oil in reservoir (Geetha, Banat and Joshi 2018).
The biosurfactants could be produced by bacteria, filamentous
fungi and yeasts. The production of various biosurfactants from
bacterial sources and their effectiveness in MEOR have been
reported using Pseudoxanthomonas sp. (Astuti et al. 2019), Bacillus
amyloliquefaciens (Alvarez et al. 2015), Bacillus cereus and Bacillus
subtilis (Amani et al. 2010), Pseudomonas aeruginosa (He et al.
2017; Gutierrez-Gomez et al. 2018), Stenotrophomonas sp. (Sabati
and Motamedi 2018), Microbacterium maritypicum (Akbari et al.
2020) and Arthrobacter oxidans (Lima et al. 2011). The features
such as the ability to utilize diverse hydrocarbon sources, larger
cell size and the greater production of biosurfactants by yeast
strains, compared with bacteria, make them more useful as
biosurfactant-producing microorganisms (Camargo et al. 2016).
There are several reports of biosurfactant-producing yeasts and
their applications in food and agriculture industries as well
as for bioremediation purposes in environmental industries,
e.g. production of sophorolipid by Candida bombicola (Kurtzman
et al. 2010); glycolipids from Meyerozyma guilliermondii (Camargo
et al. 2018), Candida orthopsilosis, Cryptococcus luteolus, Rhodotorula
glutinis, Rhodotorula mucilaginosa, Hannaela sinensis, Dipodascus
australiensis and Metschnikowia koreensis (Camargo et al. 2016);
production of glycolipids using Pseudozyma antarctica, Pseu-
dozyma tsukubaensis, Pseudozyma graminicola (Konishi et al. 2014),
Candida sphaerica (Chaprao et al. 2015); glycolipid production
by Wickerhamiella domercqiae and Trichosporon asahii (Mnif and
Ghribi 2015); sophorolipid production by Lachancea thermotoler-
ans (Mousavi, Beheshti-Maal and Massah 2015); and lipopeptide
production by Candida tropicalis (Ashish and Debnath 2018).
The aims of this study were isolation and identification of
biosurfactant-producing yeast from oil-contaminated soil.
The chemical characterization and the resistance of purified
biosurfactant against extreme conditions also were discussed.
MATERIALS AND METHODS
Sampling, enrichment and primary isolation of
biosurfactant-producing yeast
Using a sterile glass container, a total of 500 g of oily soil was
collected from three positions inside and outside of a car repair
shop in Aligoodarz (Aligudarz) city, Lorestan province, Iran,
using a standard protocol that has been previously described
(Chaudhary, Khulan and Kim 2019). The samples were trans-
ferred to the laboratory and kept at 4◦ C for the next proce-
dure. For primary isolation of biosurfactant-producing yeasts,
the Bushnell Hass medium (BHM, Merck, Germany) [KH2 PO4, 1
g/l; K2 HPO4 , 0.2 g/l; MgSO4 7H2 O, 0.2 g/l; CaCl2, 0.02 g/l; NH4 NO3,
1 g/l; FeCl3, 0.002 g/l; NaCl, 2 g/l; crude oil, 1% (v/v); pH∼6 (HCl
1N)] was used. After autoclaving the medium at 121◦ C for 15
min, 100 mg/l of filter sterilized tetracycline was added to the
medium to inhibit bacterial growth. The collected soil samples
were mixed, crushed and worn in a sterile metal container and
then passed through a gridded net filter to be fined and homoge-
nized (Mohawesh et al. 2017). Then, 100 mg of homogenized oily
soil sample was aseptically added to the 500 ml BHM and incu-
bated at 28–30◦ C under shaking speed of 130 rpm for 1 week
(Akbari et al. 2020). The enrichment process was repeated as
a three-step subculture, and finally for purification purposes,
100 μl of soil sample-BHM was cultured on potato dextrose agar
(PDA, Merck, Germany) medium containing 100 mg/l of tetracy-
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cline using streak plate method and incubated at 28–30◦ C for
72 h. The isolated individual colonies on PDA were examined
microscopically and the budding was observed in yeast smear
after simple staining using methylene blue (Mousavi, Beheshti-
Maal and Massah 2014).
Production of biosurfactant using isolated yeast
The isolated purified yeast colony was inoculated to biosurfac-
tant induction medium (BIM) [KH2 PO4, 1 g/l; K2 HPO4 , 0.2 g/l;
MgSO4 7H2 O, 0.2 g/l; CaCl2, 0.02 g/l; NH4 NO3, 1 g/l; FeCl3, 0.002
g/l; NaCl, 2 g/l; olive oil, 1% (v/v); pH∼6 (HCl 1N)] and incubated at
28–30◦ C under shaking speed of 130 rpm for 120 h. Then, the pro-
duction of biosurfactant by isolated yeast was measured using
emulsion index test, E24% , every 24 h for clarifying the best incu-
bation time for achieving the maximum biosurfactant activity
(Akbari et al. 2020).
Screening of biosurfactant-producing yeast using oil
displacement assay
The yeast isolate with the highest production of biosurfactants
was selected based on oil displacement assay. A sterile 10 cm
Petri dish was filled with 20 ml of distilled sterile water. Then,
100 μl of crude oil was added to the top of water. One milliliter
of BIM was aseptically collected and centrifuged at 6000 × g for
10 min. Then, 10 μl of supernatant was gently dropped on the
top of crude oil at the center of Petri dish and the clear zone
diameter of oil displacement was measured (Astuti et al. 2019).
Measurement of biosurfactant activity using surface
tension test
Fifty milliliters of BIM was aseptically collected and centrifuged
at 6000 × g for 10 min. To determine the surface tension reduc-
tion of the culture medium, 20 ml of oil-free supernatant was
poured into a sample tensiometer and the platinum ring was
gently immersed in the liquid. The force required to separate
the ring from the liquid surface was recorded as mN/m and con-
sidered as surface tension. Distilled water was used as negative
control (Salehizadeh and Mohammadizad 2009).
Measurement of biosurfactant activity using emulsion
index (E24% ) test
Ten milliliters of BIM was aseptically collected and centrifuged
at 6000 × g for 10 min. Then, 2 ml of supernatant was added to
2 ml of kerosene in a test tube and vortexed for 2 min, and incu-
bated at 30◦ C for 24 h. The E24% index was calculated by divid-
ing the height of the emulsified layer by the total height of the

mixture using a millimeter ruler and the result was multiplied
by 100. The height of the emulsified layer to the total height of
the mixture shows the emulsion index, that is, the emulsifying
strength of biosurfactant (Sen et al. 2017).
Biosurfactant extraction and purification procedure
The BIM was inoculated by biosurfactant-producing yeast, and
then incubated at 30◦ C under shaking speed of 130 rpm for
72 h. To isolate the yeast biomass, the medium contents were
centrifuged at 4◦ C, 6000 × g for 10 min and supernatant was
collected. Using HCl (6 N), the pH of cell-free supernatant was
adjusted to 2, and incubated at 4◦ C for 24 h. The chloroform
and methanol with the ratio of 2:1 (v/v) were added to liquid.
The resulting suspension was centrifuged at 4◦ C, 10 000 × g for
20 min and the organic phase was collected and incubated at
60◦ C for 20 h. Finally, the brownish and oily liquid was used
for biosurfactant chemical characterizations (Salehizadeh and
Mohammadizad 2009).
Molecular identification of biosurfactant-producing
yeast using 5.8s-rDNA sequencing
The DNA extraction of the best biosurfactant-producing yeast
was done using a previously described method (Mousavi,
Beheshti-Maal and Massah 2015). For amplification of Inter-
nal Transcribed Space 1, and 5.8s-rDNA of isolated yeast,
the ITS1 as the forward primer with the sequence of 5’-
TCCTCCGCTTATTGATATGCGG-3’ and ITS4 as the reverse primer
with the sequence of 5’-TCCGTAGGTGAACCTGCGG-3’ were
used, respectively (Harju, Fedosyuk and Peterson 2004). The
total volume of Polymerase Chain Reaction (PCR) reaction was
25 μl consisting of DNA template, 1 μl; ITS-1 and ITS-4 primers,
2.5 μl each; PCR master mix buffer containing Taq polymerase
and MgCl2 , 14 μl; and distilled water, 5 μl. The PCR program
was followed as 95◦ C for 5 min as initial denaturation, 95◦ C
for 1 min as denaturation, 55◦ C for 1 min as annealing, 72◦ C
for 2 min as extension and 72◦ C for 5 min as final exten-
sion. The electrophoresis was operated at 120 V for 30 min
using 5 μl of PCR amplified product mixed with 2 μl of load-
ing dye in a 1.5% agarose gel containing 10 μl of SYBR green
as visualizer. Twenty microliters of PCR amplified product with
10 μl of each ITS-1 and ITS-4 primers were sent to Cinna-
clon Co., Tehran, Iran, for DNA sequencing. The results of ITS
and 5.8s-rDNA sequences were analyzed using Mega-X soft-
ware. The BLASTN software, http://www.blastn.ncbi.nlm.nih.go
v, was used for detecting the biosurfactant-producing yeast
DNA similarities to GenBank records. After bioinformatics anal-
ysis, the ITS and 5.8s-rDNA sequences of isolated biosurfactant-
producing yeast were deposited in GenBank, NCBI, http://ww
w.ncbi.nlm.nih.gov/genbank/ (Akbari, Beheshti-Maal and Nayeri
2018; Amiri Fahliyani, Beheshti-Maal and Ghandehari 2018).
Chemical characterization and structural analysis of
biosurfactant using TLC
The thin layer chromatography, TLC, was done using silica gel
(Sigma, Ronkonkoma, NY, USA) with dimensions of 4 × 10 cm.
One drop of oily liquid specimen from extraction procedure was
transferred to silica gel at 1 cm distance of paper edge using a
capillary tube. The solvent system used as the mobile phase con-
sisted of chloroform/methanol/water with the ratio of 65/15/2
(v/v). After the solvent reached the specified level, the silica gel
Ganji et al. 3
was sprayed with a mixture of sulfuric acid/ethanol, 5/95 (v/v)
ratio, at 140◦ C for 10 min. The appeared bands on silica gel paper
were examined and evaluated using an ultraviolet illumination
system (Mousavi, Beheshti-Maal and Massah 2015).
Chemical characterization and structural analysis of
biosurfactant using FTIR
For determining the structure of purified biosurfactant and
approving its chemical category, the Fourier-transform infrared
spectroscopy, FTIR, was fulfilled. Ten milligrams of the extracted
biosurfactant was mixed with 400 mg of pure and dry potas-
sium bromide and pressed with high pressure for 30 s so that a
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thin, semitransparent tablet was obtained. The tablet was then
inserted into the FTIR device (Mattson 1000 FT, England) and the
infrared spectra in the range of 450–4000 cm−1 wave length were
evaluated (Akbari et al. 2020).
The effects of different temperatures, pH and salinity
levels on biosurfactant stability
The yeast colony was inoculated to BIM and incubated at 28–
30◦ C under shaking speed of 130 rpm for 72 h. One hundred
milliliters of BIM was aseptically collected and centrifuged at
6000 × g for 10 min. To measure the thermal stability of the bio-
surfactant, 45 ml of the supernatant was divided into nine sterile
tubes, 5 ml each, and incubated at 40, 50, 60, 70, 80, 90, 100, 110
and 120◦ C for 30 min. Then, 2 ml from each tube was collected
and the emulsification index, E24% , was measured as previously
described. For measuring the pH effects on biosurfactant stabil-
ity, 40 ml of the supernatant was divided into eight sterile tubes,
5 ml each, and their pH were adjusted to 2, 4, 5.5, 7.2, 8.5, 9.3,
10.5 and 12 using HCl (1 N) and NaOH (1 N) and incubated at
room temperature for 30 min. Then, 2 ml of each tube was col-
lected and the E24% was measured. For measuring the salinity
effects on biosurfactant stability, 25 ml of the supernatant was
divided into five sterile tubes, 5 ml each, and their salinity con-
centrations were adjusted to 2, 4, 6, 8 and 10% (w/v) using NaCl
and incubated at room temperature for 30 min. Then, 2 ml of
each tube was collected and the E24% was measured (Astuti et al.
2019).
RESULTS
Primary isolation of biosurfactant-producing yeast
The BHM after inoculation with oily soil sample and incubation
at 28–30◦ C under shaking speed of 130 rpm for 1 week showed
probable presence of biosurfactant-producing yeasts. The PDA
containing 100 mg/l of tetracycline after incubation at 28–30◦ C
for 72 h showed purified large, ≤6 mm, colonies with white
creamy color macroscopically. The simple staining of individual
colonies with methylene blue confirmed the presence of bud-
ding yeasts on smear.
Screening of biosurfactant-producing yeast using oil
displacement assay
The three different yeast colonies were isolated from collected
soil samples among which the yeast isolate with the highest
production of biosurfactants was selected based on oil displace-
ment assay. The supernatant of BIM containing one of the iso-
lated yeasts showed positive result in oil displacement assay
 4 FEMS Microbiology Letters, 2020, Vol. 367, No. 17
as the first screening test for evaluation of biosurfactant pro-
duction. The clear zone diameter performed after dropping the
supernatant on the top of crude oil at the center of Petri dish
was 9 cm (Fig. 1). So, the purified isolated yeast from oily soil was
considered as a biosurfactant-producing yeast and selected for
next experiments. The two other yeast isolates showed negative
results in oil displacement assay and were failed to be passed for
further examinations.
Measurement of biosurfactant activity using surface
tension test
The biosurfactant containing supernatant of isolated yeast
showed a very significant decrease of 42.9 mN/m. The afore-
mentioned supernatant could decrease the surface tension from
72 to 29.1 mN/m. So, the surface tension test as the second
screening test for evaluation of biosurfactant production con-
firmed that the isolated yeast could be considered as an excel-
lent biosurfactant-producing yeast.
Production of biosurfactant using isolated yeast and
measurement of its activity using emulsion index
(E24% ) test
The E24% of biosurfactant after 24-, 48-, 72-, 96- and 120-h
incubation was 27, 43, 60, 59 and 58%, respectively. The results
showed that the maximum production and activity of biosurfac-
tant was achieved after 72-h incubation and was equal to 60%
(Fig. 2).
Molecular identification of biosurfactant-producing
yeast using 5.8s-rDNA sequencing
The expected size of product after PCR amplification was
∼680 bp as it has been shown in Fig. 3A. The analysis of
5.8s-rDNA sequence using BLASTN software showed 88% of
query coverage and 98.21% of similarity to the yeast Can-
dida keroseneae strain IMI 395606 (GenBank accession num-
ber: FJ235127.1). According to bioinformatics analysis, the
biosurfactant-producing yeast was named C. keroseneae strain
GBME-IAUF-2 and its ITS/5.8s-rDNA sequence was deposited in
GenBank, NCBI under the accession number of MT012957.1. The
phylogenetic tree of current isolated yeast has been indicated in
Fig. 3B.
Chemical analysis of C. keroseneae GBME-IAUF-2
biosurfactant using TLC
The TLC result showed a band with a Rf of 0.66 (Fig. 4). The mea-
sured Rf of 0.66 indicated that the biosurfactant produced by
C. keroseneae GBME-IAUF-2 was a lactonic form of sophorolipids
that are related to glycolipid biosurfactants (Mousavi, Beheshti-
Maal and Massah 2015).
Chemical analysis of C. keroseneae GBME-IAUF-2
biosurfactant using FTIR
The FTIR spectra of purified biosurfactant produced by C. kerose-
neae GBME-IAUF-2 were recorded in the range of 450–4000 cm−1
and shown in Fig. 5. The broad absorption band between 3000
and 3600 cm−1 represents OH stretching and confirms the pres-
ence of hydroxyl group in the sugar moiety of extracted bio-
surfactant. The peaks at 2926 and 2855 cm−1 correspond to the
stretching vibration of C–H band of aliphatic hydrocarbon part
of biosurfactant. Also, the C–H bending shows an absorption
peak at 1431 cm−1 . The C = O bands appear at 1744 and 1711
cm−1 . The carbonyl bands in addition to the strong peaks in
the range of 1000–1300 cm−1 due to the C–O stretching vibration
establish the contributions of lactones, esters or acids in purified
biosurfactant. Based on these results obtained from FTIR spec-
trum of C. keroseneae GBME-IAUF-2 biosurfactant and compari-
son with similar FTIR spectra obtained from previous researches
(Chen et al. 2006; Akbari et al. 2020), the glycolipid structure of
the extracted biosurfactant from C. keroseneae GBME-IAUF-2 was
identified as sophorolipid.
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The effects of different temperatures, pH and salinity
levels on C. keroseneae GBME-IAUF-2 sophorolipid
stability
The measured E24% of purified sophorolipid after 30-min incu-
bation at 40, 50, 60, 70, 80, 90, 100, 110 and 120◦ C was 32.41,
31.58, 34.73, 40.73, 55.46, 53.47, 52.86, 55.24 and 58.35%, respec-
tively (Fig. 6A). These results indicated that the most stabil-
ity of sophorolipid was recorded at 120◦ C as 58.35%. Also, the
extracted sophorolipid had considerable stability against high
temperatures of 80–120◦ C with E24% of 52.86–58.35%. The mea-
sured E24% of purified sophorolipid after 30-min incubation at
pH of 2, 4, 5.5, 7.2, 8.5, 9.3, 10.5 and 12 was 24.31, 15.85, 35.72,
40.6, 55.46, 53.47, 50.87 and 55.23%, respectively (Fig. 6B). These
results indicated that the most stability of sophorolipid was
recorded at pH of 8.5 as 55.46%. Also, the extracted sophorolipid
had considerable stability against alkaline pH of 8.5–12 with
E24% of 50.87–55.46%. The measured E24% of purified sophorolipid
after 30-min incubation at NaCl concentrations of 2, 4, 6, 8 and
10% was 59, 60, 60, 58 and 59%, respectively (Fig. 6C). These
results indicated that the most stability of sophorolipid was
recorded at 4 and 6% of salinity concentrations as 60%. Also the
extracted sophorolipid had considerable stability against a wide
range of 2–10% of NaCl concentrations with E24% of 58–60%.
DISCUSSION
The glycolipid biosurfactants, e.g. sophorolipids, are the most
popular and promising surface active agents because of their
multiple applications in various industries as well as their high
level of production by microorganisms (Konishi et al. 2014).
While there are several reports of sophorolipid production
by yeasts, to our best knowledge, this is the first worldwide
report of sophorolipid biosurfactant production by C. kerose-
neae isolated from oily soil in Aligoodarz, Lorestan province,
Iran. According to molecular and bioinformatics analysis, we
named this sophorolipid-producing yeast, C. keroseneae GBME-
IAUF-2, and its 5.8s-rDNA sequence was deposited in Gen-
Bank, NCBI, under the accession number MT012957.1. In sev-
eral reports, the YMG culture medium containing glucose, yeast
extract, peptone and malt extract has been used for screening
the sophorolipid-producing yeasts (Kurtzman et al. 2010; Kon-
ishi et al. 2014; Mousavi, Beheshti-Maal and Massah 2015). We
used BHM and then BIM for primary screening and production
of sophorolipid by C. keroseneae GBME-IAUF-2. For evaluating
the biosurfactant activity, reduction of surface tension is one
of the most useful indexes that have been applied in several
researches. Thaniyavarn et al. (2008) reported that sophorolipid
from Pichia anomala reduced surface tension from 70 to 28 mN/m.
Fontes et al. (2010) reported that the biosurfactant produced by
 Ganji et al. 5

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Figure 1. The oil displacement test of supernatant obtained from biosurfactant-producing yeast isolated from repair shop oily soil, Aligoodarz, Lorestan province, Iran,
after 72-h incubation at 28–30◦ C, 130 rpm using Bushnell Hass medium.

Figure 2. The E24% test of supernatant provided from biosurfactant-producing yeast isolated from repair shop oily soil, Aligoodarz, Lorestan province, Iran, after 120-h
incubation at 28–30◦ C, 130 rpm using Bushnell Hass medium.

Yarrowia lipolytica IMUFRJ50682 reduced the surface tension from
70 to 20.9 mN/m. In a research, the sophorolipid produced by
Rhodotorula babjevae YS3 decreased the surface tension from 70
to 32.6 mN/m (Sen et al. 2017). In another research, the biosurfac-
tant of C. sphaerica UCP0995 decreased the surface tension from
72 to 25.25 mN/m (Luna et al. 2011). Almeida et al. (2017) showed
that the biosurfactant of C. tropicalis UCP0996 decreased the sur-
face tension from 72 to 29.98 mN/m. In the present study, the
sophorolipid extracted from C. keroseneae GBME-IAUF-2 could
decrease the surface tension from 72 to 29.1 mN/m. The compar-
ison among various glycolipid-producing yeast spp. suggested
that C. keroseneae GBME-IAUF-2 isolated in this research could
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Figure 3. Molecular analysis of isolated biosurfactant-producing C. keroseneae GBME-IAUF-2. (A) Gel electrophoresis of 5.8s-rDNA PCR amplification using ITS-1 and
ITS-4 universal primers. 1: 681 bp band; N.g1: negative control. (B) Phylogenetic distance tree.

be considered as an excellent biosurfactant-producing yeast

strain. Camargo et al. (2018) showed that the biosurfactant E24%
of Rhodotorula glutinis and Meyerozyma guilliermondii was 52.5%
and 42.8%, respectively. The yeast spp. of Rhodotorula mucilagi-
nosa, Dipodascus australiensis and Metschnikowia koreensis showed
E24% of ≥35% and C. orthopsilosis, Cryptococcus luteolus and Han-
naela sinensis had no emulsification activity. Ashish and Deb-
nath (2018) have reported the biosurfactant extracted from C.
tropicalis MTCC230 had E24% index of 55%. In this research, the
maximum E24% of C. keroseneae GBME-IAUF-2 sophorolipid was
recorded as 60%, which was a very amenable amount of biosur-
factant activity compared with other reports. Usually, the crude
oil reservoirs have extreme thermal, pH and salinity conditions.
So, the resistance of biosurfactants against extreme conditions
is important once a specified biosurfactant was selected practi-
cally. It was shown that the biosurfactants of Rhodotorula gluti-
nis and Meyerozyma guilliermondii in pH of 2–4 have maintained
87.3% and 91.38% of their original activity, respectively (Camargo
et al. 2018). Ashish and Debnath (2018) indicated that the C. trop-
icalis MTCC230 biosurfactant in different pH of 2–12 has pre-
served 67.74–86.33% and at 30–90◦ C preserved 70–90% of its pri-
mary activity. In the present research, the extracted sophorolipid
at 120◦ C had E24% of 58.35% and regarding the maximum E24%
of 60%, it preserved 97.25% of its original activity. According to
its resistance against autoclave sterility condition, it could be
highly recommended for applications in MEOR as well as food
industries as an excellent emulsifier. The purified sophorolipid
of C. keroseneae GBME-IAUF-2 could preserve 92.43%, 89.11%,
84.73% and 92.05% of its original activity in pH of 8.5, 9.3, 10.5
and 12, respectively. While the crude oils have a wide range of
pH from acidic to alkaline according to their reservoir geochem-
ical basis, in the EOR process the sea water along with chelating
agents, e.g. EDTA, is used for increasing the crude oil recovery
that results in the pH increase of crude oil as well as oil field
to 9–11 (Mahmoud, Elkatatny and Abdelgawad 2017; Song et al.
2020). According to the considerable stability of 84.73–92.43%
against alkaline pH of 8.5–12, the sophorolipid examined in this
study was suggested as a potential agent in MEOR. The emul-
sification index of purified sophorolipid in salinity levels of 2–
10% was measured as 58–60%, i.e. it preserved 96.66–100% of its
Figure 4. The TLC of biosurfactant extracted from C. keroseneae GBME-IAUF-2 iso-
original activity in saline extreme conditions that would occur
lated from oily soil in Aligoodarz, Lorestan, Iran. The Rf value of 0.66 is indicative
of sophorolipid lactonic form.
in EOR and MEOR (Song et al. 2020), so the sophorolipid pro-
duced by C. keroseneae GBME-IAUF-2 because of great stability
 Ganji et al. 7

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Figure 5. The FTIR analysis of biosurfactant extracted from C. keroseneae GBME-IAUF-2 isolated from oily soil in Aligoodarz, Lorestan, Iran. The chemical structure is
indicative of sophorolipid biosurfactant.

Figure 6. The stability of C. keroseneae GBME-IAUF-2 sophorolipid against extreme conditions of (A) temperatures of 40–120◦ C, (B) pH of 2–12 and (C) salinity levels of
2–10%.

in high concentrations of NaCl was proposed as an excellent
active agent for crude oil MEOR as well as a suitable emulsify-
ing agent in food industries. In conclusion, this is the first report
of biosurfactant production by the yeast species of C. keroseneae
we currently isolated from a repair shop oily soil sample col-
lected from Aligoodarz, Lorestan, Iran. According to the high
thermal, pH and saline stability conditions, the sophorolipid
produced by C. keroseneae GBME-IAUF-2 could be highly recom-
mended for applications in MEOR as well as food industries as
an excellent emulsifying agent. While the biosurfactant produc-
tion by this strain was studied in laboratory flasks, the pilot plant
production and scale up procedures of sophorolipid produc-
tion in semi-industrial fermenter would support the use of new
biosurfactant-producing microorganisms in industrial microbi-
ology. Also, the investigation of sophorolipid effects in labora-
tory scale or pilot plant MEOR simulator would be the next sig-
nificant experiments that could introduce a new merit biotech-
nological product to petroleum industry practically.
 8 FEMS Microbiology Letters, 2020, Vol. 367, No. 17
ACKNOWLEDGEMENT
The authors thank to the Dean of Graduate Studies, Falavarjan
Branch, Islamic Azad University, Isfahan, Iran, for their techni-
cal support. The authors of this manuscript have no conflict of
interest to declare.
Conflicts of interest. None declared.
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