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Biosurfactant production from newly isolated Rhodotorula sp.YBR and its

great potential in enhanced removal of hydrocarbons from contaminated soils

Article  in  World Journal of Microbiology and Biotechnology · January 2021

DOI: 10.1007/s11274-020-02983-3

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World Journal of Microbiology and Biotechnology (2021) 37:18
https://doi.org/10.1007/s11274-020-02983-3

ORIGINAL PAPER

Biosurfactant production from newly isolated Rhodotorula sp.YBR

and its great potential in enhanced removal of hydrocarbons
from contaminated soils
Louiza Derguine‑Mecheri1,2 · Salima Kebbouche‑Gana2 · Djamel Djenane1 

Received: 7 May 2020 / Accepted: 20 December 2020

© The Author(s), under exclusive licence to Springer Nature B.V. part of Springer Nature 2021

Abstract 
One of the very promising methods in the field of bioremediation of hydrocarbons is the application of biosurfactant- produc-
ing microorganisms based on the use of wastewater as renewable substrates of culture media, contributing to the reduction
of costs. With this aim, the production, characterization and properties of the yeast strain YBR producing a biosurfactant
newly isolated from an oilfield in Algeria, using wastewater from olive oil mills (OOMW) as a substrate for a low-cost and
effective production, have been investigated. Screening of biosurfactant production was carried out with different tests,
including emulsification index test (E24), drop collapse test, oil spreading technique and measurement of surface tension
(ST). The isolated yeast strain was found to be a potent biosurfactant producer with E24 = 69% and a significant reduction
in ST from 72 to 35 mN m ­ −1. The study of the cultural, biochemical, physiological and genetic characteristics of the isolate
allowed us to identify it as Rhodotorula sp. strain YBR. Fermentation was carried out in a 2.5 L Minifors Bioreactor using
crude OOMW as culture medium, the E ­ 24 value reached 90% and a reduction of 72 to 35 mN m ­ −1 in ST. A biosurfactant
−1
yield = 10.08 ± 0.38 g ­L was recorded. The characterization by semi-purification and thin layer chromatography (TLC)
of the crude extract of biosurfactant showed the presence of peptides, carbohydrates and lipids in its structure. The crude
biosurfactant exhibited interesting properties such as: low critical micellar concentration (CMC), significant reduction in
ST and strong emulsifying activity. In addition, it has shown stability over a wide range of pH (2–12), temperature (4–100
°C) and salinity (1–10%). More interestingly, the produced biosurfactant has proven to be of great potential application in
the remobilization of hydrocarbons from polluted soil with a removal rate of greater than 95%.

* Djamel Djenane
djenane6@yahoo.es
1
Laboratory of Food Quality and Food Safety. Mouloud,
MAMMERI University, P.O. Box 17, 15000 Tizi‑Ouzou,
Algeria
2
Laboratoire de Conservation et Valorisation des Ressources
Biologiques, Université M’Hamed Bougara de Boumerdès,
Boumerdès, Algeria

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 18   Page 2 of 18 World Journal of Microbiology and Biotechnology (2021) 37:18

Graphic Abstract

13
World Journal of Microbiology and Biotechnology (2021) 37:18 Page 3 of 18  18
Keywords Oilfield · Rhodotorula sp.YBR · Olive oil mill wastewater · Biosurfactant · Removal hydrocarbons · Polluted
soils
Introduction
Surfactants are chemical compounds, which contain both
hydrophobic and hydrophilic moieties (amphiphilic nature)
derived from the petroleum industry. These compounds act
between fluids of different polarities, allowing access to
hydrophobic substrates and causing changes such as surface
tension (ST) reduction, and raising the area of contact of
insoluble compounds (such as hydrocarbons), their mobility,
bioavailability, and therefore leading to their biodegradation
(Xue et al. 2019; Gupta et al. 2020). However, the residual
synthetic surfactants in soils and groundwater have a poten-
tial toxicity risk to human health and environment due to
their toxicity and non-biodegradability. Unlike chemical sur-
factants, biosurfactants are biological surfactants, are sec-
ondary metabolite compounds produced by microorganisms
including yeasts, moulds, and bacteria. These molecules are
implicated in cell survival under unfavorable circumstances
(Sambanthamoorthy et al. 2014; Santos et al. 2016). They
are either extracellular or bound to parts of the cell (intracel-
lular). The different classes of biosurfactants are glycolipids,
phospholipids, lipopeptides, neutral lipids and fatty acids
or lipopolysaccharides (Santos et al. 2016; Perfumo et al.
2018a). The production of biosurfactants was first confirmed
in 1941 by Bushnell and Haas (1941). They have recently
been named as sustainable and multifunctional biomolecules
of the 21st Century (Santos et al. 2016).
The great advantages of versatile biosurfactants over syn-
thetic products have drawn the attention of researchers all
over the world. This is mainly due to the many advantages
that they present such as: structural diversity, better environ-
mental compatibility, biodegradability, low toxicity, resist-
ance to extreme conditions of temperature, pH and salinity,
and their ability to be produced by inexpensive and renew-
able substrates compared to their synthetic counterparts
derived from the petroleum industry (Banat et al. 2014; San-
tos et al. 2016; Nitschke and Silva 2018; Gaur et al. 2019). In
recent decades, consumers have become more aware of the
environmental problem, combined with new environmental
laws, which have given new impetus to serious consideration
of biosurfactants as possible alternatives to existing syn-
thetic surfactants. Biosurfactant molecules are classically
recognized as emulsifier compounds and are extensively
applied in several fields including food processing, cosmet-
ics, pharmaceuticals, pest control management, medicine,
nanotechnology, commercial laundry detergents, textiles,
and in enhanced microbial oil recovery (EMOR) (Pacwa-
Pocociniczak et al. 2011; Fracchia et al. 2015; Alizadeh-Sani
et al. 2018; Perfumo et al. 2018b; Jimoh and Lin 2019; de
Andrade Teixeira Fernandesa et al. 2020; Sajid et al. 2020).
One of several promising application areas of biosurfactants
is the enhancement of hydrocarbon degradation in contam-
inated soil or water. The advantage of producing biosur-
factants from yeasts is that most of them are generally rec-
ognized as safe (GRAS), which facilitates the applicability
of their products in different industries (Fontes et al. 2008;
Alizadeh-Sani et al. 2018). In addition, biosurfactants are
an emerging technology for the remediation of heavy met-
als from contaminated soils and water (Araújo et al. 2019;
Farias et al. 2019; Jimoh and Lin 2019; Posada-Baquero
et al. 2019; Sun et al. 2019). This broad spectrum of applica-
tions translates into a growing global market estimated at $
43.6.6 × 1­ 09 in 2017; and is expected to reach more than $
66.4 × ­109 by 2025, recording a compound annual growth
rate of 5.4% from 2018 to 2025 (Shasttri et al. 2018).
The biosurfactants secreted by the microorganisms and
released into the hydrophobic medium, increase the bioavail-
ability of the hydrocarbons for the same microorganisms. In
other words, the degradation of hydrocarbons in the presence
of microorganisms is enhanced by the production of biosur-
factants to modify the physicochemical properties and aid in
the recovery of the contaminant (Francis and Nancharaiah
2015; Xue et al. 2019; Gupta et al. 2020; He et al. 2020).
Nevertheless, despite their remarkable properties, the use
and commercial availability of biosurfactants remain lim-
ited and they are not able to compete economically with
their synthetic counterparts due to their low yields and high
production costs, which hinder their commercial and eco-
nomic success (Santos et al. 2016; Nitschke and Silva 2018).
Therefore, a growing number of studies have successfully
investigated the production of biosurfactants using certain
raw materials and wastes from agricultural and industrial
processes as an alternative to low-cost production and are
expected to have a significant impact on the sustainability
of their production processes (Llori et al. 2008; Banat et al.
2014; Rivera et al. 2019).
According to the International Olive Oil Council (IOOC),
up to 95% of the olive trees cultivated in the world are
located in the Mediterranean region (Algeria, Greece, Italy,
Palestine, Spain, Tunisia, and Turkey) whose climate is the
most favorable for the cultivation of the olive tree (IOOC
2017). Olive oil-producing countries still have the prob-
lem of disposing of wastewater from the oil mills where
the olives are processed and the oil is extracted. When dis-
charged into the environment, OOMW creates serious envi-
ronmental problems such as pollution of surface and ground
water, coloring of natural waters, alterations in soil quality,
phytotoxicity, odor nuisance and a serious threat to aquatic
13
 18   Page 4 of 18
life, microorganisms, invertebrates and vertebrates due to
their acidity and their richness in organic matter, in par-
ticular polyphenols which exert a very high polluting activ-
ity (Jaouani et al. 2003, 2005; Dermeche et al. 2013; Babić
et al. 2019). The inorganic content of OOMW was mainly
composed of metals (Chatzistathis and Koutsos 2017; El-
Abbassi et al. 2017; Babić et al. 2019). The composition of
OOMW varies and depends on the climate, soil conditions,
type of olive, degree of ripeness of the fruit, region of origin,
cultivation system and on the milling method applied for the
olive oil production (traditional and modern processes) (Jus-
tino et al. 2012; Pardo et al. 2017). Moreover, Cossu et al.
(1993) reported that OOMW has been estimated to be 200
to 400 times more toxic than municipal wastewater and to
be readily fermentable. Thus, treatment and/or valorization
of OOMW are a major challenge for Mediterranean coun-
tries. Furthermore, if the residues used are environmentally
hazardous and their disposal is problematic, their valoriza-
tion as substrates for biosurfactants production represents
an additional advantage. Until now, the majority of the solu-
tions proposed for OOMW treatment have been based on
chemical or biological approaches. However, there is a need
to develop cost effective and practical solutions for the man-
agement of OOMW, especially for small producers. Various
attempts have been made to develop more efficient treat-
ment methods (chemical, physical or biological approaches).
None of these individual methods have been shown to be
fully effective for OOMW, especially in terms of reducing
toxicity (Oz and Uzun 2015; Selçuk Kuşçu and Eke 2020).
In addition to biological treatment, several chemical or
physical treatments have been tested for OOMW (Pulgarin
et al. 1999; Fiorentino et al. 2004; Marco et al. 1997; Justino
et al. 2009; Chouchene et al. 2010; Kavvadias et al. al. 2010;
Domingues et al. 2020).
The search for new surfactant molecules has driven
the study of microorganisms unexplored for this purpose,
mainly from oil reservoirs environments. In this perspective,
the OOMW agro-industrial by-products were evaluated to
develop a sustainable culture medium for the production of
biosurfactant by a new strain of yeast YBR isolated from
crude oil in the Sahara from Algeria. The kinetics of biosur-
factant production, its characterization and its properties (ST
reduction, E24, CMC and stability studies) were evaluated.
In addition, the study provides assays in the applications of
the produced biosurfactant as hydrocarbon-remobilization
in contaminated soils.
13
World Journal of Microbiology and Biotechnology (2021) 37:18
Materials and methods
All chemicals and solvents were of analytical grade purity
or greater purchased from Sigma-Aldrich and Merck (Ger-
many), and Oxoid, Basingstoke (U.K) represented by Alge-
rian Chemical Society (Algeria).
Sampling and isolation of yeast
Microorganisms living in extreme environments have gained
much attention for the last few decades, as they possess dif-
ferent properties by producing certain useful compounds
(Perfumo et al. 2018b). Yeast strain coded YBR was iso-
lated from an oilfield located in Haoud Berkaoui (Hassi
Massaoud Provence; Algeria). This region represents one
of the main hydrocarbon-producing zones of the Algerian
Sahara. It is located about 800 km southeast of the capi-
tal Algiers. The geographic coordinates of collection sites
were Latitude 31°40′49″ N, Longitude 6°04′22″ E, elevation
above sea level 152 m, and 800 km from the Mediterranean
Sea. Samples contaminated with hydrocarbons including
sand samples were collected. The collected samples were
transported to the laboratory aseptically in sterile polythene
bags/bottles for screening and isolation. So, the yeast isola-
tion was enriched by inoculation of 10 g samples in 90 mL
of yeast extract-glucose-chloramphenicol (YGC) (0.1 g ­L−1)
medium (YGC Oxoid, Basingstoke, U.K.), pH 4.5, incubated
at 30 °C for 24 h with constant shaking of 120 rpm. For
isolation, we used the diluting and plating methods. From
the enriched samples, 0.1 mL of each dilution was spread
onto sterile Sabouraud chloramphenicol agar (SCA; Oxoid,
Basingstoke, U.K.) and YGC agar plates. Then, the plates
were incubated at 30 °C for 48-72 h. After that, morphologi-
cally distinct colonies were isolated by transfer and streaking
on fresh oxytetracycline glucose agar (OGA; Oxoid, Bas-
ingstoke, U.K.) plates until obtaining pure cultures. A pure
culture of yeast strain YBR was maintained at 4 °C in YGC
agar containing (w/v) yeast extract (0.5%), D-glucose (2.0%)
and agar (1.5%). Transfers were made to fresh agar plates
each month to maintain viability.
Screening for biosurfactant production
The pure isolate was grown on YPG supplemented by 2%
of crude oil (2.0%, w/v) as carbon source for 72 h at 30 °C
with constant shaking of 200 rpm. The broth was centri-
fuged at 5000 rpm for 20 min. The resulting supernatant
was subjected to the following determinations: E ­ 24 test, drop
collapse test, oil displacement test and ST measurement
using a digital tensiometer (Gibertini, Italy) as described by
(Bodour and Miller-Maier 1998; Youssef et al. 2004; Pallas
and Pethica 1983), respectively.
World Journal of Microbiology and Biotechnology (2021) 37:18 Page 5 of 18  18
Molecular and phylogenetic analyses of the yeast
strain YBR
The genomic DNA of the selected isolate yeast strain YBR
was extracted following the method described by Harju
et  al. (2004). The universal primers used for amplifica-
tion of 5.8S-rDNA of the isolated yeast were as follows:
the forward primer was ITS1 (5′-TCC​TCC​GCT​TAT​TGA​
TAT​GC-3′) and the reverse primer was ITS4 (5′-TCC​GTA​
GGT​GAA​CCT​GCG​G-3′). The purified PCR products were
sequenced using the BigDye Terminator Cycle Sequencing
Kit (Applied Biosystems) with an ABI PRISM 310 Genetic
Analyzer (Applied Biosystems). The obtained sequence
of the gene was analyzed using BLASTN software (http://
www.blast​n.ncbi.nlm.nih.gov). Related sequences were
aligned and the phylogenetic tree was visualized using Mega
version 6.06 software. After bioinformatic analysis, the iso-
lated yeast was identified and its annotated sequence was
deposited in the EMBL database (http://www.ebi.ac.uk/).
Biosurfactant production from OOMW
OOMW was supplemented from an olive oil mill located
in the north of Algeria (Bouira), which uses a continuous
three-phase centrifugation process for olive oil extraction.
It was stored at 4 ºC until use. The concentration of total
carbohydrates in crude OOMW was determined using the
phenol–sulfuric method and appropriate dilutions were made
in order to adjust carbohydrates initial concentrations to 2%
(v/v). The medium based on crude OOMW was filtered,
diluted to 2% (v/v) of carbohydrates, and then was sterilized
at 120 ºC for 20 min.
Inoculum preparation
A pure culture of yeast strain YBR was maintained at 4 °C
in YGC agar. Transfers were made to fresh agar plates each
month to maintain viability. One loop of culture from the
yeast culture were added to 100 mL YGC broth media in
250 mL Erlenmeyer flasks and incubated at 30 °C in a rotary
shaker at 120 rpm overnight. For seed culture preparation,
5% inoculum was transferred to a flask contains 100 mL of
OOMW based medium, and then incubated under the same
growth conditions.
Cultivation in minifors bioreactor
Biosurfactant production by yeast strain YBR isolate was
conducted in a 2.5 L Minifors Bioreactor (Infors HT), con-
taining 1000 mL of the OOMW based medium. In this
sense, the use of agro-industrial wastes as substrates for the
production of different biomolecules has increased in the
last years. Agro-industrial wastes are an excellent source
of nutrients for microorganisms, even for the production of
biosurfactants. The Minifors bioreactor was inoculated from
the preculture to a resulting ­OD600 of 1 to 2. Temperature,
agitation and pH were set at 30 ºC, 200 rpm and 5.5. Dur-
ing fermentation process, aliquots were collected in regular
time for the determination of ­OD600 nm, ­E24 (%), ST (mN
­m−1) and yield (g ­L−1). All the experiments were performed
in triplicate.
Recovery of crude biosurfactant
The culture broth was centrifuged in a refrigerated cen-
trifuge at 10,000 rpm for 20 min at 4 °C. The supernatant
obtained was filtered through 0.2 µm filter (pH of the filtered
supernatant was adjusted to 2 using 1M HCl), then subjected
to acid precipitation at 4 °C overnight and followed by liq-
uid–liquid extraction method. The supernatant obtained was
extracted thrice with an equal volume of ethyl acetate and
methanol (2/1, v/v), shaking vigorously and allowing the
two layers to separate. The biosurfactant was concentrated
using a rotary evaporator at 40 °C to remove the solvent.
Biosurfactant yield and biomass were expressed in g ­L−1
(Derguine-Mecheri et al. 2018).
Purification of biosurfactant produced by yeast
strain YBR
Produced biosurfactant was semi-purified by preparative
chromatography on a thick layer of silica gel. The plates
used are 60A silica gel type (Merck). They were activated at
120 °C before being used. About 40 to 50 μL of concentrated
extract were deposited and then developed in the solvent sys-
tem (chloroform/methanol/distilled water, 80/18/2, v/v/v).
The silica gel portions corresponding to the active bands
visualized under UV light (280 nm), are scraped and put in
20 mL of methanol in order to desorb the biosurfactant. The
mixture was agitated continuously for 30 minutes, at room
temperature, and the silica removed by filtration. Then, the
fractions obtained were tested for surface activities using
the qualitative drop collapse test, the active fractions were
then pooled and vacuum dried at 40 °C. The purified biosur-
factant was dissolved in methanol.
Biosurfactant characterization
Thin layer chromatography characterization
Biosurfactant was separated by Thin Layer Chromatography
(TLC) using different solvent systems; acetone/acetic acid/
water (70/20/10) for amino acids; chloroform/methanol/
water (60/30/10) for sugars and chloroform/methanol/water
(65/25/10) for lipids. Once dry, the plate was developed
13
 18   Page 6 of 18
in a chamber saturated with iodine fumes for detection of
lipids and sprayed with ninhydrin reagent (0.5 g ninhydrin
in 100 mL anhydrous acetone) to detect peptide content, and
Molisch reagent (1 g in 5 mL sulfuric acid mixed with 95 mL
ethanol) for detection of sugar compounds. Isolated fractions
were eluted in methanol and subjected to macromolecule
quantification. Carbohydrate estimation was done by phenol-
sulfuric acid method using glucose as standard (Dubois et al.
1956). Protein estimation was done by the Bradford method
(Bradford 1976) using bovine serum albumin as standard.
Lipid content was estimated using the method described by
Sadasivam and Manickam (2004) with olive oil as standard.
Fourier transform infrared spectroscopy
Fourier transform infrared spectroscopy (FT-IR) was
employed to explore the functional groups and the chemical
bonds present in a crude extracts. Samples were prepared
by dispersing the solid uniformly in a matrix of potassium
bromide (KBr; FT-IR grade ≥ 99%; Sigma-Aldrich). IR
absorption spectra were obtained using a built-in plotter.
Infrared absorption spectra of the sample were measured
over the wave number range of 400–4000 cm−1 with a reso-
lution of 4 cm−1.
Stability studies
Stability studies in terms of ST and emulsification activity
were performed using cell-free broth from crude OOMW
medium. Effect of pH was studied over a range of pH (2 to
12). Effect of temperature was studied by heating the cell-
free broth at high temperatures from (60 to 100 ºC) for 1 h
and by incubating the cell-free broth at low temperature (4
ºC). The effect of salinity concentrations (2–10% NaCl) on
the surface and emulsifying activities of the crude biosur-
factant was also investigated.
Crude biosurfactant in enhanced motor oil recovery
from contaminated sand
Potential of the crude biosurfactant in enhanced motor oil
recovery was evaluated as described by Derguine-Mecheri
et al. (2018). 20 g of sand impregnated with 2 g of motor
oil were transferred to 250 mL Erlenmeyer flasks, followed
by the addition of 40 mL of the cell-free broth or 40 mL
of distilled water, 40 mL of Sodium Dodecyl Sulfate (SDS
≥ 95%) and 40 mL of Tween 80 as negative and positive
controls. The samples were incubated for 24 h at 27 °C with
constant shaking of 150 rpm, and then centrifuged at 10000
g for 15 min to eliminate sand. The amount of oil in the
sand after contact with the biosurfactant was gravimetrically
measured after extraction by hexane and solvent evaporation.
13
World Journal of Microbiology and Biotechnology (2021) 37:18
The experiences were conducted in triplicate and the results
were given as average values.
Statistical analysis
All experiments were repeated three times and the results
were analyzed by one-way ANOVA test (p < 0.05) using
STATISTICA version 6. The standard errors for mean
(SEM) values, at 95% confidence level, were calculated and
represented as error bars in all the graphical representations.
Results
Screening for biosurfactants producing yeast strain
YBR
Figure 1a shows that the YBR yeast strain isolate was found
to be a potent biosurfactant producer. In general, changes
in surface and interfacial tensions of hydrocarbon mixtures
are considered to be significant markers of biosurfactant
activity for potential application in bioremediation. Another
approach used for screening the potential of biosurfactant-
producing microorganisms is the estimation of ­E24. Thus, the
efficiency of the yeast strain YBR for the production of bio-
surfactants was confirmed by a significant reduction in ST
(from 72 to 35 mN ­m−1) and a higher ­E24 (69.00 ± 0.70%), a
flattened drop (positive drop collapse test: +++) and spread
oil positive with a displacement zone diameter = 2.50 cm.
Yeast strain identification
After 2 days at 25 ºC on YPG (yeast extract 1%, Glucose 2%,
Peptone 2%, Agar 1.5%) , the cells were ovoid, globular to
spherical, they can appear singly, in pairs, in short chains or
small clusters and reproduction was achieved by multilateral
budding (Fig. 2Bc). After one month there was a light pink-
to strawberry pink-colored ring with moderate sediment. At
the same time of growth on 5% malt extract agar at 25 ° C,
the color of the colony varied from salmon pink to deep
coral. The surface varied from smooth to rough and dull to
shiny. The margin was full. After one month at 19 °C on
potato dextrose agar (PDA), neither pseudomycelium nor
true mycelia were present. Besides the morphological char-
acterization, biochemical and physiological properties were
also studied; the fermentation, the assimilation of carbon (C)
and nitrogen (­ N2) compounds by yeast strain YBR isolate are
indicated in Table 1. Growth in vitamin-free medium was
negative. Growth in the presence of 20% NaCl was posi-
tive; and there was no growth in the presence of 25% NaCl.
Hydrolysis of urea was positive and starch formation was
negative. The partial 5.8s-rDNA sequence (677 pb) of the
isolated yeast strain YBR was submitted to GenBank under
World Journal of Microbiology and Biotechnology (2021) 37:18 Page 7 of 18  18
accession number MF141006 and aligned with all sequences
obtained from the GenBank database using the BLAST
search program (http://www.ncbi.nlm.nih.gov/BLAST​/).
The phylogenetic tree was visualized using the neigh-
bor-joining method using MEGA version 6.06 software
(Fig. 2). The 5.8s-rDNA gene sequence of the yeast strain
YBR allowed us to affiliate the isolate to the genus of Rho-
dotorula as it was nearly identical to the strains Rhodotorula
mucilaginosa (DQ386306) and Rhodotorula mucilaginosa
strain ATCC 32763 with a similarity value by 99%. Com-
parison of biochemical tests for the isolated yeast strain YBR
demonstrated that the isolated strain and Rhodotorula muci-
laginosa were closely identical; with special properties for
isolate yeast strain YBR as it tolerates 20% salinity and can
grow at 42 °C.
Production kinetics of biosurfactants
by Rhodotorula sp .YBR
The agro-industrial by-products such as OOMW were evalu-
ated to develop a sustainable culture medium for the pro-
duction of a new biosurfactant by a Rhodotorula sp .YBR
Fig. 1  a Screening results for yeast strain YBR isolate from left to
right; Emulsification index (­E24) obtained on YPG medium after 72
h, Drop collapse: Microtiter-plate under photonic microscope × 10
and spread oil test; b: Micrographs of Rhodotorula sp. YBR colony,
strain isolated from the Algerian oilfield. Figure 3 displays
the kinetics of batch fermentation of Rhodotorula sp. YBR
strain conducted on crude OOMW-based medium. The
increase in E ­ 24 was proportional to the biomass, reach-
ing the maximum (90%) after 54 h. A reduction in TS was
recorded (35 mN m ­ −1). The yield of biosurfactant was 10.08
−1
± 0.38 g L­ . In contrast, the production of a biosurfactant
by Pseudomonas aeruginosa UCP0992 cultivated in a low-
cost medium favored the biosurfactant yield to 26 g L ­ −1 and
−1
the reduction in ST to 26.5 mN ­m (Silva et al. 2018). The
production of biosurfactants started in the exponential phase
of growth and the pH of fermentation medium was 5.5 and
did not vary throughout the experiment (Fig. 4).
Characterization of biosurfactant crude extract
The biosurfactant crude extract was first semi-purified
by preparative chromatography on a thick plate of silica
gel. Thus, the bands corresponding to well-revealed spots
were scraped off and recovered separately. Then they were
resorbed from silica gel using methanol, then concen-
trated and tested by drop collapse method to evaluate their
cultures were grown for 72 h at 30 °C on YPG broth and YPGA
media (a, b), Micrographs of cells of Rhodotorula sp. YBR under
photonic microscope × 40 (c)
13
 18   Page 8 of 18
Fig. 2  Phylogenetic tree of Rhodotorula sp.YBR . The partial
5.8s-rDNA sequence (677 pb) of the isolate YBR was submitted to
GenBank under the accession number MF141006 and aligned with
all sequences obtained from the GenBank database using the BLAST
search algorithm. The phylogenetic tree was constructed using the
neighbor-joining method using MEGA version 6.06 software
activities. Active fractions were then characterized by ana-
lytical TLC using different solvent systems. Figuire 4 dis-
plays the TLC analysis of biosurfactant produced by Rhodo-
torula sp. YBR, and shows three spots in chromatogram of
each molecule after chemical revelation as described above
: carbohydrate ­(Rf = 0.62), protein ­(Rf = 0.48) and lipid
­(Rf = 0.76). The estimation of macromolecules revealed the
following concentrations: 1.50 ± 0.04 g L ­ −1 of lipids, 1.61
± 0.01 g ­L of carbohydrates and 1.46 ± 0.04 g ­L−1 of pro-
−1
teins. According to the macromolecules estimation and TLC
analysis, the compound produced by Rhodotorula sp.YBR
was partially characterized as glycolipoprotein.
The chemical nature of partially purified biosurfactant has
been further studied using infrared FTIR spectroscopy. The
infrared absorption spectrum of the Rhodotorula sp. YBR
extract, given in Fig. 5, showed the following bands: at 3339
­cm−1 (indicates the presence of a OH (hydrogen stretch), at
Table 1  Biochemical and
physiological characteristics of
YBR yeast isolate
13
World Journal of Microbiology and Biotechnology (2021) 37:18
1643 ­cm−1 indicates the presence of C=O which represents
an ester bond, at 1377 ­cm−1 (C–N bond), from 1273 to 1015
­cm−1 (C−O and C−O−C bonds of the carboxylic acids). IR
spectroscopy revealed the presence of aliphatic chains and
peptide moieties. FTIR spectroscopy results were in agree-
ment with those obtained with TLC and the estimation of
macromolecules by spectrometric methods. However, the
detailed structure of the produced biosurfactant requires
further analysis.
Microbial enhanced oil recovery from contaminated
sand
The treatment of soils contaminated by hydrocarbons has
been performed using physical and mechanical methods,
while chemical methods involving the application of chemi-
cal surfactants have also been largely used. However, besides
being inefficient, these methods can constitute another
source of contamination by accumulation of other toxic
compounds in the environment. The use of biosurfactants
in remediation of contaminated sites appears to be a prom-
ising strategy that favors the bioavailability of hydrophobic
products. Biosurfactants emulsify hydrocarbons by improv-
ing their solubility, decreasing ST, and increasing the move-
ment of oily substances from soil particles. Therefore, in
this study we were interested in studying the effects of bio-
surfactants produced by Rhodotorula sp.YBR cultivated on
crude OMW medium on the solubilization of hydrocarbons
present in sand contaminated by motor oil. One of the big
advantages of using biosurfactants is the possibility of pro-
ducing them from different substrates, especially renewable
sources. The selection of low-cost substrates is important to
the overall economy of the process, about 60% of the pro-
duction costs are due to the culture medium (Makkar et al.
2011; Gudiña et al. 2016).
World Journal of Microbiology and Biotechnology (2021) 37:18 Page 9 of 18  18

Fig. 3  Biosurfactant production profile; ­OD600 nm ,biomass (g ­L−1), 200 rpm in 2 L bioreactor. Error bars illustrate experimental errors
pH, ST (mN ­m−1), and emulsification index ­(E24%) of Rhodotorula (standard deviations), calculated from three independent experiments
sp. YBR grown on crude OOMW at 30 ºC, pH 5.5 and agitation of

Fig. 4  TLC Analysis of semi-

purified biosurfactant produced
by Rhodotorula sp.YBR grown
on OOMW medium after chem-
ical revelation, (a) Proteins, (b)
Carbohydrates and (c) Lipids

A comparison was made under the same conditions,
with synthetic surfactant SDS and Tween 80 as positive
controls and with distilled water as a negative control. The
results showed a great potential of the biosurfactant pro-
duced by Rhodotorula sp.YBR in removing motor oil from
sand sample with a recovery rate of 98 ± 0.28%. Our results
demonstrate a high efficiency of the biosurfactant produced
by the Rhodotorula sp.YBR strain in the enhanced removal
of hydrophobic contaminants from polluted soils which
make it a promising and potential candidate for environ-
mental applications.

13
 18   Page 10 of 18
Fig. 5  Fourier transform infrared spectroscopy (FTIR) spectra of the biosurfactant produced by Rhodotorula sp.YBR
Stability studies and properties of the biosurfactant
produced
Stability studies
Environmental factors such as temperature, pH and salinity
could affect the activity and stability of biosurfactant from
Rhodotorula sp. YBR cultivated on crude OMW medium.
However, the effect of temperature, pH and NaCl concentra-
tions was studied (Fig. 6a–c). The results indicate that the
pH has an effect on the stability of the emulsions. According
to Fig. 6b, it can be clearly observed that E­ 24 changes with
pH, and the effect is best when pH is 4–8. The stability of the
crude extract over different temperatures from 4 to 100 °C
was also determined (Fig. 6a). The ­E24 values showed a very
high stability in a temperature range of 4 to 80 °C. However,
20% of the emulsifying activity was lost at 100 °C.
The effect of the addition of sodium chloride (NaCl) on
the biosurfactants crude was studied (Fig. 6c). Regarding the
emulsifying and surface activities, the stability of the bio-
surfactant was observed at all salt concentrations (1 to 10%).
Reduction of ST is a very important parameter in the evalu-
ation of an efficient biosurfactants. Stability studies results
showed that the produced biosurfactant maintained stable
ST values at all conditions tested, extreme pH, temperature
and salinity (Fig. 6a–c). According to Fig. 6a and b, it can
be clearly observed that ­E24 changes with temperature and
13
World Journal of Microbiology and Biotechnology (2021) 37:18
pH, and the effects are best when temperature is 4-60 and
pH is 4-8.
As concluding remarks: the biosurfactants produced by
Rhodotorula sp.YBR showed a high stability under extremes
conditions of pH, temperature and salinity, which make
them potential candidates for environmental or industrial
applications.
Surface tension and critical micellar concentration
of biosurfactant isolated from Rhodotorula sp. YBR
An accurate digital tensiometer was used to obtain pre-
cise values of ST, using the method described by Du Noüy
(1919). The ST value of the biosurfactant crude extract iso-
lated from Rhodotorula sp. YBR and cultivated on crude
OMW medium was 30.16 mN ­m−1, showing a great effi-
ciency in reducing ST. A value of CMC equal 180 mg L ­ −1
was obtained for the same biosurfactant. The concentration
of the biosurfactant, above which the surface tension does
not change, is defined as the CMC (Fig. 7).
Discussion
Despite being recognized as a truly dangerous residue,
OOMW is still often released onto soil or into water courses,
in particular by small oil mills, which causes considerable
harmful effects on the environment. In this work, innovative
World Journal of Microbiology and Biotechnology (2021) 37:18 Page 11 of 18  18
Fig. 6  Effect of temperature (a), pH (b) and salt concentrations (c)
on stability of crude biosurfactant produced by Rhodotorula sp.YBR
grown on crude OOMW regarding ST and emulsifying activity. Error
bars illustrate experimental errors (standard deviations), calculated
from three independent experiments
approaches have been developed and applied as strategies
to valorize OOMW as a substrate for low-cost biosurfactant
production by the yeast strain Rhodotorula sp.YBR newly
isolated from an oilfield in Algeria.
Meneses et al. (2017) evaluated OOMW as low-cost alter-
native substrates. The composition of the culture medium
was optimized and the highest biosurfactant yield produc-
tion = 139 ± 16 mg ­L−1 was achieved. The partially puri-
fied biosurfactant showed a CMC value = 550 mg ­L−1 and
a reduction in the ST of water to 31.2 mN ­m−1. Joshi et al.
(2008) and Ruggeri et al. (2009) affirmed that the most
potent biosurfactants are those capable of reducing the ST of
the medium to values < 40 mN m ­ −1. The effectiveness of the
biosurfactant is also determined by CMC, the point at which
micelles start to form, in other words, a smaller amount of
Fig. 7  Critical micellar concentration of biosurfactant produced by
Rhodotorula sp.YBR grown on OOMW medium. Error bars illustrate
experimental errors (standard deviations), calculated from three inde-
pendent experiments
biosurfactant is needed to decrease ST, showing greater effi-
ciency (Desai and Banat 1997; Aparna et al. 2012).
Surfactin, one of the most proficient biosurfactants
known, reduces ­H2O ST from 72 to 27 mN ­m−1, which is
close to the minimum detectable value (Seydlová and Svo-
bodová 2008). In this research work, we found that Rho-
dotorula sp. YBR strain efficiently reduced ST. This strain
can be used specifically for biosurfactant production and the
glycolipoprotein biosurfactant produced by this strain has
potential for remediation of oil-contaminated soil. Yeasts
from soils polluted with petroleum hydrocarbons were tested
for their capacities for biosurfactant production using drop
collapse assay, determination of ST reduction, and emul-
sion index. The results indicated that some yeasts are able
to produce biosurfactants forming stable emulsions ­(E24 >
50%). In addition, the STs of these biosurfactants produced
by yeast strains were reduced to around 35 mN ­m−1 (Kaur
et al. 2017; Yalçin et al. 2018). Also, Jemil et al. (2016)
found that Bacillus sp. producing-biosurfactant isolated from
polluted soils in Tunisia possess high surface activity by
lowering the ST to 31 mN m ­ −1 and the CMC value of 100
−1
mg ­L . The produced biosurfactant has shown better stabil-
ity in wide range of temperature, pH and salinity.
Over the past decades, the evaluation of the biosurfactant
produced by yeasts has increased and several non-pathogenic
yeast strains have been reported as producers (Faria et al.
2015; Eldin et al. 2019). In a previous study, the yeast Wick-
erhamomyces anomalus was identified as a biosurfactant
producer in that it was able to reduce the ST to 36 mN ­m−1
in 72 h and remained stable under extreme conditions of
salinity, temperature, and pH (Souza et al. 2018). Likewise,
13
 18   Page 12 of 18
various strains of Rhodotorula glutinis isolated from Roma-
nian soils polluted by hydrocarbons have been shown to be
of potential for biosurfactant producers (Csutak et al. 2012).
Mnif and Ghribi (2015a, b) found that the produced bio-
surfactant exhibited high environmental compatibility and
high activity under extreme conditions (salinity, temperature
and pH). In addition, renewable resources through microbial
fermentation could easily be used to produce these biosur-
factants. On the basis of these studies, other authors have
shown that the biosurfactant has the ability to successfully
mobilize organic compounds adsorbed on soil constituents
thus improving their degradation (Whang et al. 2009; Silva
et al. 2018). On the other hand, Eddouaouda et al. (2012)
isolated from an Algerian crude oil contaminated soil a
Staphylococcus sp. producing a biosurfactant. The results
indicated that the biosurfactant showed better reduction of
ST and the highest ­E24.
The kinetics of the batch fermentation of Rhodotorula
sp. YBR strain was conducted on a medium based on crude
OMW. The value of ST = 35 mN m ­ −1 obtained was similar
to those described for other biosurfactants produced by yeast
strains cultured on media based on agro-industrial by-prod-
ucts: Candida lipolytica = 31 mN m ­ −1 (Rufino et al. 2011),
−1
Candida antartica = 35 mN m ­ (Bednarski et al. 2004). In
addition, the ST result obtained here was similar to those
reported by Sen et al. (2017) and Chandran and Das (2011)
for biosurfactants from Rhodotorula babjevae YS3 and Rho-
dotorula mucilaginusa, respectively. Colak and Kahraman
(2013) reported that Pseudomonas aeruginosa produced
a maximum of 400 mg ­L−1 of rhamnolipids using OMW
diluted up to 50% as the culture medium. In addition, Ji
et al. (2016) studied the production of rhamnolipids by Pseu-
domonas aeruginosa isolated from petroleum-contaminated
soil using OMW as the culture medium (sole C source) and
they reported that this pathogenic microorganism produced
12.6 g ­L−1 of rhamnolipids after optimization of the culture
conditions, with ST reduction = 27 mN ­m−1 and an emulsion
capacity of 90%. Llori et al. (2008) studied the production
of biosurfactant by Candida albicans and they reported that
this microorganism appeared to be a better biosurfactant-
producer ­(E24 = 64%).
The results of our study concerning the yield of biosur-
factant and ­E24 are better than those reported by previous
studies, although the experimental conditions were dif-
ferent. However, the yeast Candida utilis has been shown
to be able to produce a higher yield (24.22 ± 0.23 g ­L−1),
using frying oil from canola waste as substrates (Ribeiro
et al. 2020). Amaral et al. (2010) and Jimoh and Lin (2019)
found that most biosurfactants were produced in the station-
ary growth phase, although some species may produce these
biomolecules during their exponential growth phase during
nutrient depleting in the medium, stress, varying pH, tem-
perature, aeration, or naturally. According to Bednarski et al.
13
World Journal of Microbiology and Biotechnology (2021) 37:18
(2004), the constant acidity found in the culture medium was
a parameter correlated with the efficiency of the synthesis
of glicolipid by yeasts such as Candida antarctica and Can-
dida apicola. Greater glycolipid production is achieved if
the pH value is controlled and kept stable during cultivation.
However, when the pH was not adjusted in the medium, a
negative effect was exerted on the efficiency of the synthesis
of these products. In the same context, Sen et al. (2017),
studied the biosurfactant production profile of Rhodotorula
babjevae YS3 and they reported that the growth of the strain
started without a lag phase and that the exponential phase
was extended up to 168 h. The maximum biomass value
(16.61 g L ­ −1) was recorded after 192 h of growth. The yield
of biosurfactant was (19 g ­L−1) at 72 h, however it dropped
significantly after 216 h (12.23 g ­L−1), the pH of the fer-
mentation medium was 7 and did not vary significantly (p
> 0.05) throughout the experiment. The TS of the culture
was reduced from 70 to 35 mN m ­ −1 after 24 h of culture at
the start of the exponential phase and remained stable dur-
ing the incubation period (240 h). Csutak et al. (2012) also
found that biosurfactants produced by Rhodotorula glutinis
strains showed promise as pH-dependent emulsifiers for oil
degradation.
In our study, Rhodotorula sp. YBR has successfully used
OOMW as a substrate for the production of biosurfactant,
this residue is characterized by high organic matter content
(chemical oxygen ­(O2) demand: COD = 220 g L ­ −1). It also
contains toxic substances such as polyphenols, and sugars,
nitrogen compounds, organic acids and residual oils which
enhance microbial growth, but also make their treatment
difficult, so their disposal becomes a critical environmental
problem. Thus, the use of these materials is important for
both environment and the economy (Saharan et al. 2011).
Based on macromolecule estimation and TLC analysis, the
compound produced by Rhodotorula sp. YBR has been par-
tially characterized as a glycolipoprotein. The high molecu-
lar weight of biosurfactants makes them efficient emulsify-
ing agents (Markande et al. 2013). They are often applied
as a preservative to support bioremediation and removal of
hydrophobic substances from soils and water (Kavitha et al.
2014; Sajna et al. 2015; Varjani 2017). The origin of biosur-
factants and their production/purification processes strongly
influence their molecular characteristics and their success-
ful practical application. Sen et al. (2017) reported that the
chemical nature of the biosurfactant produced by Rhodo-
torula babjevae YS3 was predicted as sophorolipid using
a solvent system composed of chloroform/methanol/water
(65/15/2, v/v/v). The crude extract of Rhodotorula babjevae
YS3 showed R ­ f values of 0.56, 0.13 and 0.18. Pseudomonas
sp. was found to be a potential producer of biosurfactant able
to degrade hydrocarbons (47 to 97%). High performance
liquid chromathography–mass spectrometry (HPLC-MS)
and nuclear magnetic resonance spectroscopy (NMRS)
World Journal of Microbiology and Biotechnology (2021) 37:18 Page 13 of 18  18
revealed the glycolipid nature of this biosurfactant (Sharma
et al. 2015).
The chemical natures of partially purified biosurfactants
have been further studied using infrared FTIR spectroscopy.
These studies indicated that the biosurfactants produced
were lipo-, glyco- and phospholipid (Alizadeh-Sani et al.
2018). Our results corroborate with other studies, which
reported the same IR spectrum (Pele et al. 2018). FTIR spec-
troscopy results are in agreement with those obtained with
TLC and the estimation of macromolecules by spectrometric
methods. However, the detailed structure of the produced
biosurfactant requires further analysis.
Biosynthesis of biosurfactants is affected by a number
of factors including nutrients, temperature, salinity, and pH
(Ribeiro et al. 2020). There is an important tool to study
the influence of environmental factors on the activity and
stability of the biosurfactant of Rhodotorula sp. YBR grown
on crude OOMW medium. All these factors control biosur-
factant emulsion behavior, due to their impact on biosur-
factant molecular characteristics (Han et al. 2015; Long et al.
2017; Ribeiro et al. 2020). Our results indicate that pH has
no effect on the stability of the mulsions over a wide range.
Similar results have been reported by Mouafi et al. (2016)
and Khopade et  al. (2012) for biosurfactants produced
by Bacillus brevis and Nocardiopsis sp. B4, respectively.
Staphylococcus sp. producing a biosurfactant isolated from
an Algerian crude oil-contaminated soil has shown stability
over a wide range of pH (2–12), temperature (4-55 °C) and
salinity (0-300 g ­L−1) (Eddouaouda et al. 2012). In addition,
an emulsifier produced by Yarrowia lipolytica NCIM 3589
grown in n-hexadecane has also been shown to be stable
and active over a wide pH range (2 to 8). However, a con-
siderable loss of emulsifying activity has been observed at
pH values above 10 (Zinjarde and Pant 2002). Furthermore,
Techaoei et al. (2011) reported that the emulsifying activity
of Pseudomonas aeruginosa SCMU106 was stable over a
pH range of 6 to 12, but decreased at pH 2-4. The stability of
the crude extract at different temperatures was also studied.
We observed that the E ­ 24 values showed high temperature
stability. Similar results have been reported by Mouafi et al.
(2016) where the effect of heat treatment on the emulsifying
activity of the culture of Bacillus brevis showed that there
was no change in the emulsification capacity in the tempera-
ture range of 30 to 80 °C. In addition, Khopade et al. (2012)
showed that the biosurfactant produced by Nocardiopsis sp.
B4 was thermostable; heating the biosurfactant to 100 °C
had no significant effect on its performance. The stability of
the biosurfactant has been observed at all salt concentrations
for emulsifying and surface activities. Kiran et al. (2010)
reported that the biosurfactant produced by Actinobacterium
was stable at a high concentration of NaCl (up to 5% NaCl).
ST is a very important parameter in the evaluation of an effi-
cient biosurfactants. Our results showed that the produced
biosurfactant maintained stable ST values at all conditions
tested. Similarly, biosurfactants produced by other micro-
bial strains have indicated stability over a wide range of pH,
temperature and salinity (Joshi et al. 2008; Luna et al. 2013;
Sen et al. 2017; Kumar Gaur et al. 2019; Araújo et al. 2019)
for the following strains, respectively: Bacillus sp., Candida
sphaerica UCP0995, Rhodotorula babjevae YS3, Candida
sp., Serratia marcescens UCP 1549. In conclusion, the bio-
surfactant produced by Rhodotorula sp. YBR showed high
stability under extremes conditions of pH, temperature and
salinity, which make them potential candidates for environ-
mental or industrial applications.
The use of biological surfactants in the remediation
of contaminated sites appears to be a promising strategy
that promotes the bioavailability of hydrophobic products
(Banat et al. 2014). Biosurfactants emulsify hydrocarbons
by improving their solubility, decreasing ST, and increas-
ing movement of oily substances from soil particles. This
work showed a great potential of the biosurfactant produced
by Rhodotorula sp.YBR in removing motor oil from sand.
Different biosurfactants have been tested for the remobiliza-
tion of petroleum products in contaminated soil and water.
Chaprão et al. (2015) studied the potential application of
two biosurfactants produced by the yeast Candida sphaerica
and by the bacterium Bacillus sp. grown in low-cost sub-
strates for enhanced removal and biodegradation of motor oil
contaminated sand. Both biosurfactants have shown excel-
lent efficiency in removing motor oil from contaminated
sand (70–90%). Four bacterial strains, Stenotrophomonas
rhizophila BG32, Pseudomonaspoae BA1, Bacillus thur-
ingiensis BG3 and Acinetobacter bouvetii BP18, isolated
from petroleum hydrocarbons polluted soils were used to
test their bioremediation capacities under different condi-
tions. In this context, this study has demonstrated the effect
of natural surfactants on the increase in the apparent deg-
radation of hydrocarbons (74.60% to 96.07%) (Ali Khan
et al. 2017). Batista et al. (2010) also reported that Candida
tropicallis exhibited an 80% recovery rate of residual crude
oil adsorbed to sand. Further, Pele et al. (2018) reported that
the biosurfactant produced by Rhizopus arrhizus UCP 1607
removed 79.4% of the diesel impregnated in marine soil.
Biosurfactants can improve the mobility and removal of
hydrophobic molecules from the contaminated environment
and therefore their biodegradation (Franzetti et al. 2009;
Pacwa-Plociniczak et al. 2011). Candida antarctica has been
shown to produce an extracellular biosurfactant that could
be used to enhance the degradation of hydrophobic con-
taminants such as petroleum compounds (Hua et al. 2004).
Our results demonstrate the high efficacy of the Rhodotorula
sp.YBR biosurfactant in the enhanced removal of hydropho-
bic contaminants from polluted soils, making it a promis-
ing and potential candidate for environmental applications.
It is also interesting to note that the microorganisms that
13
 18   Page 14 of 18
produce biosurfactants can remove hydrophobic contami-
nants, thus avoiding the accumulation of biomass through
biofilter mechanisms (He et al. 2020). They act to mobilize
the hydrocarbons on the one hand by reducing the interfacial
tension between the environmental components (­ H2O, soil)
and on the other hand to increase the solubilization of the
trapped contaminants in the aqueous phase by incorporating
the hydrophobic compounds in the center of the surfactant
micelles that are formed. Therefore, they can positively
contribute to the practice of bioremediation. Additionally,
Karlapudi et al. (2018) found that biosurfactant-producing
microorganisms create their own micro-environment and
promote emulsification through various mechanisms such as
quorum sensing. It has been suggested by Soberón-Chávez
and Maier (2010), that when biosurfactants are released,
their monomers organize under micellar form, in a way that
the linear group (hydrophobic portion) is turned towards
the center, forming the nucleus, and the hydrophilic part
is turned to the sphere surface (head group), composing an
interface with ­H2O. Thus, the biosurfactant reduces the ST
between ­H2O and the oil and contributes to the micelle for-
mation, increasing hydrocarbon exposure to bacteria and ­O2
and helping hydrocarbon biodegradation.
The biosurfactant produced by Rhodotorula sp. YBR, has
been shown to be highly effective in reducing ST. Similar
results have been reported for biosurfactants produced by
different species of the genus Candida, which have been
shown to produce highly effective biosurfactants capable of
reducing water ST to low values (Santos et al. 2016). In
the same context, Kiran et al. (2009) and Silva et al. (2014)
reported that the biosurfactant produced by Aspergillus sp.
MSF and Mucor circinelloides could reduce the ST of water
to 28 mN m ­ −1 and 26 mN m ­ −1, respectively. Recently, a
study revealed that the biosurfactant produced by Rhizopus
arrhizus UCP 1607 reduced the ST of water to 26.5 mN m ­ −1
(Pele et al. 2018). A biosurfactant with hydrocarbon-degrad-
ing property produced by microorganisms was characterized
from diesel-contaminated soils. The highest reduction in ST
was obtained (41 and 46 mN ­m−1) and the emulsifying activ-
ity was also better (­ E24 = 74%) (Menezes Bento et al. 2005).
CMC value of biosurfactant obtained from Rhodotorula
sp. YBR grown on crude OOMW medium was similar than
to that obtained by Sen et al. (2017) who indicated a value of
150 mg ­L−1 for the Rhodotorula babjevae YS3 biosurfactant,
while Luna et al. (2013) obtained a CMC of 250 mg ­L−1 for
Candida sphaerica UCP0995. However, it should be noted
that the CMC of a surfactant varies with its structure, the
temperature of the solution, the presence of electrolytes or
organic compounds. The variation in the size of the hydro-
phobic region is an important factor and in general the CMC
decreases as the hydrophobicity of the surfactant increases
(Pacwa-Płociniczak et al. 2011).
13
World Journal of Microbiology and Biotechnology (2021) 37:18
Conclusion
The results of this investigation are of great interest, as it
provides a low cost effective medium for the production of
biosurfactants from a strain of yeast newly isolated from
an oilfield in Algeria identified as Rhodotorula sp .YBR..
The latter used OOMW as a substrate for the production
of biosurfactant, as the most economical source available
that can be used for biosurfactants production, especially in
Mediterranean countries exposed to this hazardous waste.
Thus, the use of this residue resolves both environmental
and economic problems. The biosurfactant produced by
Rhodotorula sp.YBR from crude OOMW has been isolated
and partially characterized as a complex of glycolipoprotein
groups. In addition, produced biosurfactant was found to
be stable under extreme conditions of temperature, pH and
salinity and showed great properties like low CMC, higher
emulsification activity and great reduction of ST. Further-
more, the produced biosurfactant showed a higher potential
for improved oil recovery from polluted soil with a recovery
rate greater than 95%. The properties of the biosurfactant
make them of major importance and open up new future
prospects for its use in industrial applications in the field of
oil recovery. However, the use of large scale biosurfactants is
still limited by competitive production costs. There are two
possible strategies to make their production more profitable.
One way would be to reduce their cost and the other would
be to increase the yield. On the other hand, a more in-depth
analysis of the composition of biosurfactants should be car-
ried out as part of future research.
Acknowledgements  This work was supported with the Grant from
Ministry of Higher Education and Scientific Research with University
M’Hamed Bougara of Boumerdes under agreement on scientific coop-
eration– CNEPRU Project No. F00320130030.
Authors contribution  The experimental, analytical of data and drafting
of the manuscript were done by LDM. SKG and DD were involved in
the designing of the experiment, data analysis, and interpretation of
the results and reviewed the manuscript. All authors read and approved
the final manuscript.
Compliance with ethical standards 
Conflict of interest  The authors declare that they have no conflict of
interest.
Ethical approval  The experiment does not include any animal or human
testing.
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