1) The document describes a study that used sunflower acid oil (SAO), a byproduct of vegetable oil refining, as a new substrate for producing rhamnolipid biosurfactant via fermentation with Pseudomonas aeruginosa. 2) The study was able to produce rhamnolipid at a maximum concentration of 4.9 g/L using 60 g/L of SAO as the carbon source. 3) Characterization of the rhamnolipid produced confirmed its structure and showed it had good surface and emulsification properties, indicating SAO's potential as an alternative substrate for rhamnolipid production.

1. ORIGINAL ARTICLE
Sunflower Acid Oil-Based Production of Rhamnolipid Using
Pseudomonas aeruginosa and Its Application in Liquid
Detergents
Jagruti V. Jadhav1
· Padmini Anbu2
· Sneha Yadav2
· Amit P. Pratap1
· Sandeep B. Kale3
Received: 21 June 2018 / Revised: 14 November 2018 / Accepted: 10 December 2018
© 2019 AOCS
Abstract Utilization of industrial waste as substrates for
the rhamnolipid synthesis by Pseudomonas aeruginosa is a
worthy alternative for conventionally used vegetable oils
and fatty acids to reduce the production cost of rhamnoli-
pid. Sunflower acid oil (SAO), a by-product of the oil
industry, contains 70% 18:0 fatty acid, with oleic acid as a
major component. In this scope, production and analysis of
rhamnolipid was successfully demonstrated using SAO as a
new substrate. Pseudomonas aeruginosa produced rhamno-
lipid (a glycolipid biosurfactant) at a maximum concentra-
tion of 4.9 g L−1
with 60 g L−1
of SAO in the medium.
Structural properties of rhamnolipid biosurfactant are con-
firmed using thin layer chromatography (TLC), high perfor-
mance liquid chromatography (HPLC), and fourier
transformed infrared spectroscopy (FTIR) analysis. Further
surface-active properties of the crude rhamnolipid were
evaluated by measuring surface tension and emulsification
properties. The synthesized rhamnolipid reduced the sur-
face tension of water to 30.12 mN m−1
and interfacial ten-
sion (against heptane) to 0.52 mN m−1
. Moreover,
rhamnolipid shows the highest emulsification index (above
80%) for vegetable oils. This study confirms the use of
SAO as a potential substrate for rhamnolipid production.
The synthesized rhamnolipid was incorporated in liquid
detergent formulation along with alpha olefin sulfonate
(AOS) and sodium lauryl ether sulfate (SLES). The perfor-
mance properties including foaming and cleaning efficiency
of liquid detergent were compared.
Keywords Rhamnolipid Sunflower acid oil Oil
refinery waste Emulsification index Biosurfactant
Detergent
J Surfact Deterg (2019) 22: 463–476.
Introduction
Surfactants are organic molecules that orient themselves at
interfaces and reduce the interfacial tension. Their surface-
active properties such as foaming, detergency, emulsifica-
tion, and wettability make them prominent molecules in
many fields (Desai and Banat, 1997). Biosurfactants are
amphiphilic molecules produced by microbial cells. Grow-
ing environmental concern and increasing consumer aware-
ness associated with the use of bio-based products are
anticipated to be a key driver for the growth of utilization
of biosurfactants in industrial and household applications.
The most known biosurfactants are glycolipids. The
common way to produce a glycolipid biosurfactant is by
fermentation of vegetable oils, particularly soybean oil,
corn oil, coconut oil, sunflower oil, and olive oil with
Supporting information Additional supporting information may be
found online in the Supporting Information section at the end of the
article.
* Amit P. Pratap
amitpratap0101@rediffmail.com
1
Department of Oils, Oleochemicals and Surfactants Technology,
Institute of Chemical Technology (University under Section 3 of
UGC Act 1956; Formerly UDCT/ UICT), Nathalal Parekh Marg,
Matunga (East), Mumbai, 400 019, India
2
Department of Chemistry, K. J. Somaiya College of Science and
Commerce, Vidyavihar, Mumbai, 400 077, India
3
DBT–ICT Centre for Energy Biosciences, Department of
Chemical Engineering, Institute of Chemical Technology
(University under Section 3 of UGC Act 1956; Formerly UDCT/
UICT), Nathalal Parekh Marg, Matunga (East), Mumbai, 400
019, India
Published online: 28 February 2019
J Surfact Deterg (2019) 22: 463–476
J Surfact Deterg (2019) 22: 463–476
DOI 10.1002/jsde.12255

2. different carbohydrates such as sorbitol, glucose, and
sucrose. The fermentation is commenced by different yeast
and bacterial strains under sterile conditions. Utilization of
vegetable oil as a substrate, which is essential for the food
consumption, is not advisable due to the importance of con-
servation of food sources. Many vegetable oils with noned-
ible values are already being studied for biosurfactant
production (Pratap et al., 2011). Residues obtained in the
vegetable oil processing were also used previously as feed-
stock to produce biosurfactants (Sidal and Ozkale-Taskin,
2003; Wadekar et al., 2011a). Apart from nonedible oils,
the by-products resulting from vegetable oil processing are
also studied to yield biosurfactants.
Rhamnolipid (RL) is one of the glycolipid biosurfactants
produced by utilizing numerous renewable substrates such
as sunflower oil, soybean oil, corn oil, glucose, and so
on. The rhamnolipid can also be obtained from waste mate-
rials such as palm fatty acid distillate (FAD), glycerol resi-
due, olive oil mill effluent, soapstock (SS), and molasses.
The major obstacle in rhamnolipid commercialization is the
high cost of their production. To overcome this barrier, a
process for rhamnolipid production using inexpensive feed-
stock with the productive microbial strain needs to be
developed. Oil processing and agricultural wastes are con-
sidered as promising substrates for production of rhamnoli-
pid, which can also help to alleviate the waste disposal
issue. Rhamnolipids are the multifaceted compounds with a
wide range of possible applications from cleaning agents to
high-value biological and medicinal agents (Randhawa and
Rahman, 2014). Different microbial strains are used for the
microbial production of rhamnolipid such as
P. aeruginosa, Pseudomonas fluorescens, Pseudomonas
putida, Acinetobacter calcoaceticus, and Burkholderia glu-
mae. Rhamnolipids are mainly produced in the form of
monorhamnolipid and dirhamnolipid that depend on the
presence of rhamnose molecules in the structure. The type
of rhamnolipid produced depends on the carbon substrate,
microbial strain, C/N ratio, and fermentation conditions.
The demand for vegetable oil is increasing as a conse-
quence of world’s rising population. Vegetable oil con-
sumption will increase tremendously as population
continues to expand. The crude vegetable oil contains many
impurities from expellers or solvent extraction plant, which
must be removed to make the oil stable against oxidation
upon storage and to maintain edible or more palatable qual-
ity of the oil. The process of removing these impurities and
improving quality of oil is called refining. These refining
processes produce some by-products of low commercial
value such as SS, deodorizer FAD, and acid oil (AO).
These by-products can cause significant environmental pol-
lution problems if discharged in the ecosystem. However,
these by-products can be used for other beneficial or indus-
trial activity. AO is one of the most significant by-products
of the vegetable oil-refining industry, which predominantly
contains unesterified fatty acids and acylglycerols. This by-
product is generated upon the splitting of SS produced in
the alkali neutralization step in the chemical-refining pro-
cess. AO is a source of unesterified fatty acids for different
oleochemical applications. The use of AO as a raw material
was already reported due to the presence of large amounts
of fatty acids in it (Marchetti et al., 2011).
Many researchers tried to use different industrial wastes
for rhamnolipid production, however, use of sunflower acid
oil (SAO) as a feedstock has not been reported yet. Reduc-
ing the cost of raw material in fermentative production of
rhamnolipid could be one of the approaches to make it eco-
nomically viable. The originality of this work is to present
a new route for valorization of SAO, with production of
rhamnolipid as a value-added product and thereby reducing
the cost of rhamnolipid. In light of this, SAO was consid-
ered as a potential carbon substrate for production of rham-
nolipid. The purpose of this work is to scrutinize the
structural and surface-active properties and possible appli-
cations of rhamnolipids produced by P. aeruginosa MTCC
2453 by utilizing SAO as a carbon source.
Experimental
Substrates and Chemicals
SAO was kindly provided by M/s Godrej Industries Ltd.,
Mumbai, Maharashtra, India. All other chemicals, salts,
and solvents (hexane and ethyl acetate) used in this study
were supplied by M/s Hi-Media, Mumbai and were of ana-
lytical reagent grade.
Culture Conditions for Production of Rhamnolipid
Pseudomonas aeruginosa MTCC 2453 strain was obtained
from the National Collection of Industrial Microorganism,
Pune, India. It was maintained on a nutrient agar slant at 4
C. Bacterial growth from the nutrient agar slant was trans-
ferred to liquid nutrient broth and incubated for 24 h at 30
C. The optical density of bacterial suspension was
adjusted to 0.78 and the same was used as the inoculum. A
2% (v/v) of seed culture was added to 50 mL of sterile
mineral salt medium as mentioned in previous work
(Jadhav et al., 2018) with following composition (g L−1
):
NaNO3, 4; K2HPO4, 1; KH2PO4, 0.5; MgSO4.7H2O, 0.01;
KCl, 0.1; FeSO4.7H2O, 0.01; and CaCl2.2H2O, 0.01; Yeast
extract, 0.01 and 0.05 mL trace element solution containing
(g L−1
): H3BO3, 0.26; CuSO4.5H2O, 0.5; MnSO4.H2O,
0.5; MoNa2O4.2H2O, 0.06; and ZnSO4.7H2O, 0.72. The
pH of the medium was adjusted to 6.8 1 before
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3. autoclaving. SAO was used as a carbon source at different
concentrations.
Shake flask fermentation batches were carried out in an
incubator shaker at 30 1
C and 220 rpm. The fermenta-
tion process was further scaled up on a 5 L stirred batch
bioreactor (BioFlow 115) equipped with a water jacket for
temperature control, a pH probe for pH maintenance, a DO
probe for dissolved oxygen measurement, a six blade Rush-
ton impeller for stirring, and a ring sparger for submerge
gassing. The agitation speed was limited to 450 rpm to
avoid cell damage. The temperature was kept constant at
30
C, and the aeration rate was maintained at 1 vvm. The
pH was maintained to 6.5 using 5 M sodium hydroxide and
5 M sulfuric acid solution. Samples were withdrawn peri-
odically for analysis of cell growth, residual substrate, and
rhamnolipid yield. Each analysis was performed in tripli-
cate to obtain statistically reliable results.
Analysis of Biomass Growth
To investigate microbial growth, 2 mL cell culture was
transferred to a microcentrifuge tube and centrifuged at
10,000 rpm for 10 min. The supernatant was disposed of
and cell pellets were separated. The cell pellets were
washed with 0.85% (w/v) salt solution followed by wash-
ing with ethyl acetate to remove rhamnolipid and other
hydrophobic content. The cell pellets were dried at 90
C
till constant weight and biomass was measured. The results
are expressed as the means of three repetitions and standard
deviation.
Residual Substrates and Rhamnolipid Yield
SAO utilization was quantified by measuring the residual
amount of SAO in the cell-free culture broth. For this,
2 mL culture broth was centrifuged and the cell-free super-
natant was extracted with hexane to remove residual oil
and subsequently extracted with ethyl acetate. The hexane
fractions were evaporated to dryness to obtain residual oil.
The ethyl acetate fractions were collected in a test tube and
evaporated to dryness at 80
C. After cooling to room tem-
perature, the residue was dissolved in 1 mL of distilled
water and the rhamnolipid content was determined by the
orcinol method (Chandrasekaran and Bemiller, 1980). A
calibration curve was prepared with different concentra-
tions of rhamnose. When rhamnose is used for building up
the calibration curve, a correction factor must be applied to
compensate for the extra mass of the lipidic portion of RL.
Deziel et al. (2000) calculated a correction factor of 2.25,
the same factor was considered for determination of the
rhamnolipid concentration. Each analysis was performed in
triplicate to obtain statistically reliable results. From the
calibration graph, rhamnolipid concentration of unknown
samples was determined.
Recovery of Rhamnolipid
Culture broth was centrifuged at 8000 × g for 20 min, to
separate the cells from broth. The cell-free broth was acidi-
fied with 2 N H2SO4 to pH 2 for precipitation of the bio-
surfactant. After acidification, the broth was first extracted
with hexane to remove the unused lipid substrate, followed
by ethyl acetate to extract rhamnolipid from broth. The sol-
vent was evaporated to obtain crude rhamnolipid as viscous
sticky brownish liquid (Abdel-Mawgoud et al., 2009). Fur-
ther crude rhamnolipid was purified by column chromatog-
raphy. The column was loaded with activated silica gel to
get a column height of 20 cm using chloroform. The col-
umn was washed and equilibrated with chloroform. The
crude product was redissolved in chloroform and applied to
a chromatography column. Different solvent ratios of chlo-
roform and methanol were used to elute the rhamnolipid
(George and Jayachandran, 2012). Fractions of 10 mL elu-
ent were collected to analyze the separation. The elution
was monitored with TLC analysis.
Characterization of Rhamnolipid
The crude product was characterized using thin layer chro-
matography in the presence of rhamnolipid congeners. The
sample was dissolved in chloroform and 50 μL samples
were applied to precoated silica gel plates (Merck DC Kie-
selgel 60 F254). The plates were developed in a previously
saturated solvent chamber containing chloroform: metha-
nol: water (65:15:2, v/v) as the solvent system (Sim et al.,
1997). After development, the air-dried plates were evenly
sprayed with orcinol reagent and dried in the oven at 110
C for 10 min. The presence of the rhamnolipid sample
was observed and compared with the previously reported
literature.
Fourier Transformed Infrared Spectroscopy (FTIR)
FTIR spectra of purified rhamnolipid were recorded on Shi-
madzu 8000 Miracle 10 equipped with attenuated total
reflection accessory. The confirmation of rhamnolipid spec-
tra was carried out using standard procedures described in
our earlier paper (Jadhav and Pratap, 2017).
1
H NMR Spectroscopy
1
H NMR spectra of purified rhamnolipid were recorded
using a 400 MHz Nuclear Magnetic Resonance (NMR)
spectrometer (Agilent Technologies). Samples were dis-
solved in 1 mL of solvent (CDCL3) and transferred to a
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4. NMR tube. The NMR spectra were recorded as chemical
shifts expressed in parts per million with respect to tetra-
methylsilane as an internal standard reference.
HPLC
HPLC analysis of rhamnolipid was performed on an Agi-
lent 1200 series module equipped with an autoinjector, an
ultraviolet (UV) variable-wavelength detector and Chem-
Station software. Reverse phase C8 (5 mm, 4.6 × 250 mm)
column (Eurospher 100) with gradient mobile phase com-
prising acetonitrile and water (30:70, v/v) was used for sep-
aration. The gradient system was as follows: 30%
acetonitrile for 5 min, 30–100% acetonitrile for 40 min,
100% acetonitrile for 6 min, and 100–30% acetonitrile for
3 min. The partially purified product was dissolved in ace-
tonitrile to achieve the concentration of 10 mg mL−1
and
injected on column. The solvent flow rate was maintained
at 1 mL min−1
and the elution was monitored at a fixed
wavelength of 225 nm (Jadhav et al., 2018).
Surface Activity Measurements
The surface tension (SFT) reduction potential of rhamnoli-
pid was determined using a Krüss K 100 tensiometer
(KRÜSS GmbH, Germany) by the Whilmey plate method.
The rhamnolipid solution of 0.1% (w/v) concentration was
prepared by dissolving crude rhamnolipid in distilled water
at pH 7. The interfacial tension (IFT) was measured against
n-heptane. The critical micelle concentration (CMC) was
measured as the surface tension at different concentrations
of biosurfactant. All measurements were performed at 25
C. The results are expressed as the means of three
repetitions.
Emulsifying Activity
The emulsifying activity of the rhamnolipid biosurfactant
was determined against various hydrocarbons and oils such
as benzene, xylene, kerosene, soybean oil, and sunflower
oil. For this, equal volume of surfactant solution (0.1%
w/v) and hydrophobic phases were mixed in test tubes
using vortex for 2 min. After mixing, the solutions were
allowed to stand for 24 h at 25
C. The measurements were
performed in triplicate. Emulsification activity was deter-
mined as the percentage of the total height of emulsion
after 24 h (Camacho-Chab et al., 2013).
Emulsification index ¼
Emulsion height
Total height
× 100 ð1Þ
Preparation of Liquid Detergent Formulations
Liquid detergent formulations were prepared using alpha ole-
fin sulfonate (AOS), sodium lauryl ether sulfate (SLES), and
rhamnolipid. Different concentrations of surfactant along with
sorbitol, urea, ethylenediaminetetraacetic acid (EDTA), and
distilled water were stirred at room temperature for 30 min to
obtain a clear solution of liquid detergent. The composition of
detergent formulations is listed in Table 1. SLES and AOS
are synthetic surfactants widely used in the detergent industry.
The feedstock for production of SLES is a fatty alcohol
whereas AOS is a petroleum product. Rhamnolipid and SLES
are considered as sustainable surfactants based on natural raw
materials compared to AOS. Hence, SLES is more sustainable
than AOS. Therefore, the formulation was optimized contain-
ing the minimum amount of AOS and more amounts of SLES
and RL to develop a greener product. To compare the effec-
tiveness of detergent formulation, foaming and cleaning effi-
ciency was measured using the Ross miles apparatus (Ross
and Miles, 1941) and the detergency test, respectively. The
analysis was performed as three independent replicates.
Detergency Tests
Coconut oil (35.8 g), carbon black (28.4 g), lauric acid (17.9 g),
and mineral oil (17.9 g) were mixed using a mortar and pestle
to form a thick paste. Artificial soil solution was prepared by
adding 2 g of this paste to 500 mL of carbon tetrachloride and
used for soiling of cloths. White cotton and polyester fabrics
(10 × 10 cm) were immersed in this soil solution for 10 min
(Chiplunkar et al., 2017). The soiled fabrics were then dried
overnight in a drying oven (temperature 50
C).
The washing was done using a Terg-O-Tometer
(Wadegati Labequip Private Limited) equipped with a con-
trolled temperature bath system as follows: speed,
100 rpm; water hardness, 250 ppm; washing detergent
solution, 1000 mL; washing time, 15 min; rinsing time,
10 min; temperature, 30
C; each detergent formulation
with 2% concentrations was used for washing. Soil removal
Table 1 Composition of liquid detergent formulation (%wt.)
Ingredients LDa
RLD1 RLD2 RLD3 RLD4 RLD5
AOS 8 7 6 5 4 3
SLES 4 4 4 4 4 4
Rhamnolipid 0 1 2 3 4 5
Sorbitol 10 10 10 10 10 10
Urea 1 1 1 1 1 1
EDTA 0.1 0.1 0.1 0.1 0.1 0.1
Water 76.9 76.9 76.9 76.9 76.9 76.9
a
LD stands for liquid detergent formulation without rhamnolipid.
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5. from the washed fabrics was determined by reflectance
measurement (Premier Colorscan Instrument). After wash-
ing, the detergency (%) was calculated using the formula:
Detergency% ¼ Rw−Rs
ð Þ × 100= Ro−Rs
ð Þ ð2Þ
where Rw, Rs, and Ro are the reflectance measured on
washed fabrics, soiled fabrics (before washing), and
unsoiled fabrics, respectively. Each measurement was per-
formed in triplicate.
Results and Discussion
Analysis of Substrates
Physicochemical properties of SAO were analyzed as per the
AOCS official methods. In general, hydrophobic substrates
comprising a fatty acid chain length up to 18 carbons is neces-
sary to yield rhamnolipid with good productivity (Zhang et al.,
2014). The physicochemical properties of SAO are shown in
Table 2. The moderate iodine value indicates the presence of
unsaturation in the SAO. In addition, SAO was found to con-
tain a higher amount of unesterified fatty acids due to a high
acid value. The gas chromatography analysis reveals that,
SAO contains both saturated (16:0, 18:0) and unsaturated
(18:1, 18:2) fatty acids. Furthermore, SAO also composed of
oleic acid as the major fatty acid component. Considering the
fatty acid profile and the presence of unsaturation, SAO was
selected as a carbon substrate for production of rhamnolipid.
Rhamnolipid Production by P. aeruginosa at Various
Concentrations of SAO
Substrate concentration plays a vital role in the cell growth
and the following stage in the production of rhamnolipid.
P. aeruginosa can utilize both hydrophilic as well as hydro-
phobic substrates to produce rhamnolipid, although the
water-immiscible substrates give higher production com-
pared to the water-soluble substrate (Sim et al., 1997;
Wadekar et al., 2011a).
In the present study, we studied different concentrations
of the SAO to optimize cell growth and rhamnolipid pro-
duction. The cellular growth with different patterns of pro-
ductivity at varying concentrations of the SAO is shown in
Fig. 1. An increase in the SAO concentration up to
60 g L−1
resulted in a significant increase in rhamnolipid
production with a notable increase in biomass. Rhamnoli-
pid yield (Yp/s) using SAO as a carbon source was around
0.092 g g−1
with 30 g L−1
of the initial SAO concentration
at 96 h, with an increase in (Yp/s) to 0.119 g g−1
when
60 g L−1
SAO was initially present. Whereas a further
increase in the SAO concentration did not affect the rham-
nolipid productivity and substrate consumption was also
reduced.
The ability of P. aeruginosa to consume substrates
depends on direct cell-substrate contact. During bacterial
growth on hydrophobic substrate, the forces interfering
with direct cell-substrate contact need to be overcome by
cell surface adaption. In the case of P. aeruginosa (gram-
negative bacteria), the cell-substrate contact is dependent
on the outer membrane of cell and is also affected by cell
culture conditions, like temperature, pH, and nutrient avail-
ability. Hence, bacterial adaption of the outer membrane is
crucial in attachment and utilization of hydrophobic sub-
strates (Norman et al., 2002). Talaiekhozani et al. (2015)
have studied the reaction rate for production of rhamnolipid
using crude oil and observed an increase in the reaction rate
with an increase in the oil concentration at a low concentra-
tion of crude oil (below 1000 g m−3
). Whereas, at high
concentration of oil, bacterial growth slows down due to
low aqueous solubility of oil. Also researchers reported
rhamnolipid production (Abalos et al., 2001; Haba et al.,
Table 2 Physicochemical properties of sunflower acid oil
Properties AOCS method
of analysis
(Firestone 1994)
Sunflower
acid oil
Acid value (mg KOH g−1
) Te-la-64 128.5 0.50
Saponification value
(mg KOH g−1
)
Tl-la-64 178.8 0.60
Iodine value (mg I2 g−1
) Tg-la-64 120.1 0.40
Unsaponifiable matter (%) Tk la-64 1.8 0.09
Peroxide value (mEq kg−1
) Cd-8-53 13.4 0.25
Unesterified fatty acids (%
as oleic acid)
Ca-5a-40 63.2 0.23
Viscosity (cP) at 28
C Ja-10-87 45.4 0.40
Specific gravity (28
C)
(g cm−3
)
0.93 0.07
Data is expressed as mean standard deviation and represents mean
value of three replicates.
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
0
0.02
0.04
0.06
0.08
0.1
0.12
0.14
30 40 50 60 70 80
Cell
growth
(g
L
-1
)
Yp/s
(g
g
-1
)
Sunflower acid oil (g L-1
) as a substrate
Fig. 1 Cell growth ( ) and rhamnolipid production ( ) at different
concentrations of sunflower acid oil by Pseudomonas aeruginosa
MTCC 2453 at 30
C and 220 rpm in 96 h. Error bars represent the
standard deviation of the mean
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6. 2000; Pratap et al., 2011) using different concentrations of
hydrophobic compounds in the range of 30–50 g L−1
.
Moreover, Ramirez et al. (2016) have mentioned
100 g L−1
concentration of olive mill waste for rhamnoli-
pid production. Thus, the substrate concentration depends
on its fatty acid composition, phytotoxic, and other chemi-
cal constituents. Indeed, upon increasing the SAO concen-
tration, the reduction in yield may be due to the substrate
inhibitory effect (Eswari et al., 2016).
However, few studies have reported the effect of the vis-
cous nature of the fermentation broth on microbial growth
and productivity. An increase in the concentration of
hydrophobic substrates causes an increase in broth viscos-
ity, a decrease in dissolved oxygen, and product inhibition
in the fermentation medium (Zhang et al., 2017). In case of
SAO, increasing the concentration from 60 to 80 g L−1
leads to a decrease in biomass due to oxygen limitation
hence reducing the productivity. It is reported that the pres-
ence of two separate phases significantly reduces the oxy-
gen mass transfer coefficient due to resistance of mass
transfer at the air/liquid interface increasing the viscosity of
the medium resulting in oxygen limitation and nonhomo-
geneity of broth (Dolman et al., 2017). The inhibitory effect
was ascribed to problem linked to the difficulty of the
microbe to gain access to the nutrients at a high concentra-
tion of hydrophobic substrates in the batch mode. Addition
of an excessive amount of hydrophobic substrate led to an
increase in viscosity of the culture broth due to accumula-
tion of oil; thus restricting the cells from converting the
substrate into product showing the product inhibition effect
(Chen et al., 2007). This indicates that SAO can be utilized
as a carbon source at a concentration of 60 g L−1
for
microbial growth and maximum RL production without
any pretreatment.
Rhamnolipid Production by SAO
The ability of SAO to produce rhamnolipids is highly sig-
nificant as SAO did not diminish the cell growth. The pro-
duction profile for optimized substrate concentration was
further studied in shake flasks as well as in a 5 L bioreac-
tor. Use of SAO from oil-refining industries as a sole car-
bon source had a dramatic effect on cell growth and
rhamnolipid productivity. The data of rhamnolipid yield
(g L−1
), cell growth (g L−1
), and substrate utilization
(g L−1
) are shown in Fig. 2a. The SAO concentration
dropped significantly from 60 to 35.6 g L−1
and rhamnoli-
pid yield of 3.5 g L−1
was achieved at the shake flask
level.
In addition, rhamnolipid production was also performed
on the lab scale bioreactor to achieve better productivity
with oxygenation (Kronemberger et al., 2008). Because
rhamnolipid production is an aerobic bioprocess, aeration
condition affects the cell growth as well as secondary
metabolite production. Transfer of process from the shake
flask level to the fermenter level augmented cell growth
and rhamnolipid yield. The cell culture in the bioreactor
achieved higher growth compared to the shake flask as the
stationary phase in the bioreactor is accomplished in 48 h,
whereas the shake flask takes 72 h to reach the stationary
phase (Fig. 2b). Controlled aeration and pH in the bioreac-
tor exceed the level of rhamnolipid production to 4.9 g L−1
by using SAO. Volumetric productivity in the bioreactor
was about 0.051 g L−1
h−1
with 60 g L−1
initial SAO con-
centration at 96 h.
The data of product yield based on the initial substrate
concentration (Yp/si, g g−1
), substrate consumption (Yp/s,
g g−1
), and biomass production (Yp/x, g g−1
) along with
productivity (g L−1
h−1
) at shake flask and fermenter levels
are presented in Table 3. An increase in overall yield was
due to higher biomass produced at the bioreactor level
under controlled pH and aeration.
0
0.5
1
1.5
2
2.5
3
3.5
4
0
10
20
30
40
50
60
70
(a)
(b)
Cell
growth
(g
L
–1
)
and
yield
(g
L
–1
)
Cell
growth
(g
L
–1
)
and
yield
(g
L
–1
)
0
1
2
3
4
5
6
0
10
20
30
40
50
60
70
0 24 48 72 96
Time (h)
0 24 48 72 96
Time (h)
Sunflower
acid
oil
(g
L
–1
)
Sunflower
acid
oil
(g
L
–1
)
Fig. 2 Time course of cell growth ( ), yield ( ), and sunflower acid
oil consumption ( ) by Pseudomonas aeruginosa MTCC 2453. (a)
Fermentation in 250 mL shake flask with 50 mL working volume
using 60 g L−1
sunflower acid oil as carbon source with initial
medium pH 6.5 at 30
C and 220 rpm for 96 h. (b) Fermentation in a
5 L bioreactor with 3 L working volume using 60 g L−1
sunflower
acid oil as carbon source at 30
C and 450 rpm for 96 h, controlled
pH 6.5 during fermentation with aeration 1 vvm. Error bars represent
the standard deviation of the mean
468 J Surfact Deterg
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7. Some previously reported rhamnolipid yields obtained
by different microbial strain using different industrial sub-
strate as feedstock is summarized in Table 4. The product
yield based on substrate utilization (yield g g−1
substrate)
and volumetric productivity is compared along with the
previously available data. Volumetric productivity was cal-
culated based on the time required to achieve the maximum
rhamnolipid yield. The rhamnolipid yield in this study was
lower compared with those obtained in other studies using
different waste materials as substrates and different micro-
bial strains. Avoiding the variation in microbial strain, the
rhamnolipid yield was comparable to that of sugarcane
molasses and higher than that of waste frying oil, glycerol
residue, and olive mill waste. In our study, a hydrophobic
substrate such as SAO using pseudomonas species yields
maximum 4.9 g L−1
RL, whereas the same strain gives
lower yield when hydrophilic sources are used as carbon
substrates (Wadekar et al., 2011a). The long alkyl chain of
hydrophobic substrates compared to hydrophilic sources
that supply carbon sources to the cells is the key attribute
of higher production yield (Singh et al., 2013). In addition,
rhamnolipid production by different microbial strains can
be increased by increasing water availability of water-
immiscible substrates through emulsification. The variation
in yield proves that microbial strains and substrates play a
crucial role in biosurfactant production.
Fatty Acid Composition of Residual Oil
The fatty acid composition of residual SAO was analyzed
using gas chromatography (Table 5). In rhamnolipid
Table 3 Comparison of rhamnolipid yield produced in a shake flask
and at a 5 L bioreactor level
Yielda
Shake flask 5 L bioreactor
Yp/si 0.05 0.08
Yp/s 0.14 0.15
Yp/x 1.86 2.35
P 0.03 0.05
Biomass (g L−1
) 1.88 2.08
a
Yp/si, g yield per g initial substrate concentration; Yp/s, g yield per
g substrate consumed; Yp/x, g yield per g biomass; P, productivity
(g L-1
h-1
).
Table 4 Rhamnolipids produced on various waste materials as carbon substrates
Substrate Microbial
strain
Fermentation
scale
RL
max (g L−1
)
Yield
(g g−1
)
Yield*
(g g−1
substrate)
Productivity
(g L−1
h−1
)
Source
Glycerol residue (5%) P. aeruginosa
ATCC10145
Shake flask 2.5 N/P 0.05 0.026 Wadekar
et al. (2011a)
Soybean oil refinery waste
(5%)
P. aeruginosa
AT10
Shake flask 9.5 N/P 0.19 0.098 Abalos
et al. (2001)
Waste frying oil (4%) P. aeruginosa
47 T2 NCIB
40044
Shake flask 2.7 N/P 0.34 0.033 Haba
et al. (2000)
Soybean oil soapstock (2%) P. aeruginosa
LBI
Shake flask 11.7 N/P 0.585 0.081 Nitschke
et al. (2010)
Molasses (7%) P. aeruginosa
GS3
Shake flask 0.24 N/P 0.003 0.002 Patel and Desai
(1997)
Orange fruit peeling (3%) P. aeruginosa
MTCC 2297
Shake flask 9.18 N/P 0.306 0.095 George and
Jayachandran
(2009)
Sunlower oil soapstock
(3%)
P. aeruginosa
LBI
Shake flask 7.3 0.22 — 0.104 Benincasa and
Accorsini
(2008)
Olive mill waste (2%) P. aeruginosa Shake flask 0.012 0.013 0.0006 0.00008 Ramirez
et al. (2016)
Olive mill waste (5%) 0.03 0.018 0.0006 0.0002
Olive mill waste (10%) 0.2 0.058 0.002 0.001
Sugarcane molasses and
corn steep liquor (10%)
P. aeruginosa
112
Shake flask 3.19 N/P 0.032 0.022 Gudina
et al. (2016)
Bioreactor 2.23 N/P 0.022 0.023
Sunflower acid oil (6%) P. aeruginosa
(ATCC
10145)
Shake flask 3.5 0.1434 0.058 0.036 This study
Bioreactor 4.9 0.1556 0.081 0.051
N/P, not provided in the reference.
Yield (g g − 1) is the product yield based on the substrate consumed.
Yield* (g g − 1) is the product yield based on the initial substrate fed.
469
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8. production using SAO, the microbe consumes more lino-
leic acid (77.5%) compared to other fatty acids. It has been
reported that linoleic acid was the most favorable source
with higher percentage utilization (Nitschke et al., 2005;
Pratap et al., 2011). After linoleic acid, oleic acid (72.3%)
was the second preferential source consumed by the
microbe followed by palmitic acid (7.69%). Rhamnolipids
are typically produced on triacylglycerols. The maximum
utilization of linoleic and oleic acids indicates that C18 fatty
acids are also preferred by bacteria in the synthesis of
rhamnolipid. On the other hand, some unknown peaks were
also detected in gas chromatography (GC), which need to
be studied further. These peaks may be due to the degrada-
tion fractions of fatty acids. The preference for fatty acid
also depends on the type of substrate used. In particular,
the fatty acid consumption depends on the differences in
triacylglycerol composition and the specificity of the bacte-
rial lipase. When corn oil refinery waste was used as sub-
strate, palmitic acid was the preferential fatty acid. Whereas
in the case of soybean oil refinery waste, linolenic acid con-
sumption was higher followed by linoleic acid, palmitic
acid, and oleic acid (Nitschke et al., 2005). Pratap
et al. (2011) also reported a similar trend for fatty acid con-
sumption when sunflower oil was used as a carbon
substrate.
SAO is acidulated SS, consisting of unesterified fatty
acids along with other impurities like mineral acids, triacyl-
glycerol, phospholipids, and sterols (Chiplunkar et al.,
2017). Whereas original vegetable oils (soybean oil, sun-
flower oil, and corn oil) that are used as carbon sources for
efficient production of rhamnolipid do not contain such
components. Additionally, SAO also contains some
amounts of peroxides (Table 2). Wadekar et al. (2012)
reported the effect of peroxides present in waste frying oil
on production of rhamnolipid and found improved forma-
tion of rhamnolipids by reducing the peroxide value from
102 to 8 mEq kg−1
. In present study, the peroxide content
in SAO is 13.4 mEq kg−1
, which is much less and does not
interfere in microbial metabolism. The study proves the use
of SAO as a newer hydrophobic feedstock for production
of rhamnolipid.
Structural Characterization of Rhamnolipid Produced
on SAO
The biosurfactant formed was characterized using thin layer
chromatography to confirm the presence of product rham-
nolipid. The TLC analysis of crude product reveals the
presence of different components based on the Rf value.
The spot at lower Rf value (0.2) corresponds to dirhamnoli-
pids while a major spot at higher Rf value 0.51 and 0.4
shows the presence of monorhamnolipids (Fig. 3). TLC
results signify that the isolated products comprise rhamnoli-
pid showing a similar retention factor to that mentioned in
the previous literature (Jadhav et al., 2018; Pratap
et al., 2011).
To analyze the rhamnolipid structure, the crude product
was purified using column chromatography. The structure
of purified rhamnolipid was characterized using different
analytical techniques like FTIR, HPLC, and NMR. The
FTIR spectrum of prepared rhamnolipid is shown in
Fig. S1, Supporting information. The broad peak at
3419.79 cm−1
confirms the presence of a hydroxyl moiety.
The peak at around 2927.94–2854.65 cm−1
corresponds to
aliphatic C H stretching vibration. Carbonyl C O stretch
of the ester group is at 1747.51 cm−1
. The stretch of ether
Table 5 Percentage utilization of fatty acid in sunflower acid oil dur-
ing rhamnolipid production by Pseudomonas aeruginosa (MTCC
2453) after 96 h with 60 g L−1
of initial concentration of acid oil as a
carbon source
Fatty acids
composition
(Ce-1-62)
Original
sunflower acid
oil composition
(% w/w)
Residual
sunflower acid
oil composition
(% w/w)
Utilization
(% w/w)
Palmitic
16:0
6.50 1.55 12.89 1.55 7.69
Stearic 18:0 3.50 0.98 6.89 0.98 6.66
Oleic 18:1 68.22 1.21 39.72 1.21 72.39
Linoleic
18:2
9.50 0.56 4.50 0.56 77.50
Linolenic
18:3
0.80 0.33 1.10 0.33 35.41
Other 10.50 1.55 33.21 1.25
Data represent the mean values of three independent measurements,
and are expressed as relative percentage of the total GC peak areas.
Fig. 3 Thin layer chromatography of rhamnolipid produced on sun-
flower acid oil. Stationary phase- silica gel, solvent system- chloro-
form: Methanol:Water (65:15:2, v/v), and visualizing reagent- orcinol
reagent
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9. linkage appears at 1078.21 and 1240.23 cm−1
. The stretch
of the C O bond of the carbonyl group from acid is absent
in the given spectrum hence it is concluded that rhamnolipid
could form from AO and hence show characteristic peaks.
The purified product was analyzed using HPLC.
Figure 4 shows the HPLC chromatogram with character-
istic peaks at retention times 29.91 and 35.22 min repre-
senting the presence of dirhamnolipid and
monorhamnolipid, respectively (Jadhav et al., 2018). It
has been reported in the literature that HPLC peaks at
29 and 35 min correspond to anions of dirhamnolipids
(m/z 649) and monorhamnolipids (m/z 504), respectively
(Pratap et al., 2011; Wadekar et al., 2012). According to
the fragmentation patterns mentioned by Wadekar
et al. (2012), the structural assignment of these anions
resembles the rhamnolipid structure.
The rhamnolipid structure was confirmed by 1
H NMR
analysis and the chemical shifts are presented in Table 6.
The chemicals shifts observed in the NMR spectra were in
accordance with the rhamnolipid structure and were similar
to those reported in the literature (Pratap et al., 2011; Sim
et al., 1997; Wadekar et al., 2012).
Surfactant Properties of Rhamnolipids
Rhamnolipid comprises a mixture of different homologs
such as monorhamno-monolipidic, dirhamno-monolipidic,
monorhamno-dilipidic, and dirhamno-dilipidic. The surfac-
tant property of rhamnolipid depends on the distribution of
these homologs in the crude product and it varies depend-
ing on the microbial strain, culture conditions, medium
compositions, and the carbon substrate used in fermentation
(Abalos et al., 2001; Deziel et al., 2000). The other parame-
ters affecting the surfactant properties are the presence of
residual substrates and the presence of salts in the culture
broth (Mata-Sandoval et al., 2001).
Tensiometric measurements were performed to deter-
mine the effectiveness and efficiency of crude rhamnolipid.
The produced rhamnolipid notably reduces the surface ten-
sion of water from 70.1 0.02 mN m−1
to 30.12 0.05
mN m−1
, at 0.1% RL concentration. Whereas interfacial
tension against n-heptane was about 0.52 0.02 mN m−1
at the same concentration. The reduction in surface tension
(SFT) and interfacial tension (IFT) indicates the presence
of the surface-active molecule. The crude biosurfactant is a
viscous oily liquid soluble in water at pH 4 with optimum
solubility at pH 7–7.5 (Abdel-Mawgoud et al., 2009). Sim-
ilar results of SFT for rhamnolipid solution are mentioned
in the literature (Abalos et al., 2001; Benincasa and Accor-
sini, 2008). Many authors reported the surface tension of
rhamnolipid up to 26–31 mN m−1
and interfacial tension
between 0.5–2 mN m−1
depending on the different carbon
substrates used (Benincasa and Accorsini, 2008; Nitschke
Fig. 4 HPLC chromatogram of rhamnolipid produced on sunflower acid oil
Table 6 1
H NMR chemical shift data for rhamnolipids produced by
P. aeruginosa using sunflower acid oil
Moiety Proton location Chemical
shift (ppm)
Rhamnose CH O C 5.27 d
OH group 4.81 s
CH OH 3.63 m
CH3 1.18, 1.20 d
Hydroxy fatty
acid
(CH2) CH( O C O)
CH2COO
4.13 m
(CH2) CH(O Rha)
CH2COO
3.39 m
CH(O) CH2COO 2.59 m
(CH2) CH(O) CH2COO 1.56 m
(CH2)5 1.29 m
CH3 0.88 t
t, triplet; s, singlet; m, multiplet; d, doublet.
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10. et al., 2010; Wadekar et al., 2011b). The rapid decrease in
surface tension of water with increasing rhamnolipid con-
centration is shown in Fig. 5. It indicates that rhamnolipid
solution reduces the surface tension up to minimum of 29.5
mN m−1
until the critical micelle concentration was
attained. From the intercept of two straight lines of the sur-
face tension curve, the critical micelle concentration was
estimated to be about 70.109 0.5 mg L−1
. Benincasa and
Accorsini (2008) reported a critical micelle concentration
(CMC) of 120 mg L−1
for rhamnolipid synthesized from
sunflower oil refinery waste, which is higher than the CMC
value reported herein. However, Wadekar et al. (2012)
found a comparatively lower CMC of 39 mg L−1
for crude
rhamnolipid synthesized using waste frying oil. This dis-
crepancy could be due to variation in composition and dis-
tribution of homologous molecules in rhamnolipid
(Nitschke et al., 2010).
The emulsifying ability is one of the most important
properties for application of surfactant in laundry detergent.
The emulsification degree of produced rhamnolipid was
analyzed in comparison with nonionic chemical surfactant
polysorbate 20. The emulsification index of rhamnolipid
and polysorbate 20 against different hydrocarbons is shown
in Fig. 6. It revealed that rhamnolipids and polysorbate
20 had almost equivalent ability to emulsify two immisci-
ble phases. The emulsification index for hydrocarbons like
kerosene was 51.3% and for mineral oil it was 65.2%,
whereas polysorbate 20 has a slightly lower emulsification
index for the same hydrocarbons. A similar tendency of
rhamnolipid with lesser emulsification indices for kerosene
and other hydrocarbons was reported previously
(Benincasa and Accorsini, 2008; Nitschke et al., 2010;
Sifour et al., 2007). Comparatively, rhamnolipid was more
effective than polysorbte 20 to emulsify vegetable oils, as
the emulsions formed with vegetable oil were more stable
than that of other hydrocarbons evaluated. Patel and Desai
(1997) also observed a better emulsion stability with vege-
table oil using rhamnolipid by P. aeruginosa from molas-
ses. The excellent emulsification properties of rhamnolipids
seemed to be useful for hydrocarbon recovery as well as
for biodegradation of hydrocarbons (Cheng et al., 2017).
The higher emulsifying index of a rhamnolipid solution
with vegetable oils demonstrates its potential for the deter-
gent, pharmaceutical, cosmetic, and food industry applica-
tions. As a result of high emulsification index, the
rhamnolipid has shown favorable results in removal of oily
stain from cotton cloth (Bafghi and Fazaelipoor, 2012).
Nitschke et al. (2005) demonstrated variations in SFT, IFT,
and CMC, as well as the emulsification index of rhamnoli-
pid when different carbon substrates were used for produc-
tion of rhamnolipid under the same fermentation
conditions. The variations observed in the surface-active
properties of biosurfactants obtained from the oil wastes
are probably due to differences in individual homologous
concentrations in the crude product.
Rhamnolipid-Based Liquid Detergent
Based on the superior emulsification properties, synthe-
sized rhamnolipid was incorporated in liquid detergent for-
mulation. Liquid detergents with different combinations of
AOS and SLES were formulated and evaluated for their
performance properties like foaming and detergency. AOS
and SLES are widely used in detergents as foaming and
cleaning agents to remove dirt from fabrics. As rhamnolipid
is a low-foaming surfactant, AOS and SLES are used in
combination to enhance the foaming as well as cleaning
performance. EDTA was used as a water softener and pre-
servative. Urea was used as a hydrotrope to prevent gel for-
mation in the liquid detergent formulation.
Liquid detergent formulations were evaluated for foam-
ability. The foam volume was measured in the Ross-Miles
apparatus with respect to time. All formulations containing
rhamnolipid exhibited low-foaming properties compared to
formulation (LD) containing only AOS and SLES
(Table 7). Although the foam volume was less but the foam
stability was significant for rhamnolipid-containing
25
30
35
40
45
50
55
60
65
70
75
0 50 100 150 200 250
Surface
tension
(mN
m
–1
)
SFT (mN m–1
)
Concentration (mg L–1
)
Fig. 5 CMC and minimum surface tension reduction by rhamnolipid
produced using sunflower acid oil
0
10
20
30
40
50
60
70
80
90
100
Benzene Sunflower
oil
xylene Kerosene Mineral oil Soyabean
oil
Emulsification
index
(%)
Hydrocarbons
Rhamnolipid Polysorbate 20
Fig. 6 Emulsification index (EI) of the rhamnolipid and polysorbate
20 with various hydrocarbons after 24 h. error bars represent the stan-
dard deviation of the mean
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11. formulations. When SLES was replaced by rhamnolipid,
the foamability was reduced but the stability of foam was
not affected. The foam stabilization of detergent formula-
tion was due to rhamnolipid molecules. The hydrogen
bonding created by rhamnose molecules and carboxyl
groups enhanced closer packing of surfactant molecules
increasing the film viscosity at the air interface. As a result,
rhamnolipid shows less foaming but it helps to stabilize the
foam (Wadekar et al., 2011b).
The cleansing action of detergent is due to emulsification
and micelle formation. Rhamnolipid exhibited a higher
emulsifying index with vegetable oils, therefore rhamnoli-
pid can enhance the cleaning power of detergent. Percent
detergency of nonrhamnolipid-containing (LD) formulation
was the highest as compared to other formulations
(Table 7). Whereas the rhamnolipid-containing formula-
tions show potential to remove oily stains with a similar
cleaning efficiency for cotton as well as polyester fabric.
Although the LD formulation shows highest detergency, its
high foaming property makes it unacceptable for machine
wash laundry applications. As high amount of suds limits
the cleaning efficiency in washing machine. The formula-
tions RLD4 and RLD5 showed a better detergency effect
with moderate foam compared to other formulations.
Accordingly, liquid detergent formulations containing
rhamnolipid are suitable for machine wash laundry applica-
tion as well as for hand wash laundry. The liquid detergents
formulated by using rhamnolipid biosurfactants are biode-
gradable and environmentally friendly. Additionally, less-
foaming detergents provide a better cleaning efficiency
with a low water usage. The conventional detergent formu-
lations contain petroleum-based products like linear alkyl
benzene sulfonate as an active ingredient and a silicone-
based antifoaming agent. The antifoaming compounds are
effective at specific concentration, below which they are
less effective and at higher concentration they act as foam
stabilizers, hence the concentration is important. The
antifoaming agents are insoluble in water thus it need to be
formulated for inclusion in the liquid formulation. Further-
more, upon long-term storage and variation of storage tem-
perature, the antifoam activity may get impaired due to
migration of some antifoam active substances. It causes
accumulation of antifoam floccules at the surface of the liq-
uid detergent. The liquid detergent containing rhamnolipid
with other surfactant will not require additional antifoaming
additives to control foam. Hence, the use of rhamnolipid as
a surfactant in a liquid detergent with moderate foam will
be beneficial.
Economics of Rhamnolipids
Biosurfactant market is dramatically rising due to its biode-
gradable, specific, nontoxic, and eco-friendly properties
(Randhawa and Rahman, 2014). Although the biosurfac-
tants are efficient, they have a serious limitation in commer-
cialization, mainly related to their high production cost.
The industrial utilization of biosurfactant depends on its
economic production. Many pharmaceutical and food
industries are currently keen to substitute synthetic surfac-
tants with biosurfactants like rhamnolipid. The high pro-
duction cost is a major drawback in the commercialization
of the rhamnolipid biosurfactants. Cost reduction of biosur-
factants prompted research to utilize industrial waste and
by-product as raw materials. The economics of rhamnolipid
production is complicated but favorable owing to demand
of sustainable and environmentally friendly (green) chemi-
cals. The raw material value involved in the production of
rhamnolipid production should be studied to estimate the
effects of varying feedstock prices and rhamnolipid yield.
The raw material cost includes the cost of medium compo-
nents and the cost of substrates. The cost of various sub-
strates for rhamnolipid production based on substrate
conversion rate is presented in Table 8. Maximum rhamno-
lipid yields were calculated using substrate concentration as
Table 7 Performance properties of liquid detergent formulation at 0.1% concentration of detergent in water
Samples Foam height (cm) Detergency (%)
0 min 5 min 10 min 15 min 20 min Cotton
fabric
Polyester
fabric
Rhamnolipid based liquid
detergent
RLD1 15.2 0.35 14.2 0.35 14.2 0.35 14.2 0.35 13.2 0.35 63.4 0.56 65.1 0.49
RLD2 14.5 0.70 14.2 0.35 13.2 0.35 13.5 0.70 13.5 0.70 64.3 0.63 65.5 0.56
RLD3 12.2 0.35 12.5 0.70 12.5 0.70 11.2 0.35 11.5 0.70 65.4 0.70 67.9 1.20
RLD4 10.2 0.35 10.5 0.70 9.2 0.35 9.5 0.70 9.2 0.35 67.7 0.91 69.4 0.70
RLD5 9.5 0.70 9.2 0.35 8.5 0.70 8.5 0.70 8.5 0.70 68.3 0.35 70.2 0.55
RLD6 8.5 0.70 8.2 0.35 8.2 0.35 7.5 0.70 7.5 0.70 61.2 0.42 64.2 0.42
Liquid detergent without
rhamnolipid
LD 25.5 0.70 24.2 0.35 24.5 0.70 23.2 0.35 22.2 0.35 70.2 0.28 72.1 0.56
Results are expressed as the means of three repetitions standard deviation.
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12. indicated in references assuming no by-products and the
absence of other limiting factors (Chen et al., 2007; Mar-
sudi et al., 2008; Patel and Desai, 1997; Radzuan et al.,
2017; Wadekar et al., 2012). The low-cost substrates for
rhamnolipid production are glucose, palm oil, and palm
fatty distillate, but the conversion rate is less for these sub-
strates. Compared with other substrate cost, molasses con-
taining sugar are the low-priced substrates for rhamnolipid
production. Apart from sugar-containing waste, SAO used
in the present study with the lowest price of 232.26 ₹ kg−1
rhamnolipid may compensate the high production cost of
rhamnolipid. Hence oil refinery waste serves as a signifi-
cant feedstock for rhamnolipid production. Rhamnolipid
production from industrial waste is economical but has not
been reported in detail. As discussed, the use of SAO as an
inexpensive substrate will dramatically reduce the cost of
rhamnolipid.
Conclusions
The vegetable oil-refining process generates a great amount
of waste as SS, AO, and acidic waste water. The utilization
of waste AO to produce a biosurfactant with commercial
value will help to minimize waste treatment cost in the oil
industry with economical rhamnolipid production. SAO,
waste from oil-refining industries, was successfully
employed as a feedstock for production of rhamnolipid by
P. aeruginosa. The concentration of SAO was optimized to
find out the optimal concentration for the maximum pro-
duction (4.9 g L−1
) of rhamnolipid. Thus AO is efficiently
used for rhamnolipid production, which can reduce the
substrate cost. As AO is waste from oil refinery industries,
it is available at a cheaper rate compared to vegetable oils
that are used for rhamnolipid production without any pre-
treatment. The main emphasis of this study is to increase
the rhamnolipid production by reducing the cost. This
study looks at the future perspectives of large-scale profit-
able production of biosurfactants by using oil refinery
waste. Additionally, the synthesized product exhibited a
very low CMC of 70.109 mg L−1
. The synthesized rham-
nolipid shows excellent emulsification properties, hence
would be possibly suitable for applications in detergents,
pharmaceuticals, and cosmetic industry. The rhamnolipid-
based liquid detergents, RLD4 and RLD5, showed moder-
ate foaming properties with a better detergency compared
to other formulations. As a result of low foaming attributes,
rhamnolipid-based liquid detergents can be utilized as laun-
dry detergents for washing machines.
Acknowledgements The authors are thankful to Rajiv Gandhi Sci-
ence and Technology Commission, Government of Maharashtra, for
financial support.
Conflict of Interest The authors declare that they have no conflict
of interest.
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of rhamnolipid on different carbon substrates in shake flask cultures
Substrate Raw
material
price
(₹ kg−1
)
Max. yield
(g rhamnolipid g−1
substrate)
Substrate cost
(₹ kg−1
rhamnolipid)
Sunflower
oil
66.5† 0.18 369.44
Glucose 32* 0.06 533.33
Palm oil 25** 0.061 409.84
Glycerol 37* 0.09 411.11
Oleic acid 70.5† 0.009 7833.33
Palm fatty
acid
distillate
21.6†† 0.019 3789.47
Molasses 25* 0.24 104.17
Sunflower
acid oil
36* 0.155 232.26
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