J Surfact Deterg (2019)

DOI 10.1002/jsde.12255

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).

Supporting information Additional supporting information may be Introduction

found online in the Supporting Information section at the end of the
article.
Surfactants are organic molecules that orient themselves at
interfaces and reduce the interfacial tension. Their surface-
* Amit P. Pratap active properties such as foaming, detergency, emulsifica-
amitpratap0101@rediffmail.com
tion, and wettability make them prominent molecules in
1
Department of Oils, Oleochemicals and Surfactants Technology, many fields (Desai and Banat, 1997). Biosurfactants are
Institute of Chemical Technology (University under Section 3 of amphiphilic molecules produced by microbial cells. Grow-
UGC Act 1956; Formerly UDCT/ UICT), Nathalal Parekh Marg, ing environmental concern and increasing consumer aware-
Matunga (East), Mumbai, 400 019, India
ness associated with the use of bio-based products are
2
Department of Chemistry, K. J. Somaiya College of Science and anticipated to be a key driver for the growth of utilization
Commerce, Vidyavihar, Mumbai, 400 077, India
of biosurfactants in industrial and household applications.
3
DBT–ICT Centre for Energy Biosciences, Department of The most known biosurfactants are glycolipids. The
Chemical Engineering, Institute of Chemical Technology
common way to produce a glycolipid biosurfactant is by
(University under Section 3 of UGC Act 1956; Formerly UDCT/
UICT), Nathalal Parekh Marg, Matunga (East), Mumbai, 400 fermentation of vegetable oils, particularly soybean oil,
019, India corn oil, coconut oil, sunflower oil, and olive oil with

J Surfact Deterg (2019)


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
J Surfact Deterg
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
J Surfact Deterg (2019)
J Surfact Deterg
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
J Surfact Deterg (2019)
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
 J Surfact Deterg

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).
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
Surface Activity Measurements
Coconut oil (35.8 g), carbon black (28.4 g), lauric acid (17.9 g),
The surface tension (SFT) reduction potential of rhamnoli-
and mineral oil (17.9 g) were mixed using a mortar and pestle
pid was determined using a Krüss K 100 tensiometer
to form a thick paste. Artificial soil solution was prepared by
(KRÜSS GmbH, Germany) by the Whilmey plate method.
adding 2 g of this paste to 500 mL of carbon tetrachloride and
The rhamnolipid solution of 0.1% (w/v) concentration was
used for soiling of cloths. White cotton and polyester fabrics
prepared by dissolving crude rhamnolipid in distilled water
(10 × 10 cm) were immersed in this soil solution for 10 min
at pH 7. The interfacial tension (IFT) was measured against
(Chiplunkar et al., 2017). The soiled fabrics were then dried
n-heptane. The critical micelle concentration (CMC) was
overnight in a drying oven (temperature 50  C).
measured as the surface tension at different concentrations
The washing was done using a Terg-O-Tometer
of biosurfactant. All measurements were performed at 25
 (Wadegati Labequip Private Limited) equipped with a con-
C. The results are expressed as the means of three
trolled temperature bath system as follows: speed,
repetitions.
100 rpm; water hardness, 250 ppm; washing detergent
solution, 1000 mL; washing time, 15 min; rinsing time,
Emulsifying Activity 10 min; temperature, 30  C; each detergent formulation
with 2% concentrations was used for washing. Soil removal
The emulsifying activity of the rhamnolipid biosurfactant
was determined against various hydrocarbons and oils such Table 1 Composition of liquid detergent formulation (%wt.)
as benzene, xylene, kerosene, soybean oil, and sunflower Ingredients LDa RLD1 RLD2 RLD3 RLD4 RLD5
oil. For this, equal volume of surfactant solution (0.1%
w/v) and hydrophobic phases were mixed in test tubes AOS 8 7 6 5 4 3
using vortex for 2 min. After mixing, the solutions were SLES 4 4 4 4 4 4
allowed to stand for 24 h at 25  C. The measurements were Rhamnolipid 0 1 2 3 4 5
performed in triplicate. Emulsification activity was deter- Sorbitol 10 10 10 10 10 10
mined as the percentage of the total height of emulsion Urea 1 1 1 1 1 1
after 24 h (Camacho-Chab et al., 2013). 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
Emulsion height
Emulsification index ¼ × 100 ð1Þ a
LD stands for liquid detergent formulation without rhamnolipid.
Total height

J Surfact Deterg (2019)

J Surfact Deterg

from the washed fabrics was determined by reflectance
measurement (Premier Colorscan Instrument). After wash-
ing, the detergency (%) was calculated using the formula:
Detergency% ¼ ðRw − RsÞ × 100=ðRo− RsÞ 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.
Table 2 Physicochemical properties of sunflower acid oil
Properties AOCS method Sunflower
of analysis acid oil
(Firestone 1994)
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.,
0.14 1.6

0.12 1.4
Acid value (mg KOH g−1) Te-la-64 128.5  0.50 1.2
Cell growth (g L-1)

0.1
Saponification value Tl-la-64 178.8  0.60
Yp/s (g g-1)

1
(mg KOH g−1) 0.08
0.8
Iodine value (mg I2 g−1) Tg-la-64 120.1  0.40 0.06
0.6
Unsaponifiable matter (%) Tk la-64 1.8  0.09 0.04
0.4
Peroxide value (mEq kg−1) Cd-8-53 13.4  0.25 0.02 0.2
Unesterified fatty acids (% Ca-5a-40 63.2  0.23
0 0
as oleic acid) 30 40 50 60 70 80
Viscosity (cP) at 28  C Ja-10-87 45.4  0.40 Sunflower acid oil (g L-1) as a substrate
Specific gravity (28  C) 0.93  0.07
(g cm−3) Fig. 1 Cell growth ( ) and rhamnolipid production ( ) at different
concentrations of sunflower acid oil by Pseudomonas aeruginosa
Data is expressed as mean  standard deviation and represents mean MTCC 2453 at 30  C and 220 rpm in 96 h. Error bars represent the
value of three replicates. standard deviation of the mean

J Surfact Deterg (2019)

 J Surfact Deterg

2000; Pratap et al., 2011) using different concentrations of (a)

hydrophobic compounds in the range of 30–50 g L−1. 70 4

Cell growth (g L–1) and yield (g L–1)

Moreover, Ramirez et al. (2016) have mentioned 60 3.5

Sunflower acid oil (g L–1)

100 g L−1 concentration of olive mill waste for rhamnoli- 50
3
pid production. Thus, the substrate concentration depends 2.5
40
on its fatty acid composition, phytotoxic, and other chemi- 2
30
cal constituents. Indeed, upon increasing the SAO concen- 1.5
tration, the reduction in yield may be due to the substrate 20
1
inhibitory effect (Eswari et al., 2016). 10 0.5
However, few studies have reported the effect of the vis-
0 0
cous nature of the fermentation broth on microbial growth 0 24 48 72 96
and productivity. An increase in the concentration of Time (h)
hydrophobic substrates causes an increase in broth viscos-
ity, a decrease in dissolved oxygen, and product inhibition (b)

Cell growth (g L–1) and yield (g L–1)

70 6
in the fermentation medium (Zhang et al., 2017). In case of 60

Sunflower acid oil (g L–1)

5
SAO, increasing the concentration from 60 to 80 g L−1
50
leads to a decrease in biomass due to oxygen limitation 4
40
hence reducing the productivity. It is reported that the pres- 3
30
ence of two separate phases significantly reduces the oxy-
2
gen mass transfer coefficient due to resistance of mass 20

transfer at the air/liquid interface increasing the viscosity of 10 1

the medium resulting in oxygen limitation and nonhomo- 0 0

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
0 24 48 72 96
Time (h)
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
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.

J Surfact Deterg (2019)

J Surfact Deterg

Table 3 Comparison of rhamnolipid yield produced in a shake flask different waste materials as substrates and different micro-
and at a 5 L bioreactor level bial strains. Avoiding the variation in microbial strain, the
Yielda Shake flask 5 L bioreactor rhamnolipid yield was comparable to that of sugarcane
molasses and higher than that of waste frying oil, glycerol
Yp/si 0.05 0.08
residue, and olive mill waste. In our study, a hydrophobic
Yp/s 0.14 0.15
substrate such as SAO using pseudomonas species yields
Yp/x 1.86 2.35
maximum 4.9 g L−1 RL, whereas the same strain gives
P 0.03 0.05
lower yield when hydrophilic sources are used as carbon
Biomass (g L−1) 1.88 2.08
substrates (Wadekar et al., 2011a). The long alkyl chain of
a
Yp/si, g yield per g initial substrate concentration; Yp/s, g yield per hydrophobic substrates compared to hydrophilic sources
g substrate consumed; Yp/x, g yield per g biomass; P, productivity that supply carbon sources to the cells is the key attribute
(g L-1 h-1). 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-
Some previously reported rhamnolipid yields obtained
immiscible substrates through emulsification. The variation
by different microbial strain using different industrial sub-
in yield proves that microbial strains and substrates play a
strate as feedstock is summarized in Table 4. The product
crucial role in biosurfactant production.
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- Fatty Acid Composition of Residual Oil
culated based on the time required to achieve the maximum
rhamnolipid yield. The rhamnolipid yield in this study was The fatty acid composition of residual SAO was analyzed
lower compared with those obtained in other studies using using gas chromatography (Table 5). In rhamnolipid

Table 4 Rhamnolipids produced on various waste materials as carbon substrates

Substrate Microbial Fermentation RL Yield Yield* (g g−1 Productivity Source

−1
strain scale max (g L ) (g g−1) substrate) (g L−1 h−1)

Glycerol residue (5%) P. aeruginosa Shake flask 2.5 N/P 0.05 0.026 Wadekar
ATCC10145 et al. (2011a)
Soybean oil refinery waste P. aeruginosa Shake flask 9.5 N/P 0.19 0.098 Abalos
(5%) AT10 et al. (2001)
Waste frying oil (4%) P. aeruginosa Shake flask 2.7 N/P 0.34 0.033 Haba
47 T2 NCIB et al. (2000)
40044
Soybean oil soapstock (2%) P. aeruginosa Shake flask 11.7 N/P 0.585 0.081 Nitschke
LBI et al. (2010)
Molasses (7%) P. aeruginosa Shake flask 0.24 N/P 0.003 0.002 Patel and Desai
GS3 (1997)
Orange fruit peeling (3%) P. aeruginosa Shake flask 9.18 N/P 0.306 0.095 George and
MTCC 2297 Jayachandran
(2009)
Sunlower oil soapstock P. aeruginosa Shake flask 7.3 0.22 — 0.104 Benincasa and
(3%) LBI Accorsini
(2008)
Olive mill waste (2%) P. aeruginosa Shake flask 0.012 0.013 0.0006 0.00008 Ramirez
Olive mill waste (5%) 0.03 0.018 0.0006 0.0002 et al. (2016)
Olive mill waste (10%) 0.2 0.058 0.002 0.001
Sugarcane molasses and P. aeruginosa Shake flask 3.19 N/P 0.032 0.022 Gudina
corn steep liquor (10%) 112 Bioreactor 2.23 N/P 0.022 0.023 et al. (2016)
Sunflower acid oil (6%) P. aeruginosa Shake flask 3.5 0.1434 0.058 0.036 This study
(ATCC Bioreactor 4.9 0.1556 0.081 0.051
10145)
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.

J Surfact Deterg (2019)

 J Surfact Deterg

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 Original Residual Utilization

composition sunflower acid sunflower acid (% w/w)
(Ce-1-62) oil composition oil composition
(% w/w) (% w/w)

Palmitic 6.50  1.55 12.89  1.55 7.69

16:0
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 9.50  0.56 4.50  0.56 77.50
18:2
Linolenic 0.80  0.33 1.10  0.33 35.41
18:3
Other 10.50  1.55 33.21  1.25 Fig. 3 Thin layer chromatography of rhamnolipid produced on sun-
flower acid oil. Stationary phase- silica gel, solvent system- chloro-
Data represent the mean values of three independent measurements, form: Methanol:Water (65:15:2, v/v), and visualizing reagent- orcinol
and are expressed as relative percentage of the total GC peak areas. reagent

J Surfact Deterg (2019)

J Surfact Deterg

Fig. 4 HPLC chromatogram of rhamnolipid produced on sunflower acid oil

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 1H 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).
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
Table 6 1H NMR chemical shift data for rhamnolipids produced by
P. aeruginosa using sunflower acid oil
Moiety Proton location Chemical
shift (ppm)

Surfactant Properties of Rhamnolipids Rhamnose CH O C 5.27 d

OH group 4.81 s
Rhamnolipid comprises a mixture of different homologs CH OH 3.63 m
such as monorhamno-monolipidic, dirhamno-monolipidic, CH3 1.18, 1.20 d
monorhamno-dilipidic, and dirhamno-dilipidic. The surfac- Hydroxy fatty (CH2) CH( O C O) 4.13 m
acid CH2COO
tant property of rhamnolipid depends on the distribution of
(CH2) CH(O Rha) 3.39 m
these homologs in the crude product and it varies depend-
CH2COO
ing on the microbial strain, culture conditions, medium
CH(O) CH2COO 2.59 m
compositions, and the carbon substrate used in fermentation
(CH2) CH(O) CH2COO 1.56 m
(Abalos et al., 2001; Deziel et al., 2000). The other parame-
(CH2)5 1.29 m
ters affecting the surfactant properties are the presence of
CH3 0.88 t
residual substrates and the presence of salts in the culture
broth (Mata-Sandoval et al., 2001). t, triplet; s, singlet; m, multiplet; d, doublet.

J Surfact Deterg (2019)


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
75 SFT (mN m–1)
70
Surface tension (mN m–1)
65
60
55
50
45
40
35
30
25
0 50 100 150
Concentration (mg L–1)
Fig. 5 CMC and minimum surface tension reduction by rhamnolipid
produced using sunflower acid oil
J Surfact Deterg
100
Rhamnolipid Polysorbate 20
90
Emulsification index (%)
80
70
60
50
40
30
20
10
0
Benzene xylene Kerosene Mineral oil Soyabean Sunflower
oil oil
Hydrocarbons
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
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
200 250 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
J Surfact Deterg (2019)
J Surfact Deterg

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 Polyester
fabric fabric

Rhamnolipid based liquid 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
detergent 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 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
rhamnolipid

Results are expressed as the means of three repetitions  standard deviation.

J Surfact Deterg (2019)

 J Surfact Deterg

Table 8 Approximate raw material cost for fermentative production substrate cost. As AO is waste from oil refinery industries,
of rhamnolipid on different carbon substrates in shake flask cultures it is available at a cheaper rate compared to vegetable oils
Substrate Raw Max. yield Substrate cost that are used for rhamnolipid production without any pre-
material (g rhamnolipid g−1 (₹ kg−1 treatment. The main emphasis of this study is to increase
price substrate) rhamnolipid) the rhamnolipid production by reducing the cost. This
(₹ kg−1)
study looks at the future perspectives of large-scale profit-
Sunflower 66.5† 0.18 369.44 able production of biosurfactants by using oil refinery
oil waste. Additionally, the synthesized product exhibited a
Glucose 32* 0.06 533.33 very low CMC of 70.109 mg L−1. The synthesized rham-
Palm oil 25** 0.061 409.84 nolipid shows excellent emulsification properties, hence
Glycerol 37* 0.09 411.11 would be possibly suitable for applications in detergents,
Oleic acid 70.5† 0.009 7833.33 pharmaceuticals, and cosmetic industry. The rhamnolipid-
Palm fatty 21.6†† 0.019 3789.47 based liquid detergents, RLD4 and RLD5, showed moder-
acid
ate foaming properties with a better detergency compared
distillate
to other formulations. As a result of low foaming attributes,
Molasses 25* 0.24 104.17
rhamnolipid-based liquid detergents can be utilized as laun-
Sunflower 36* 0.155 232.26
acid oil dry detergents for washing machines.

Prices of the raw materials are according to †Chemical Weekly, 2017;
*India Mart, 2018; **Business Insider, 2018; † † Alibaba.com, 2018.
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
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.
References
Abalos, A., Pinazo, A., Infante, M. R., Casals, M., Garcia, F., &
Manresa, A. (2001) Physicochemical and antimicrobial properties
of new rhamnolipids produced by Pseudomonas aeruginosa AT10
from soybean oil refinery wastes. Langmuir, 17:1367–1371.
Abdel-Mawgoud, A. M., Aboulwafa, M. M., & Hassouna, N. A. H.
(2009) Characterization of rhamnolipid produced by Pseudomonas
aeruginosa isolate Bs20. Applied Biochemistry and Biotechnology,
157:329–345.
Alibaba.com. (2018) Global trade starts here: Current market prices
for palm fatty acid distillate, and waste cooking oil. Retrieved from
http://www.alibaba.com
Bafghi, M. K., & Fazaelipoor, M. H. (2012) Application of rhamnoli-
pid in the formulation of a detergent. Journal of Surfactants and
Detergents, 15:679–684.
Benincasa, M., & Accorsini, F. R. (2008) Pseudomonas aeruginosa
LBI production as an integrated process using the wastes from
sunflower-oil refining as a substrate. Bioresource Technology, 99:
3843–3849.
Business Insider. (2018) Green approach for production of rhamnoli-
pid using acid oil. Retrieved from https://markets.
businessinsider.com
Camacho-Chab, J. C., Guézennec, J., Chan-Bacab, M. J., Ríos-
Leal, E., Sinquin, C., Muñiz-Salazar, R., … Ortega-Morales, B. O.
(2013) Emulsifying activity and stability of a non-toxic bioemulsi-
fier synthesized by microbacterium sp. MC3B-10. International
Journal of Molecular Sciences, 14:18959–18972.
Chandrasekaran, E. V., & Bemiller, J. N. (1980) Methods in carbohy-
drate chemistry. In R. L. Whistler (Ed.), (Vol. 8, p. 89). New York,
NY: Academic Press.
Chemical Weekly. (2017, January 10) A weekly trade journal catering
to the Indian chemical industry. Chemical Weekly 62(23).

J Surfact Deterg (2019)

J Surfact Deterg
Chen, S. Y., Lu, W. B., Wei, Y. H., Chen, W. M., & Chang, J. S.
(2007) Improved production of biosurfactant with newly isolated
Pseudomonas aeruginosa S2. Biotechnology Progress, 23:
661–666.
Cheng, T., Liang, J., He, J., Hu, X., Ge, Z., & Liu, J. (2017) A novel
rhamnolipid-producing Pseudomonas aeruginosa ZS1 isolate
derived from petroleum sludge suitable for bioremediation. AMB
Express, 7:120.
Chiplunkar, P. P., Shinde, V. V., & Pratap, A. P. (2017) Synthesis
and application of palm fatty acid distillate based alkyd resin in liq-
uid detergent. Journal of Surfactants and Detergents, 20:137–149.
Desai, J. D., & Banat, I. M. (1997) Microbial production of surfac-
tants and their commercial potential. Microbiology and Molecular
Biology Reviews, 61:47–64.
Deziel, E., Lepine, F., Milot, S., & Villemur, R. (2000) Mass spec-
trometry monitoring of rhamnolipids from a growing culture of
Pseudomonas aeruginosa strain 57RP. Biochimica et Biophysica
Acta (BBA) - Molecular and Cell Biology of Lipids, 1485:145–152.
Dolman, B. M., Kaisermann, C., Martin, P. J., & Winterburn, J. B.
(2017) Integrated sophorolipid production and gravity separation.
Process Biochemistry, 54:162–171.
Eswari, P., Kavitha, S., Kaliappan, S., Yeom, I. T., & Banu, J. R.
(2016) Enhancement of sludge anaerobic biodegradability by com-
bined microwave-H2O2 pretreatment in acidic conditions. Environ-
mental Science and Pollution Research International, 23:
13467–13479.
Firestone, D. (1994) Official methods and recommended practices of
the American oil chemists’ society (4th ed.). Champaign, IL: AOCS
Press.
George, S., & Jayachandran, K. (2009) Analysis of rhamnolipid bio-
surfactants produced through submerged fermentation using orange
fruit peelings as sole carbon source. Applied Biochemistry and Bio-
technology, 158:694–705.
George, S., & Jayachandran, K. (2012) Production and characteriza-
tion of rhamnolipid biosurfactant from waste frying coconut oil
using a novel Pseudomonas aeruginosa D. Journal of Applied
Microbiology, 114:373–383.
Gudina, E. J., Rodrigues, A. I., Freitas, V. A., Azevedo, Z.,
Teixeira, J. A., & Rodrigues, L. R. (2016) Valorization of agro-
industrial wastes towards the production of rhamnolipids. Biore-
source Technology, 212:144–150.
Haba, E., Espuny, M. J., Busquets, M., & Manresa, A. (2000) Screen-
ing and production of rhamnolipids by Pseudomonas aeruginosa
47T2 NCIB 40044 from waste frying oils. Journal of Applied
Microbiology, 88:379–387.
India Mart. (2018) India’s Online B2B Marketplace, connecting
buyers with suppliers, India. Retrieved from http://www. dir.
indiamart.com
Jadhav, J., Dutta, S., Kale, S., & Pratap, A. P. (2018) Fermentative
production of Rhamnolipid and purification by adsorption chroma-
tography. Preparative Biochemistry and Biotechnology, 48:
234–241.
Jadhav, J., & Pratap, A. P. (2017) Enzymatic synthesis and characteri-
zation of sucrose erucate. Tenside Surfactants Detergents, 54:
539–545.
Kronemberger, F. A., Santa Anna, L. M., Fernandes, A. C.,
Menezes, R. R., Borges, C. P., & Freire, D. M. (2008) Oxygen-
controlled biosurfactant production in a bench scale bioreactor.
Applied Biochemistry and Biotechnology, 147:33–45.
Marchetti, J. M., Pedernera, M. N., & Schbib, N. S. (2011) Production
of biodiesel from acid oil using sulfuric acid as catalyst: Kinetics
study. International Journal of Low-Carbon Technologies, 6:
38–43.
Marsudi, S., Unno, H., & Hori, K. (2008) Palm oil utilization for the
simultaneous production of polyhydroxyalkanoates and
J Surfact Deterg (2019)
rhamnolipids by Pseudomonas aeruginosa. Applied Microbiology
and Biotechnology, 78:955–961.
Mata-Sandoval, J. C., Karns, J., & Torrents, A. (2001) Effect of nutri-
tional and environmental conditions on the production and compo-
sition of rhamnolipids by P. aeruginosa UG2. Microbiological
Research, 155:249–256.
Nitschke, M., Costa, S. G., & Contiero, J. (2010) Structure and appli-
cations of a rhamnolipid surfactant produced in soybean oil waste.
Applied Biochemistry and Biotechnology, 160:2066–2074.
Nitschke, M., Costa, S. G., Haddad, R., Gonçalves, G., Lireny, A.,
Eberlin, M. N., & Contiero, J. (2005) Oil wastes as unconventional
substrates for rhamnolipid biosurfactant production by Pseudomo-
nas aeruginosa LBI. Biotechnology Progress, 21:1562–1566.
Norman, R. S., Frontera-Suau, R., & Morris, P. J. (2002) Variability
in Pseudomonas aeruginosa lipopolysaccharide expression during
crude oil degradation. Applied and Environmental Microbiology,
68:5096–5103.
Patel, R. M., & Desai, A. J. (1997) Biosurfactant production by Pseu-
domonas aeruginosa GS3 from molasses. Letters in Applied Micro-
biology, 25:91–94.
Pratap, A., Wadekar, S., Kale, S., Lali, A., & Bhowmick, D. N.
(2011) Non-traditional oils as newer feedstock for rhamnolipids
production by Pseudomonas aeruginosa (ATCC 10145). Journal
of the American Oil Chemists’ Society, 88:1935–1943.
Radzuan, M. N., Banat, I. M., & Winterburn, J. (2017) Production
and characterization of rhamnolipid using palm oil agricultural
refinery waste. Bioresource Technology, 225:99–105.
Ramirez, I. M., Altmajer Vaz, D., Banat, I. M., Marchant, R., Jurado
Alameda, E., & García Román, M. (2016) Hydrolysis of olive mill
waste to enhance rhamnolipids and surfactin production. Biore-
source Technology, 205:1–6.
Randhawa, K. K., & Rahman, P. K. (2014) Rhamnolipid
biosurfactants—Past, present, and future scenario of global market.
Frontiers in Microbiology, 5:454.
Ross, J., & Miles, G. D. (1941) An apparatus for comparison of foam-
ing properties of soaps and detergents. Journal of the American Oil
Chemists’ Society, 18:99–102.
Sidal, U., & Ozkale-Taskin, E. (2003) Rhamnolipid production from
olive oil mill effluent (OOME) using the rotating biodisc reactor.
Biologia, 58:1081–1086.
Sifour, M., Al-Jilawi, M. H., & Aziz, G. M. (2007) Emulsification
properties of biosurfactant produced from Pseudomonas aerugi-
nosa RB 28. Pakistan Journal of Biological Sciences, 10:
1331–1335.
Sim, L., Ward, O. P., & Li, Z. Y. (1997) Production and characterisa-
tion of a biosurfactant isolated from Pseudomonas aeruginosa UW-
1. Journal of Industrial Microbiology and Biotechnology, 19:
232–238.
Singh, S. P., Bharali, P., & Kumar Konwar, B. (2013) Optimization
of nutrient requirements and culture conditions for the production
of Rhamnolipid from Pseudomonas aeruginosa (MTCC 7815)
using Mesua ferrea seed oil. Indian Journal of Microbiology, 53:
467–476.
Talaiekhozani, A., Jafarzadeh, N., Fulazzaky, M. A., Talaie, M. R., &
Beheshti, M. (2015) Kinetics of substrate utilization and bacterial
growth of crude oil degraded by Pseudomonas aeruginosa. Journal
of Environmental Health Science and Engineering, 13:1–8.
Wadekar, S. D., Kale, S. B., Lali, A. M., Bhowmick, D. N., &
Pratap, A. P. (2012) Microbial synthesis of rhamnolipids by Pseu-
domonas aeruginosa (ATCC 10145) on waste frying oil as low cost
carbon source. Preparative Biochemistry and Biotechnology, 42:
249–266.
Wadekar, S. D., Patil, S. V., Kale, S. B., Lali, A. M.,
Bhowmick, D. N., & Pratap, A. P. (2011a) Study of glycerol resi-
due as a carbon source for production of rhamnolipids by

Pseudomonas aeruginosa (ATCC 10145). Tenside Surfactants
Detergents, 48:16–22.
Wadekar, S. D., Patil, S. V., Kale, S. B., Lali, A. M.,
Bhowmick, D. N., & Pratap, A. P. (2011b) Structural elucidation
and surfactant properties of Rhamnolipids synthesized by Pseudo-
monas aeruginosa (ATCC 10145) on sweet water as carbon source
and stabilization effect on foam produced by sodium lauryl sulfate.
Tenside Surfactants Detergents, 48:286–292.
J Surfact Deterg
Zhang, L., Pemberton, J. E., & Maier, R. M. (2014) Effect of fatty
acid substrate chain length on Pseudomonas aeruginosa ATCC
9027 monorhamnolipid yield and congener distribution. Process
Biochemstry, 49:989–995.
Zhang, Y., Jia, D., Sun, W., Yang, X., Zhang, C., Zhao, F., & Lu, W.
(2017) Semicontinuous sophorolipid fermentation using a novel
bioreactor with dual ventilation pipes and dual sieve-plates coupled
with a novel separation system. Microbial Biotechnology, 11:
455–464.
J Surfact Deterg (2019)