Bioprocess Biosyst Eng (2015) 38:1629–1637
DOI 10.1007/s00449-015-1404-9
ORIGINAL PAPER
Optimization of liquid–liquid extraction of biosurfactants
from corn steep liquor
X. Vecino1 • L. Barbosa-Pereira1 • R. Devesa-Rey1,2 • J. M. Cruz1
A. B. Moldes1
Received: 9 February 2015 / Accepted: 17 April 2015 / Published online: 26 April 2015
Ó Springer-Verlag Berlin Heidelberg 2015
Abstract In this work, the optimization of the operational
conditions for the chloroform-based extraction of surface-
active compounds from corn steep liquor (CSL) was car-
ried out and the nutritional properties of the remnant
aqueous phase (CSL-less biosurfactant) was evaluated as
microbial fermentation medium. The optimal conditions to
obtain biosurfactants from CSL were as follows: chloro-
form/CSL ratio 2 (v/v), 56 °C at extraction times[30 min.
At the optima conditions, 100 % of biosurfactant extract
can be obtained from CSL, obtaining 12.0 ± 0.5 g of
biosurfactant extract/Kg of CSL. The critical micelle con-
centration (CMC) of the biosurfactant extract was
399.4 mg L-1. This value is similar to the CMC of cetri-
monium bromide (CTAB), a cationic surfactant used in the
formulation of nanoparticles. The extraction of biosurfac-
tant can be also carried out at room temperature although in
this case, the extraction yield decreased about 15 %. The
extraction of surface-active compounds from agroindustrial
streams can suppose important advances for the bio-based
surfactants industry. Biosurfactants obtained in this work
are not only more eco-friendly than chemical detergents
but also can be cost competitive with its chemical coun-
terparts. Furthermore, after the extraction of surface-active
compounds, CSL-less biosurfactant was found to be suit-
able as nutritional supplement for lactic acid bacteria,
& A. B. Moldes
amoldes@uvigo.es
1 industry, the residual stream produced at the early stages of
Chemical Engineering Department, School of Industrial
Engineering (EEI), University of Vigo, Campus As Lagoas-
Marcosende, 36310 Vigo-Pontevedra, Spain
2
Defense University Center, Naval Academy, University of
Vigo, Plaza de España 2, 36920 Marı́n-Pontevedra, Spain
•
maintaining its nutritional properties in comparison with
regular CSL.
Keywords Corn steep liquor  Industrial stream  Liquid–
liquid process  Biosurfactant  Nutritional medium
Introduction
Biosurfactants are surface-active compounds released by
microorganisms, with several advantages over the chemical
surfactants as lower toxicity, higher biodegradability, bet-
ter environmental compatibility, higher foaming, high se-
lectivity and specific activity at extreme pH, temperatures
and salinity. In addition, biosurfactants can be synthesized
from renewable agroindustrial feed stocks [1]. Natural
detergents have been shown to have important roles in
biomedical and therapeutic areas; pharmaceutical, cos-
metic and food industries as well as in the environmental
field [2]. Furthermore, due to self-assembly properties of
biosurfactants, new and fascinating applications in nan-
otechnology are predicted for these natural detergents [3].
In the food industry, various extraction procedures are
used to recover valuable soluble components from raw
materials. This is usually done by dissolving the raw ma-
terial in a liquid solvent to separate the soluble compo-
nents, which can then be recovered from the solvent in an
additional step. Raw materials that are suitable for ex-
traction may comprise solids only, solids and a solution, or
solids and a liquid. For example, in the corn wet-milling
corn processing contains large amounts of solids and water-
soluble components [4]. Corn is soaked in water with sulfur
dioxide (SO2) or sodium bisulphite (NaHSO3), for around
36 h at an elevated temperature (45–52 °C), in a process
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known as steeping where raw starch is the input and pro-
cessed water is the output [4, 5]. During the steeping
process of corn, readily available carbohydrates are re-
leased favoring the growth of lactic acid bacteria that can
produce up to 50–150 g L-1 of lactic acid as primary
metabolite and biosurfactants as secondary metabolite [4,
6, 7].
In this study, attention was focused on the liquid in
which the corn is steeped, which after evaporation is
converted into a residual stream referred to as corn steep
liquor (CSL) [6]. CSL is a soluble solid that constitutes
nearly 40–50 % (w/w) of the dry weight of corn and con-
sists of a mixture of reducing sugars and amino acids [8].
CSL has various industrial applications, e.g., it can be
mixed with fiber and after dewatering and drying used as
corn feed [9], or it can be used directly as a nutritional
medium for industrial fermentations [10–13]. Therefore,
this residual stream has been proposed by Bustos et al. [14]
as nitrogen supplement during fermentation with lactic acid
bacteria or Gao and Yuan [15] has used CSL to improve
the biotechnological production of 2-keto-l-gulonic acid.
Some authors have proposed the utilization of organic
solvents for the extraction of biosurfactants. Thus, Camil-
ios et al. [16] extracted an extracellular rhamnolipid bio-
surfactant produced by Pseudomonas aeruginosa, in a
solid-state fermentation, by washing with water the solid
culture and then undertaking a liquid–liquid extraction into
chloroform–methanol 3:1 (v/v). Most of the biosurfactants
considered in the literature are produced extracellularly;
thus, they are recovered from the fermentation media by
liquid–liquid extraction. However, some microorganisms
such as Lactobacillus pentosus produce cell-bound bio-
surfactant, which are extracted from cells by solid–liquid
extraction. For instance, Vecino et al. [17] proposed a
solid–liquid extraction with an aqueous phosphate buffer in
the absence of salt to extract the biosurfactant from L.
pentosus cells.
A novel application of CSL, discovered recently by
Vecino et al. [7], is based on the possibility of extracting
biosurfactants from this corn wet-milling byproduct. This
is an interesting finding because if the liquid–liquid ex-
traction process is optimized, this biosurfactant could be
cost competitive with the surfactants obtained by chemical
synthesis. In a previous work, Vecino et al. [7] have tested
various organic solvents for undertaking the liquid–liquid
process of biosurfactants contained in CSL. Among the
solvents evaluated, chloroform gave the best extraction
yields. The extraction conditions used in this preliminary
study compromised the utilization of CSL:solvent ratio of
1:10 (v/v), for 4 h at room temperature (25 °C). These
conditions have also been used by Jamal et al. [18] during
the extraction of biosurfactants produced by Pseudomonas
aeruginosa. However, from an industrial point of view,
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Bioprocess Biosyst Eng (2015) 38:1629–1637
more studies are needed to optimize the extraction condi-
tions of biosurfactants from CSL. For this purpose, it will
be interesting to study the interaction of extraction tem-
perature with other extraction conditions like amount of
solvent or extraction time.
The main objective of this work it is optimize the con-
ditions for the extraction of low-cost biosurfactant from
CSL, using chloroform in a single stage. The extraction
conditions evaluated in this study consist of temperature,
amount of solvent and extraction time. In addition, after the
extraction of biosurfactants, the nutritional properties of the
remnant aqueous phase of CSL, as nutritional supplement
for lactic acid bacteria, will be evaluated.
Experimental section
Reagents
Chloroform (stabilized with 50 ppm of amylene as
preservative), CHCl3 (99.8 %); sulphuric acid, H2SO4
(96 %, PA-ISO); methanol, MeOH (99.9 %, HPLC-gradi-
ent grade); MRS broth; yeast extract; precipitated calcium
carbonate, CaCO3 (99.0 %, PA) and anhydrous D(?)-
glucose, C6H12O6 (PA-ACS) were purchased from Panreac
(Barcelona, Spain).
Corn steep liquor (Lot #MKBN0183 V, liquid brown,
50 % solid content and pH 4.4) and surfactin, a commercial
surfactant (C98 %), from Bacillus subtilis, were supplied
by Sigma-Aldrich (St. Louis, MO, USA).
Preparation of microorganisms and inocula
Lactobacillus pentosus (L. pentosus) CECT-4023T
(ATCC-8041) was obtained from the Spanish collection of
type cultures (Valencia, Spain).
The strain was grown in Petri dishes containing com-
plete medium MRS Agar, at 31 °C for 24 h. The microbial
biomass in the inocula was determined indirectly, by
measuring the optical density at 600 nm, and adjusted by
dilution with water to 3.0 g of dry cells per liter. One
milliliter of this suspension was added to 150 mL of each
fermentation medium to achieve an initial biomass con-
centration of 0.02 g of dry cells per liter.
Extraction of biosurfactant from CSL
The biosurfactant was extracted from CSL (15 g L-1) at
different ratios of chloroform/CSL (2, 1.25 and 0.5 (v/v))
and different temperatures (30.5, 39, 47.5 and 56 °C) up to
200 min at 200 rpm in 1 L Pyrex bottles with screw caps
(for avoid chloroform evaporation). After extraction, the
mixture chloroform–CSL was decanted and the
Bioprocess Biosyst Eng (2015) 38:1629–1637
biosurfactant was separated from the chloroform by rotary
evaporation (Buchi Rotavapor R-210, equipped with a
digital controller V-850, heating bath B-491, vacuum pump
V-700, and closed-circuit chiller F-105, Switzerland). The
evaporation was carried out at 474 mbar and 60 °C. Finally
the biosurfactant extract was dissolved in water or metha-
nol (MeOH) by vortex and sonication process.
Extraction yield of biosurfactant from CSL
After extraction of the biosurfactant from the CSL, the
chloroform was evaporated to dryness and the biosurfactant
extract was redissolved in water or MeOH. Two mL of this
solution containing the biosurfactant extract, at concen-
tration of 2000 mg L-1, was dried at 105 °C for 24 h for
calculation of the dry weight and the extraction yield. All
measurements were carried out in triplicate.
Determination of critical micelle concentration
of the biosurfactant extracted from CSL
The critical micelle concentration (CMC) is the concen-
tration of biosurfactant over which it is impossible to re-
duce the surface tension of water in more units. The CMC
of the biosurfactant extract obtained from CSL was deter-
mined by diluting the extract in water (from a concentra-
tion of 2033.3 up to 10.2 mg L-1). The surface tension of
the solutions was measured by the Wilhelmy plate method
in a force tensiometer with a platinum plate (Easy Dyne
K20, KRUSS GmbH), at room temperature. All determi-
nations were carried out in triplicate.
Fourier transform infrared spectroscopy analysis
of biosurfactant
The biosurfactant extracted from CSL or surfactin, used as
control (1 mg), were ground with 10 mg of potassium
bromide (KBr) and pressed (7500 kg for 30 s) to produce
translucent pellets.
The FTIR analyses were carried out in a FTIR spec-
trometer (Nicolet 6700, Thermo Scientific). Spectral mea-
surement was performed in transmittance mode in a range
of 400–4000 cm-1 with a resolution of 4 cm-1 with an
average range of 32 data scans. A KBr pellet was used as
background reference.
Evaluation of the nutritional properties of CSL
for lactic acid fermentation
The liquid phase obtained after the extraction of the sur-
face-active compounds from CSL (CSL-less biosurfactant)
was tested for use as nutritional supplement for the
biotechnological production of lactic acid by Lactobacillus
1631
pentosus. Experiments were run in 500 mL Erlenmeyer
flasks, with a fermentation volume of 150 mL using
30 g L-1 of glucose as carbon source and 20 g L-1 of
CSL-less biosurfactant and 10 g L-1 of yeast extract as
nutritional supplements. Fermentations were carried out in
an orbital shaker at 150 rpm, 31 °C and 30 g L-1 of
CaCO3 were added at the beginning of fermentation pro-
cess to neutralize the lactic acid produced. For comparative
purposes, two additional fermentations, with MRS broth
and regular CSL were assayed under the same operational
conditions. Both CSL (regular CSL and CSL-less biosur-
factant) were supplemented with 10 g L-1 of yeast extract
and 30 g L-1 of glucose. At given fermentation times,
samples were withdrawn from the fermentation media and
centrifuged.
Quantification of glucose and lactic acid by HPLC–
DAD–RID
The glucose and lactic acid in the supernatant were ana-
lyzed by high-performance liquid chromatography (HPLC)
with diode array detection (DAD) and refractive index
detection (RID) (Agilent Technologies 1200 Series, Ger-
many). Instrument control and data acquisition were per-
formed with OpenLAB CDS software.
A sample volume of 20 lL was injected into a Rezex
RHM-Monosaccharide H? (8 %) column (300 9 7.80 mm,
8 lm particle size) (Phenomenex, USA) maintained at a
constant temperature of 65 °C. The target compounds were
analyzed using an isocratic elution, with Milli-Q water plus
0.005 N of H2SO4, at a flow rate of 0.4 mL min-1.
Statistical analysis
The commercial Solver software (Microsoft Excel 2011)
was used to fit the experimental data to the models by least
squares non-linear regression. Lactic acid production and
glucose consumption were described by kinetic models
proposed in previous studies [19–22].
Results and discussion
Production of biosurfactant from CSL: optimization
of the extraction conditions
Figure 1a shows a general flow chart for the steeping of
corn in the wet-milling industry that results in the extrac-
tion of water-soluble components and Fig. 1b shows the
flow diagram proposed in this work for extracting surface-
active compounds from CSL. In a previous work [7], dif-
ferent organic solvents were evaluated (chloroform, ethyl
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(a)
Maize
Reception Dry cleaning
Glucose Yeast Extract
(b)
Lactic Acid
Fermentation
L. pentosus
Fig. 1 Flowchart illustrating: a corn steep liquor production (CSL) and b the extraction of biosurfactant from CSL
acetate, methyl tert-butyl ether, trichloroethylene, hexane,
heptane, and dichloromethane) for extracting the biosur-
factant contained in CSL observing that chloroform,
methyl tert-butyl ether, and dichloromethane gave 100 %
extraction yields; however, the high amount of organic
solvent used in this preliminary work, 10 mL organic sol-
vent/mL of CSL stream as well as high extraction times
(4 h), can inhibit the application of this extraction protocol
at industrial scale. Among the solvents evaluated, in this
previous study [7], chloroform has been chosen because it
is denser than water; it has a low boiling point (61.2 °C);
and in previous works [7], it has showed a good extraction
capacity. Thus, surface-active compounds can be separated
by liquid–liquid extraction from the aqueous phase and
later recovered from the organic phase with a low energy
cost.
Figure 2 shows the kinetics of the extraction of biosur-
factant from CSL. Different ratios of chloroform/CSL and
different extraction temperatures were tested. When the
chloroform/CSL ratio was fixed at 0.5 (v/v), maximum
extraction yields were obtained at 56 °C after 187.5 min;
whereas when the ratio chloroform/CSL was fixed at 1.25
and 2 (v/v), maximum extraction yields were achieved
after 82.5 and 30 min, respectively. At lower temperatures,
the extraction time had to be increased to maximize the
extraction yields. Thus, at 30.5 °C, the extraction time
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Bioprocess Biosyst Eng (2015) 38:1629–1637
Processing of the solid fraccion: First
grinding/ Degermination/ Second
grinding/Sieving/ Separation
Steeping
Liquid fraction of water soluble
compounds namely corn steep liquor (CSL)
Aqueous solution
containing
NaHSO 3 or SO 2 Evaporation H2O
Concentrated
CSL (containing
50% of solids)
Aqueous phase Liquid-Liquid Chloroform
(CSL-less biosurfactant ) Extraction
CaCO3 Organic phase
(containing BS)
Extract of Biosurfactant (BS) Distillation
should be fixed at 187.5, 135 and 82.5 min for chloroform/
CSL ratios of 0.5, 1.25 and 2, respectively. Optima con-
ditions to obtain biosurfactants from CSL can be fixed as
follows: chloroform/CSL ratio 2 (v/v), 56 °C at extraction
times [30 min.
Moreover, to determine whether all the surface-active
compounds were extracted in the first extraction process, a
second extraction was carried out at chloroform/CSL ratio
2 (v/v), 56 °C during 60 min. No more surface-active
compounds were detected after the second extraction stage.
Extraction yield of the biosurfactant from CSL
Under optimal operational conditions, once chloroform
was evaporated, the balloon containing the biosurfactant
was dried and weight; the yield in this conditions was
12.3 ± 0.3 g of biosurfactant extract per kg of CSL. In
addition, to see if the amount of water used to re-dissolve
the biosurfactant was enough to solubilize all the biosur-
factant extracted, biosurfactant also was dissolved in
methanol for extraction yield. Thus, when the dry weight
was calculated after dilution of the biosurfactant in water or
methanol, the yield was 12.3 ± 1.8 and 11.5 ± 0.7 g of
biosurfactant extract per kg of CSL, respectively. Almost
no differences were observed in the extraction percentages
calculated in 2 different solutions. The average extraction
Bioprocess Biosyst Eng (2015) 38:1629–1637
Fig. 2 Kinetics plots obtained during the extraction of biosurfactant
from CSL at different temperatures of extraction and different
chloroform/CSL ratios (v/v): a 2, b 1.25 and c 0.5
yield was 12.0 ± 0.5 g of biosurfactant extract per kg of
CSL. Some biosurfactants (such as surfactin) are poorly
soluble in water. In this case, the biosurfactant extracted
from CSL, was soluble in both aqueous and organic phases,
at concentration of 2000 mg L-1 as indicated by the
similar extraction yields obtained in both cases.
Determination of critical micelle concentration
of biosurfactant from CSL
Calibration curve was prepared using solutions containing
different concentrations of biosurfactant extracts and
measuring the surface tension of these solutions. When the
1633
Fig. 3 Critical micelle concentration (CMC) of the biosurfactant
extracted from CSL
concentration of biosurfactant extract was lower than the
CMC, the surface tension of water in the presence of bio-
surfactant was linearly related to the concentration of sur-
factant, and thus it is possible to establish a relationship
between the biosurfactant extract concentration and the
surface tension. Figure 3 shows the relationship between
the concentration of biosurfactant extract and the reduction
of surface tension in water. The CMC of biosurfactant
extract was calculated from the intersection of the tangent
to the curve at the inflection point with the horizontal
tangent through the points at low concentration (see
Fig. 3). Biosurfactant extracted from CSL was able to re-
duce the surface tension of water from 70.6 ± 0.2 to
40.9 ± 1.7 mN m-1 with a CMC of 399.4 mg L-1. Some
chemical surfactants have similar CMC values than the
biosurfactant extracted in this work; thus cetrimonium
bromide (CTAB), a cationic detergent used in the formu-
lation of nanoparticles, has a CMC of 9.2 9 10-4 mol L-1
corresponding with 335.3 mg L-1. On the other hand, the
surface tension reduction in water achieved with biosur-
factant from CSL was similar to other biosurfactants pro-
duced by lactic acid bacteria. Thus, biosurfactants from L.
acidophilus ATCC-4356, L. casei ATCC-7469 and L. fer-
mentum ATCC-14931 are able to reduce the surface ten-
sion of aqueous solution in 27 units; whereas biosurfactants
produced by L. plantarum ATCC-14917 reduced the sur-
face tension of water up to 24 units [23]. Otherwise, the
surface tension activity of the biosurfactant extract from
CSL was higher than the surface tension reduction
achieved by the biosurfactant produced by L pentosus
ATCC-8041 and L. rhamnosus ATCC-9595 that reduced
the surface tension in 19 and 18 units, respectively [24, 25].
FTIR analysis of biosurfactant from CSL vs
Surfactin
Figure 4 shows the results of the FTIR analysis of the
biosurfactant extracted from CSL in comparison with a
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commercial lipopeptide biosurfactant (surfactin). The bio-
surfactant from CSL and surfactin produced four similar
bands in the FTIR spectra: (1) a band at 3425 cm-1,
indicates the presence of peptide groups resulting from N–
H stretching and O–H stretching of carboxylic acids,
although by the size of the band this it can be deduced that
the concentration of peptides should be lower in the bio-
surfactant obtained form CSL than in surfactin; (2) bands at
3010–2855 and 1467–1378 cm-1, denoting the presence of
C–H stretching corresponding to CH2 and CH3 groups of
aliphatic chains; (3) a band at 1743–1710 cm-1, corre-
sponding to the stretching vibration of C=O bonds in car-
boxyl esters and (4) a band at 1647 cm-1, corresponding to
C=O–N bond (amide I bond) and at 1543 cm-1 denoting
the presence of N–H bonds (amide II bond).
Evaluation of nutritional properties of CSL-less
biosurfactant
Some authors have pointed out that during the biotech-
nological production of different metabolites, extra
complications can be noticed during the downstream
processing of fermentation products when the culture
media contain biosurfactants [26]. Thus, the extraction
process of biosurfactant contained in CSL not only re-
sults interesting to obtain natural bio-based detergents,
but also it is convenient if extract complications are want
to be avoided during the utilization of CSL as nutritional
supplement in biotechnological processes. However, be-
fore using CSL-less biosurfactant, without biosurfactants,
as nutritional supplement during industrial fermentations,
it is necessary to corroborate that CSL keeps its prop-
erties as nutritional supplement. Thus in this work, var-
ious fermentations were carried out with lactic acid
bacteria that are generally recognized as nutritionally
fastidious because of their high nutritional requirements
Fig. 4 Comparison of the FTIR
spectra of surfactin vs
biosurfactant extracted from
CSL
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Bioprocess Biosyst Eng (2015) 38:1629–1637
in order to corroborate this fact [27]. Figure 5 shows the
kinetic production of lactic acid and glucose consump-
tion by L. pentosus during use of the CSL-less biosur-
factant as a nutritional medium, in comparison with the
use of MRS broth or regular CSL (containing the sur-
face-active compounds). After 25 h, all the glucose was
converted into lactic acid in all three nutritional media
evaluated.
In addition, the lactic acid production was successfully
described by the kinetic model proposed by Mercier et al.
[28]. Several authors have used this kinetic model to pre-
dict the generation of lactic acid during fermentation [19–
22]. According to the aforementioned authors, the pro-
duction of lactic acid can be described by a sigmoidal
curve. The product concentration can be easily modeled by
direct integration of the corresponding differential equation
and non-linear regression with Eq. 1.
 
dP P
¼ Pr P 1  ð1Þ
dt Pm
where t is time, P is the product concentration, Pm is the
maximum product concentration, and Pr is the ratio be-
tween the initial volumetric rate of product formation (rp)
and the initial product concentration (P0). The original
equation proposed by Mercier et al. [28] for biomass
(X) modeling included a correction term for inhibition
(1 - X/Xm). As the product also causes inhibition, and to
provide a more conceptual approach, a product original
inhibition term was included in the equation (1 - P/Pm),
which presented a variation pattern closely related to the
function included in the original equation of Mercier et al.
[28] but provided a better understanding of the process.
Thus, the resulting differential equation can be easily
solved by a fourth-order Runge–Kutta method coupled
with an optimization algorithm dealing with Newton’s
method [19] to produce Eq. 2.
Bioprocess Biosyst Eng (2015) 38:1629–1637
Fig. 5 Experimental data and calculated time courses of lactic acid
and glucose concentrations during lactic acid fermentations using L.
pentosus, carried out with three different nutritional media: a MRS
broth; b Regular CSL; c CSL-less biosurfactant
P0 Pm ePr t
P¼ ð2Þ
Pm  P0 þ P0 ePr t
After fitting the lactic acid production data to the kinetic
model described above, glucose consumption was deter-
mined by using Eq. 3 [20].
1 carried out under no severe conditions (chloroform/CSL
S ¼ S0  ðP  P0 Þ ð3Þ
YP=S
where YP/S is the product yield, P and P0 are the final and
initial product concentrations, respectively (g L-1), and
S and S0 are the final and initial glucose concentrations
(g L-1).
Figure 5 illustrates the close agreement between ex-
perimental and calculated data for consumption of glucose
1635
and the production of lactic acid, for the three different
nutritional media.
During the fermentations, L. pentosus fermented glucose
by the homo-fermentative Embden-Meyerhof-Parnas
(EMP) pathway to yield lactic acid as the sole product in
also the cases, even when CSL-less biosurfactant, subjected
to a liquid–liquid process with chloroform, was used as
nutritional medium. Table 1 shows the maximum ratio
between initial volumetric rate of product formation and
initial product concentration, Pr, and the yield expressed as
g of lactic acid per g of glucose consumed, YP/S, during
fermentations carried out with the three nutritional media.
These fermentation parameters were obtained by applying
the above-mentioned kinetic modeling process. The max-
imum value of Pr (0.609 h-1) corresponded to the MRS
broth fermentation, whereas there were no differences be-
tween the Pr values for fermentations carried out with CSL
containing surface-active compounds (0.279 h-1) and CSL
where the surface-active compounds were extracted
(0.237 h-1). Regarding the maximum lactic acid produc-
tion, Pm, there were almost no differences between the
three media evaluated, achieving maxima lactic acid con-
centrations around 26.0 g L-1. Table 1 also includes the r2
values (a measure of the degree of correlation) and Fis-
cher’s ‘F’ parameter (a measure of the statistical sig-
nificance of equations).
Economical considerations
Surfactants find applications in a wide variety of industrial
processes [29–32]. However, as the environmental com-
patibility is becoming a progressively important factor in
selecting industrial chemicals, the commercialization of
biosurfactant is gaining much attention in comparison their
chemical counterparts. Thus, surfactin, produced by
Bacillus subtilis, is one of the most widely studied bio-
surfactants [33]; at a concentration of 10 mg L-1, surfactin
can reduce the surface tension of water from 72 to
27 mN m-1. Nevertheless, the high cost of surfactin ($820/
50 mg) prevents its industrial use. From this point of view,
the biosurfactant extracted from CSL could have industrial
applications, rather than surfactin, as it can be obtained
from a cheap and renewable substrate with a simple ex-
traction process using chloroform. This process can be
ratio 2 (v/v), 56 °C at extraction times[30 min) and in this
work, it has been verified 96.5 % of chloroform can be
recirculated into the system. The technology proposed in
this work, avoid the high cost and longer periods of times
associated with the fermentation process involving the
production of biosurfactants by a specific microorganisms.
Moreover, MRS broth is an expensive fermentation
medium, used in several biotechnological processes. This
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Table 1 Parameters obtained after the kinetic modeling of experimental data for glucose consumption and lactic acid production during lactic
acid fermentation of L. pentosus in the presence of three different nutritional supplements
Lactic acid production
-1 -1 -1
P0 (g L ) Pm (g L ) Pr (h ) rp (g L
MRS broth 0.004 26.039 0.609 0.002
Regular CSL 0.494 26.321 0.279 0.138
CSL-less biosurfactant 0.720 25.975 0.237 0.171
* Significance [99 % for the three different nutritional media
medium contains peptone or yeast extract as nitrogen
sources being the current price about US $2.8–4.5 and US
$1.9–2.35 kg-1, respectively. Thus, alternative nitrogen
sources such as CSL are of great interest because the
current price of CSL is about US $0.5 kg-1. However, CSL
may also be of industrial interest in the detergents and
surfactants industry because, as it has shown in this work, it
contains surface-active compounds. Therefore, before the
commercialization of CSL as nutritional fermentation
supplement, biosurfactants can be extracted without af-
fecting the nutritional properties of the product and obtain
additional profits.
Conclusions
The valorization of the aqueous stream generated in the
corn wet-milling industry is possible by extraction of sur-
face-active compounds and further use of the CSL-less
biosurfactant as a fermentation medium. It has been
demonstrated that corn steep liquor (CSL) contains sur-
face-active compounds that can be extracted by liquid–
liquid process, using a chloroform/CSL ratio of 2 (v/v), at
56 °C using extraction times above 30 min. Moreover,
after extraction of the biosurfactant, the CSL-less biosur-
factant maintains the nutritional properties required for use
as nitrogen supplement for lactic acid bacteria.
Acknowledgments The authors thank the Spanish Ministry of
Economy and Competitiveness (This work was funded by FEDER
funds under the project CTM2012-31873). X. Vecino gratefully ac-
knowledges the University of Vigo for receipt of a predoctoral
contract.
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