Colloids and Surfaces B: Biointerfaces 101 (2013) 34–43

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Colloids and Surfaces B: Biointerfaces

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Kinetic study of biosurfactant production by Bacillus subtilis LAMI005 grown in

clarified cashew apple juice
Darlane Wellen Freitas de Oliveira a , Ítalo Waldimiro Lima França a ,
Anne Kamilly Nogueira Félix a , João Jeferson Lima Martins a , Maria Estela Aparecida Giro a ,
Vânia Maria M. Melo b , Luciana Rocha Barros Gonçalves a,∗
a
Universidade Federal do Ceará, Departamento de Engenharia Química, Bloco 709, Campus do Pici, CEP. 60.455-760, Fortaleza, CE, Brazil
b
Universidade Federal do Ceará, Depto. de Biologia, LemBiotech, Laboratório de Ecologia Microbiana e Biotecnologia, Bloco 909, Campus do Pici, CEP. 60.455-760, Fortaleza, CE, Brazil

a r t i c l e i n f o a b s t r a c t

Article history: In this work a low cost medium for the production of a biosurfactant by Bacillus subtilis LAMI005 and the
Received 22 December 2011 kinetics of surfactin production considering the effect of initial substrate concentration were investigated.
Received in revised form 2 May 2012 First, cashew apple juice supplementation for optimal production of biosurfactant by B. subtilis LAMI005
Accepted 12 June 2012
was studied. The medium formulated with clarified cashew apple juice and distilled water, supplemented
Available online 21 June 2012
with 1.0 g/L of (NH4 )2 SO4 , proved to be the best among the nutrients evaluated. The crude biosurfactant
had the ability to decrease the surface tension of water to 30 dyne/cm, with a critical micelle concentration
Keywords:
(CMC) of 63.0 mg/L. Emulsification experiments indicated that this biosurfactant effectively emulsified
Biosurfactant
Biotransformations
kerosene (IE24 = 67%) and soybean oil (IE24 = 64%). Furthermore, the emulsion stability was always very
Clarified cashew apple juice high. It was shown by biochemical analysis, IR spectra, that there is no qualitative differences in the
Fructose, Glucose composition of the crude biosurfactant from a standard sample of surfactin from B. subtilis.
Submerged culture © 2012 Elsevier B.V. All rights reserved.

1. Introduction conditions of temperature and pH. Furthermore, they have lower

toxicity and can be produced from renewable and cheaper sub-
Surfactants are surface active substances, consisting of a strates [2,5–7]. However, the production of biosurfactant has some
hydrophilic (polar) and a hydrophobic (nonpolar) part on its issues associated with the expensive costs of substrates and some
molecules [1,2]. They have the ability to reduce surface and inter- inefficient product recovery methods [7]. Therefore, in order to
face tensions between liquids, solids, and gases, thus they allow enhance the industrial use of biosurfactant, the production cost
them to mix or disperse readily as emulsions in water or other should become competitive with the synthetic surfactants. A possi-
liquids [2]. Nowadays, the huge demand of surfactants is cur- ble strategy to achieve this goal is to use alternative raw materials,
rently provided by chemical surfactants derived from petroleum, such as agro-industrial residues such as cashew apple peduncle, as
but these compounds have the problem of being toxic to the envi- culture medium in submerge fermentation.
ronment and non-biodegradable. In the north coast of Brazil, the cashew agroindustry has an
A biosurfactant is a surfactant produced extracellularly or as important role on local economy and the official estimate for the
part of the cell membrane by bacteria, yeasts, and fungi from var- Brazilian cashew nut for 2011 was around 298 thousand of tons [8],
ious substrates including sugars, oils, alkanes, among others [1]. which corresponds to more than 2.6 million tons of cashew apple.
They are amphiphilic compounds with considerable potential in However, only 18% of the total peduncle is consumed in natura or
commercial applications within various industries, such as health processed industrially to produce different products from concen-
care and food processing industries, as well as enhancing oil recov- trated juice to desserts. It is estimated that about 15–20% of the
ery, crude oil drilling lubricants, bioremediation of water-insoluble pulp is exploited and 80% is wasted. Cashew apple is a pseudofruit
pollutants [1,3,4]. Biosurfactant have advantages over its chemi- rich in vitamin C, flavor and aroma, but the majority of cashew
cal counterparts in biodegradability and effectiveness at extreme apples rot in the soil. Those facts, together with its rich composition
(reducing sugar, vitamins and minerals salts), turns cashew apple
juice (CAJ) into an interesting and inexpensive culture medium for
biosurfactant production [9].
∗ Corresponding author at: Departamento de Engenharia Química, Universidade
Bacillus subtilis is considered a suitable microorganism for
Federal do Ceará, Campus do Pici, Bloco 709, Fortaleza, CE 60455-760, Brazil.
Tel.: +55 85 3366 9611; fax: +55 85 3366 9610. biosurfactant production owing to the absence of pathogenicity,
E-mail address: [email protected] (L.R.B. Gonçalves). which permits the use of its products in food and pharmaceutical

0927-7765/$ – see front matter © 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.colsurfb.2012.06.011
 D.W. Freitas de Oliveira et al. / Colloids and Surfaces B: Biointerfaces 101 (2013) 34–43 35

Table 1
Description of culture medium employed in this study using Bacillus subtilis LAMI005 in batch cultivation. TRS: total reducing sugars, mainly glucose and fructose, present in
clarified cashew apple juice and TES: trace element solution, consisting in g/L by ZnSO4 ·7H2 O: 10.95, FeSO4 ·7H2 O: 5.0, MnSO4 ·H2 O: 0.39, CuSO4 ·5H2 O: 54, Co(NO3 )2 ·6H2 O:
0.25 and Na2 B4 O7 ·10H2 O: 0.17.

Culture medium Nature of ingredients

C-source (g/L) N-source (g/L) TES (%) Other (g/L)

A TRS (46.24) (NH4 )2 SO4 (1.0) – –

B TRS (40.98) (NH4 )2 SO4 (1.0) 0.1 –
C TRS (45.66) (NH4 )2 SO4 (1.0) – NaCl (2.7)
D TRS (49.96) (NH4 )2 SO4 (1.0) – MgSO4 ·7H2 O (0.6)
E TRS (43.79) (NH4 )2 SO4 (1.0) – Na2 HPO4 ·7H2 O (7.2)
KH2 PO4 (3.0)

industries [10]. Therefore, discovery of new strains and improve- flask containing 50 mL of CCAJ, diluted with water up to 22.92 g/L
ment on culturing methods of B. subtilis can provide a safe source of of total reducing sugars (glucose and fructose), supplemented with
biosurfactant. Surfactin, one of the most effective cyclic lipopeptide 1.0 g/L of (NH4 )2 SO4 and 0.1% of trace element solution (consisting
biosurfactants produced by B. subtilis, can lower the surface tension in g/L by ZnSO4 ·7H2 O: 10.95, FeSO4 ·7H2 O: 5.0, MnSO4 ·H2 O: 0.39,
of water from 72 to 27 dyne/cm and the interfacial tension of the CuSO4 ·5H2 O: 54, Co(NO3 )2 ·6H2 O: 0.25 and Na2 B4 O7 ·10H2 O: 0.17).
water/n-hexadecane system from 43 dyne/cm to 1 dyne/cm [7]. The flasks were incubated in a rotary shaker (Tecnal-TE240, São
Therefore, the aim of this work was to evaluate a low cost cul- Paulo, Brazil) at 180 rpm, 30 ◦ C for 24 h. The OD of this culture was
ture medium, using cashew apple as carbon and energy source, to adjusted with sterile water to 0.1–0.2 at 600 nm, in order to assure
cultivate a new isolate of B. subtilis and to produce biosurfactant. that the same cell concentration was present at the seed culture.
The influence of juice concentration and its effect on biosurfactant
concentration and surface activity were also investigated.
2.5. Batch fermentation for biosurfactant production
2. Materials and methods
Biosurfactant production was conducted in Erlenmeyer flask
2.1. Microorganism (250 mL) containing 50 mL aliquots of the culture medium. An
aliquot of 5 mL (10%, v/v) of the seed culture was transferred to
B. subtilis LAMI005 used in this study was previously identified the Erlenmeyer flask and the experiments were carried out in a
as a potential producer of biosurfactant [11] and it was isolated rotary shaker (Tecnal-TE240) at 180 rpm, 30 ◦ C for 72 h. Samples
from the tank of chlorination at the Wastewater Treatment Plant were collected at defined intervals of time and submitted to analy-
on Campus do Pici (WWTP-PICI) in the Federal University of Ceará, sis of biomass. Cells were removed by centrifugation at 10,000 × g
Brazil. It was identified by 16S rRNA gene sequence, which was for 20 min and the cell-free supernatant was assayed to determine
deposited at Genbank with the accession number FJ413046. The its pH, carbohydrates, and surfactin concentrations. All assays were
strain was maintained on APGE medium (consisting of 15.0 g/L agar, performed in duplicate and the results represent means ± standard
5.0 g/L peptone, 5.0 g/L glucose, and 2.5 g/L yeast extract) slants at deviations of the two independent experiments.
4 ◦ C and transferred monthly as described in a previous work [11].

2.2. Clarified cashew apple juice preparation 2.6. Analytical methods

Cashew apple juice (CAJ) was obtained by compressing the 2.6.1. Biomass content
cashew apple (Anarcardium occidentale L.). The juice was then cen- Cell growth was determined by measuring the optical densities
trifuged at 5000 × g for 20 min (BIO ENG, BE-6000), filtered using (OD) of samples, using a UV–vis spectrophotometer (20 Genesis,
a 45 ␮m filter paper and clarified using gelatin (10–30%, w/v). The BR) at 600 nm. Biomass concentration, in g/L, was determined by
clarified cashew apple juice (CCAJ) was kept at −10 ◦ C and it con- calibration curve of dry weight (g/L) versus OD [11].
tains basically glucose, fructose, minerals and free amino acids as
described before [9].
2.6.2. Carbohydrate concentration
2.3. Culture medium Substrate concentration (glucose and fructose – Total Reduc-
ing Sugars, TRS) was measured by HPLC using a Waters
Clarified cashew apple juice (CCAJ) was diluted with water, sup- high-performance-liquid chromatographer (Waters, Milford, MA)
plemented with different nutrients (Table 1) and used in this study equipped with a refractive index detector (Model 2414, Waters), a
as culture medium for B. subtilis LAMI005 growth and biosurfac- Supelcogel C610H (30 cm × 7.8 mm) column and a Sigma–Aldrich
tant production. The pH of the medium was adjusted to 7.0 with pre-column (5 cm × 4.6 mm). Ultrapure water (Simplicity 185, Mil-
HCl (3 M) or NaOH (3 M) and it was sterilized at 110 ◦ C for 10 min. lipore, Billerica, MA) with 0.1% v/v of H3 PO4 was used as solvent
When necessary, 0.1% (v/v) of a sterile trace element solution (con- with a flow rate of 0.5 mL/min at room temperature (around 28 ◦ C)
sisting in g/L by ZnSO4 ·7H2 O: 10.95, FeSO4 ·7H2 O: 5.0, MnSO4 ·H2 O: and the sample size was 10 ␮L. The samples were identified by com-
0.39, CuSO4 ·5H2 O: 54, Co(NO3 )2 ·6H2 O: 0.25 and Na2 B4 O7 ·10H2 O: paring the retention times with those of carbohydrate standards.
0.17) was added after autoclavation.

2.4. Inoculum preparation 2.6.3. Emulsification index

Emulsifying index was performed according to Cooper and
B. subtilis LAMI005 was inoculated (spread) onto APGE plates Goldenberg [12], with slight modifications [13]: 2 mL of cell-free
and incubated at 30 ◦ C for 24 h. After this period, three loops full of supernatant were added to 2 mL of kerosene, gasoline or soybean
the strain grown on APGE were transferred to 250-mL Erlenmeyer oil, and the mixture was vortexed for 2 min. After 24 h, the height of
36 D.W. Freitas de Oliveira et al. / Colloids and Surfaces B: Biointerfaces 101 (2013) 34–43

emulsion layer was measured. The emulsification index (IE24 ) was 2.6.9. Vibrational spectroscopy in the infrared region – FT-IR
calculated using Eq. (1). The dried biomaterial was milled with KBr to form a very fine
HEL powder. This powder was then compressed into a thin pellet which
IE24 (%) = × 100 (1) could be analyzed by FT-IR spectra measurement, carried out by
Hs
using a spectrometer model FTLA 2000-ABB, with a spectral win-
where HEL is the height of the emulsion layer and Hs is the height dow from 400 to 4000 cm−1 .
of total solution. A control sample was prepared by using 2 mL of
culture medium before inoculation instead of the cell-free super- 2.7. Calculation of fermentation parameters
natant.
Substrate conversion was calculated according to Fogler [19],
2.6.4. Emulsification activity see Eq. (2):
Emulsification activity was determined according to Cirigliano
and Carman [14], with slight modifications [11]. Samples of the S0 − S
S (%) = × 100 (2)
supernatant free of cells (0.5 mL) were placed in a screw-capped S0
test tube (15 × 125 mm) and diluted with 0.5 mL of 0.1 M sodium where S0 is the initial substrate concentration (glucose plus fruc-
acetate buffer (pH 3.6); 0.25 mL of kerosene was added, the tube tose) and S the substrate concentration (glucose plus fructose) in
was capped, and the mixture was shaken for 2 min at room temper- the samples at each time interval.
ature (around 28 ◦ C). The resulting uniform emulsion was allowed The volumetric productivity (PP and PX ) was calculated as the
to sit for 10, 20, 30, 40 and 50 min, after which its absorbance (A) was ratio of maximum biosurfactant (Pmax , mg/L) or cell concentration
measured at 540 nm with a UV–vis spectrophotometer (20 Gene- (Xmax , g/L) to the fermentation time when the maximum concen-
sis, BR). One unit (U) of emulsification activity was defined as that tration was achieved (tPmax or tXmax , respectively, h):
amount of emulsifier that affected an emulsion with A of 1.0 at
540 nm. Pmax
PP = (3)
tPmax
2.6.5. Surface tension determination Xmax
Surface tension was determined by a Tensiometer (Kruss K6) PX = (4)
tXmax
at 30 ◦ C, according to the De Nöuy ring method [15]. The surface
tension determination was replicated at least three times and it The yield of biosurfactant on cell mass (YP/X , g/g) was defined as:
was performed using cell-free supernatants. Pf − P0
YP/X = (5)
Xf − X0
2.6.6. Surfactin extraction
Surfactin extraction was performed according to the literature where P0 and X0 are the initial product and cell concentration, while
[11], with slight modifications: cells were separated by centrifuga- Pf and Xf , product and cell concentration in the samples at each time
tion at 10,000 × g for 20 min at 15 ◦ C. The pH of 20 mL of cell-free interval.
supernatant was adjusted to 2.0 by adding 3 M HCl. The resultant
solution was maintained at rest for 12 h to allow surfactin to set- 3. Results and discussions
tle. The precipitate was collected by centrifugation at 10,000 × g
for 15 min at 15 ◦ C, and the crude surfactin was obtained. For fur- 3.1. Evaluation of cashew apple juice supplementation for
ther purification, the crude surfactin was dissolved in 8.0 mL of optimal production of biosurfactant by Bacillus subtilis LAMI005
deionized water and it was extracted three times by using an equal in batch cultivation
volume of dichloromethane (Vetec, São Paulo, Brazil). The solvent
layer was harvested and evaporated at room temperature (around In a previous work, the viability of biosurfactant production by
28 ◦ C). The resulting brown-colored paste was dissolved in 2.0 mL B. subtilis LAMI005 grown on a mineral medium formulated using
methanol, achieving a solution containing a semi-purified surfactin. clarified cashew apple juice and several nutrients was demon-
strated [11]. Since the type of medium and growth conditions can
2.6.7. Determination of surfactin concentration influence the type and yield of the biosurfactant [20], in this work,
Surfactin concentration was measured by HPLC using a Waters the influence of juice supplementation on biosurfactant produc-
high-performance-liquid chromatographer equipped with a UV tion was investigated, aiming at reducing costs of production. Fig. 1
detector (Model 2487, Waters), at 205 nm, and a Symmetry C18 shows the profile of substrate conversion, cell concentration, sur-
column (150 × 4.6 mm, 5 ␮m, Waters, Ireland). The mobile phase factin production by B. subtilis LAMI005 and surface tension of the
consisted of 20% v/v TFA (3.8 mM) and 80% v/v acetonitrile. The fermented broth free of cells after 30 h of fermentation using five
elution rate was 1 mL/min at 30 ◦ C and the sample size was 20 ␮L. different culture media, see Table 1.
The identity of the semi-purified surfactin was obtained by using It can be observed in Fig. 1A and B that the strain was able to
commercially available 95% pure surfactin (Sigma–Aldrich) as the grow and produce biosurfactant in all culture medium evaluated.
authentic compound [16]. The amount of surfactin varied from almost 35 mg/L to more than
175 mg/L, depending on the medium used. Furthermore, surface
2.6.8. Determination of critical micelle concentration (CMC) tensions (Fig. 1B) of the cell-free fermented broths were reduced
Different concentrations of the produced surfactin were from 53–54 dyne/cm to 29–30 dyne/cm, except for media D and
obtained by performing several dilutions of cell-free fermented E (around 36 dyne/cm). According to the literature [1,15,21], the
medium, after 48 h of fermentation [17]. Surface tension of effectiveness of a surfactant is determined by its ability in reducing
the resulting solutions was measured at room temperature, as surface tension of water below 35 dyne/cm. Therefore, the reduc-
described above. The CMC was determined by plotting the sur- tion of surface tension to those levels indicates the production of
face tensions as a function of surfactin concentration and it was surface active compounds by B. subtilis LAMI005. Fig. 1B also indi-
found from the intercept of two straight lines extrapolated from the cates a correlation between surfactin concentration and surface
concentration-dependent and concentration-independent sections tension of the fermented broth free of cells, since an expressive
[18]. decrease (below 30 dyne/cm) in surface tension is observed only
 D.W. Freitas de Oliveira et al. / Colloids and Surfaces B: Biointerfaces 101 (2013) 34–43 37

growth are pictured in Fig. 2A and B, respectively, when B. subtilis

LAMI005 was cultivated in media A and B.
A plot of Ln(cell concentration) versus time (data not shown)
allowed to observe an intense cell growth up to 48 h or 24 h of fer-
mentation, for media A and B, respectively, when stationary phase
was achieved. An experimental biosurfactant production (YP/X ) of
0.052 g surfactin/g cells (24 h) and 0.052 g surfactin/g cells (8 h)
were obtained, respectively, for media A and B. The highest sur-
factin concentration (219.99 ± 0.4 mg/L), however, was achieved
when B. subtilis LAMI005 was cultivated in medium B (Fig. 2B).
Other authors [11], who studied the production of surfactin by
the same microorganism using mineral media and clarified cashew
apple juice (MM-CAJC), achieved a surfactin concentration around
125 mg/L, which is lower than the result obtained in this work.
The volumetric production rate (PP , g/(L h)) of surfactin, how-
ever, was higher for medium A (PP = 7.3 mg/(L h)) when compared
to results in medium B (PP = 4.2 mg/(L h)). When B. subtilis LAMI005
was cultivated in medium A (Fig. 2A), maximum production of bio-
surfactant (around 177.93 ± 0.1 mg/L) was observed between 24 h
and 48 h. However, the pH of the fermented broth reached val-
ues below 5.0 after 48 h (data not shown), which may have caused
surfactin precipitation, underestimating surfactin concentration in
the supernatant. Several authors [4,23–25] observed that surfactin
precipitates at acidic pH values (≤5). Wei et al. [26], working with
B. subtillis ATCC 21332 in iron-enriched culture, observed that the
acidification of the broth caused surfactin precipitation resulting
in the disappearance of soluble surfactin at pH lower than 5.0,
whereas the insoluble surfactin was dissolved completely when
the pH was increased back to pH 6.0 by adding NaOH.
Although high concentrations of glucose and fructose were still
present in the culture medium (Fig. 2), an interruption of cellu-
lar growth and biosurfactant formation occurred. At the end of the
process (72 h), total sugar (glucose and fructose) conversion was,
respectively, 30.2% and 32.0% for media A and B. One possible expla-
nation is a limitation on the nitrogen source needed for bacterial
Fig. 1. Influence of supplementation of clarified cashew apple juice on biosurfac- metabolism. Usually the carbon to nitrogen ratio is important to
tant production by B. subtilis LAMI005 cultivated at 30 ◦ C, 180 rpm for 30 h: (A) Cell the product yield of bioactive molecules, which may have limited
concentration and (B) surfactin concentration and surface tension of the fermented
the process. Moreover, the end of the fermentation process may be
broth free of cells. Medium A: clarified cashew apple juice and (NH4 )2 SO4 , medium
B: clarified cashew apple juice, (NH4 )2 SO4 and trace element solution, medium
associated with the formation of toxic by-products accumulation
C: clarified cashew apple juice, (NH4 )2 SO4 and NaCl, medium D: clarified cashew in the medium [27].
apple juice, (NH4 )2 SO4 and MgSO4 ·7H2 O, Medium E: clarified cashew apple juice, Based on the results of surfactin concentration, and considering
(NH4 )2 SO4 , Na2 HPO4 ·7H2 O and KH2 PO4 . The surface tension of the un-inoculated that the cost of production of medium B is higher than the cost
broths incubated in the same conditions was 51–54 dyne/cm. Error bars illustrate
of medium A, which may be an obstacle on the way of industrial
experimental errors (standard deviations), calculated from two independent exper-
iments. application, medium A was selected for further studies.

3.3. Kinetics of biosurfactant production by Bacillus subtilis

for media A–C, when more than 120 mg/L of biosurfactant concen- LAMI005 considering the effect of carbon source concentration
tration was achieved. It is worthy of note that regardless of the
surfactant concentration, a further decrease in the surface tension To investigate the kinetic behavior of B. subtilis LAMI005 growth
will not be observed once the critical micelle concentration (CMC) and biosurfactant production in batch cultivation, CCAJ was diluted
has been reached [22]. Therefore, even though surfactin concen- with water, to achieve different initial concentrations of total
tration in medium A is higher than in media B and C, the surface reducing sugars (glucose and fructose), and supplemented with
tension value in these samples is almost the same probably because ammonium sulfate (1.0 g/L). Sugar concentration varied from 13 g/L
the CMC has been achieved. to 96 g/L (undiluted juice). Fig. 3 shows the experimental results of
Since the highest surfactin production (higher than 140 mg/L) cell growth, surfactin production, substrate consumption and pH
with the best tensoative characteristics (decrease in surface tension of the fermented broth along time.
to less than 30 dyne/cm) was achieved when media A and B were Fig. 4A shows a sigmoidal growth trend for the B. subtilis
used, they were selected for further studies. LAMI005 cells, in which the exponential growth phase and the
stationary phase can be observed. A plot of Ln(cell concentration)
versus time (data not shown) allowed observing that, when more
3.2. Effect of incubation time on growth and biosurfactant than 48.96 g/L of initial sugar concentration was used, the cells
production by Bacillus subtilis LAMI005 entered an exponential growth phase before 10 h and continued up
to 48 h. Moreover, a stationary phase was reached, which started
The influence of incubation time on biosurfactant production at 48 h and lasted until 72 h. When more than 12.71 g/L of initial
and surface activities of culture supernatants was investigated. The sugar concentration was used, the stationary phase was reached
profiles of substrate consumption, surfactin production and cell at 48 h. Maximum cell concentration and volumetric biomass
38 D.W. Freitas de Oliveira et al. / Colloids and Surfaces B: Biointerfaces 101 (2013) 34–43

Fig. 2. Influence of incubation time on biosurfactant production by strains of B. subtilis LAMI005 cultivated at 30 ◦ C, 180 rpm for 72 h: cell concentration (), glucose (䊉) and
fructose () consumption and surfactin production (). Medium A: clarified cashew apple juice and (NH4 )2 SO4 , medium B: clarified cashew apple juice, (NH4 )2 SO4 and trace
element solution. Error bars illustrate experimental errors (standard deviations), calculated from two independent experiments.

productivity increased with increasing sugar concentrations up to biosurfactant production was lower. The highest concentration of
65.04 g/L, when it remained almost constant, see Table 2. surfactin (319.3 ± 0.1 mg/L) was achieved after 72 h of incubation
Similar to cell concentration, maximum product (surfactin) of B. subtilis LAMI005 on CCAJ, at an initial sugar concentration of
concentration was also affected by the increase in sugar con- 65.04 g/L, supplemented with ammonium sulfate (1.0 g/L). Other
centration in the culture medium, Fig. 3B and Table 2. Product authors [28], who studied the production of surfactin by B. subtilis
concentration increased with increasing sugar concentration up ATCC 21332 using glucose as carbon source, achieved maximum
to 65.04 g/L. When sugar concentration was enhanced to 96.1 g/L, surfactin concentrations in various nutritional conditions ranging

Table 2
Main kinetic results of biosurfactant production by Bacillus subtilis LAMI005 at different initial substrate concentration (S0 ). S is substrate conversion, YP/X is the yield of
biosurfactant on cell mass, Pmax is the maximum biosurfactant concentration, Xmax is the maximum biomass concentration, PX is the volumetric biomass productivity and
PP is the volumetric biosurfactant productivity. Experiments were performed in duplicate and the results represent means ± standard deviations of the two independent
experiments.

Run S0 (g/L) pHa S (%)a YP/X (g/g)b Xmax (g/L) Pmax (mg/L) PX (g/L h) PP (mg/L h)

1 12.71 ± 0.05 3.9 ± 0.56 38.6 ± 0.06 0.0148 2.0 ± 0.16 30.7 0.09 1.3
2 48.96 ± 0.09 4.0 ± 0.16 30.2 ± 4.16 0.0523 7.5 ± 0.18 177.9 0.14 3.7
3 65.04 ± 0.29 5.0 ± 0.62 34.3 ± 0.33 0.0450 8.6 ± 0.94 319.3 0.20 4.4
4 96.10 ± 1.42 6.8 ± 0.01 28.7 ± 1.34 0.0354 8.4 ± 0.12 214.2 0.17 4.0
a
After 72 h of incubation.
b
Calculated for the growth phase.
 D.W. Freitas de Oliveira et al. / Colloids and Surfaces B: Biointerfaces 101 (2013) 34–43 39

Fig. 3. Time course profiles of Bacillus subtilis LAMI005 cell growth (A), surfactin production (B), substrate consumption (C) and pH of the fermented broth (D) at 30 ◦ C, 180 rpm
for 72 h. Culture medium was composed of clarified cashew apple juice (CCAJ) + ammonium sulfate (1.0 g/L) with different initial substrate concentrations: () 12.71 g/L, ()
48.96 g/L, () 65.04 g/L, () 96.10 g/L (undiluted juice). Error bars illustrate experimental errors (standard deviations), calculated from two independent experiments.

from 31.2 to 439.0 mg/L. Volumetric biosurfactant productivity (PP ) concentration to this parameter, S0 = 48.96. A decrease or increase
was also affected by the initial substrate concentration in the cul- in S0 was expressed in a lower YP/X . Other authors [28], working
ture medium. PP increased with increasing sugar concentrations up with B. subtilis ATCC 21332, obtained a YP/X ranging from 0.0068
to 65.04 g/L, when it remained almost constant, see Table 2. to 0.075 g/g, depending on the nutritional condition evaluated.
The production of biosurfactant can alternatively be eval- According to some authors [3,16,29,30], glucose and fructose,
uated through the yield of biosurfactant on cell mass (YP/X ), a which are present in cashew apple juice, are suitable substrates for
volume-independent parameter very useful in scale up [10]. It can the synthesis of biosurfactant. However, low substrate conversion
be observed in Table 2 that there is an optimal initial substrate (less than 40%), see Table 2, was achieved in all assays, which indi-
cated that carbon limitation did not occur. Since an increase in cell
concentration, together with higher product formation, is observed
when sugar concentration is increased, it is possible that another
nutrient present in the juice, for instance, amino-acids, favors bio-
surfactant production. Other authors [28], who used 30 g/L glucose
as carbon source and 1.0 g/L of ammonium nitrate as nitrogen
source under aerobic and nitrogen limited condition with B. sub-
tilis ATCC 21332, also observed large amounts of residual glucose
at the end of the assay. It is also important to notice that expressive
substrate consumption, see Fig. 3C, started between 8 and 24 h of
incubation, which may indicate a lag phase have occurred before
this period.
After 72 h of cultivation, the acidification of culture medium was
observed; except for run 4 (S0 = 96.1 g/L), see Table 2 and Fig. 3D. It
can also be observed that media prepared with small amounts of
juice became more acidic. This may be explained by the buffer effect
provided by protein constituents present in cashew apple juice [9].
Another hypothesis for the fast pH decrease is the change from
aerobic to anaerobic respiration. B. subtilis grows in the absence
Fig. 4. Time behavior of the ln(X/X0 ) () and biosurfactant concentration (P)
of oxygen using nitrate ammonification and various fermentation
() during the cultivation of B. subtilis LAMI005 on clarified cashew apple juice
(S0 = 65.04 g/L) supplemented with ammonium sulfate (1.0 g/L) at 30 ◦ C and 180 rpm. processes. Lactate, acetate, acetoin, ethanol, and succinate are the
40 D.W. Freitas de Oliveira et al. / Colloids and Surfaces B: Biointerfaces 101 (2013) 34–43

Fig. 5. Emulsifying activity (U) of surfactin produced by B. subtilis LAMI005 at 30 ◦ C, 180 rpm for 72 h. Culture medium was composed of clarified cashew apple juice
(CCAJ) + ammonium sulfate (1.0 g/L) with different initial substrate concentrations: (A) 12.71 g/L, (B) 48.96 g/L, (C) 65.04 g/L, (D) 96.10 g/L (undiluted juice). Error bars illustrate
experimental errors (standard deviations), calculated from two independent experiments.

main fermentation products. The change to anaerobic respiration to form a stable emulsion is not associated with surface tension
could be induced by the accumulation of acidic compounds such as reduction [36]. The rate of emulsification determines the ability
pyruvate and acetate from fermentation [31]. of the biosurfactant to form emulsions, and the ability to stabi-
Some authors suggest [24,32] that biosurfactant production by lize emulsions is also a parameter used to evaluate the surface
B. subtilis is associated with growth, while other authors observed activity of surfactants. There are some examples in the literature
that the biosurfactant production occurs mainly at the end of of biosurfactant that both lowered surface tension and stabilized
the exponential growth phase [33] or at the stationary phase of emulsions. On the other hand, some Bacillus species produce a type
biomass growth [34]. Several factors are responsible for this behav- of emulsifier that may not cause an appreciable reduction of the
ior, among which we should mention the type of organism being surface tension of water but it is able to form an excellent emul-
used and the culture medium in which it is grown. Therefore the sion with kerosene, for instance [12]. Therefore, the surface activity
analysis of the profiles of ln(X/X0 ) versus time (t) and product con- of biosurfactant produced by B. subtilis LAMI005 cultivated in CCAJ
centration (P) versus time, during biosurfactant production by B. supplemented with ammonium sulfate, at different initial substrate
subtilis LAMI005 on CCAJ, supplemented with ammonium sulfate concentrations, was characterized by measuring the surface ten-
at a concentration of 1.0 g/L, were compared in order to distin- sion, emulsifying activity and emulsification index of the fermented
guish between the three broad kinetic groups proposed by Elmer broth free of cells.
and Gaden [35]. Results are shown in Fig. 4, for S0 = 65.04.1 g/L. It Table 3 shows the emulsification index measured at the fer-
can be observed that ln(X/X0 ) × t and P × t have similar profiles, in mentation time where the highest biosurfactant concentration was
other words, there is a connection between the curves. Production achieved, which varied depending on the initial substrate concen-
kinetics is parallel to the biomass kinetics to a large extent during tration used. The biosurfactant produced by LAMI005 showed high
logarithmic growth. Similar profiles were obtained for the other ini- emulsification index (IE24 > 50%) on kerosene and soybean oil, but
tial substrate concentration studied (data not shown). Therefore, not against gasoline. Most microbial surfactants are substrate spe-
in the conditions assayed here, surfactin production by B. subtilis cific, solubilizing or emulsifying different hydrocarbons at different
LAMI005 is associated with growth. rates [37]. Best results for emulsification index (IE24 ) were obtained
by using kerosene (67%), followed by soybean oil (64%). Similar
3.4. Evaluation of the surface activity of the biosurfactant results were obtained by other authors [9], after 72 h of cultiva-
produced by B. subtilis LAMI005 cultivated in CCAJ supplemented tion, 65% of kerosene emulsification was obtained, indicating that
with ammonium sulfate this biosurfactant has an emulsifying activity.
The ability to stabilize an emulsion is an indication that the
The stability of oil/water emulsions is also widely used as an microorganism is producing biosurfactant [38]. Results of emulsi-
indicator of surface activity, although the ability of a molecule fying capacity and stability can be seen in Fig. 5. The biosurfactant
 D.W. Freitas de Oliveira et al. / Colloids and Surfaces B: Biointerfaces 101 (2013) 34–43 41

Table 3
Emulsification index (IE24 ) of the biosurfactant produced by Bacillus subtilis LAMI005 grown in clarified cashew apple juice, supplemented with ammonium sulfate (1.0 g/L)
at 30 ◦ C and 180 rpm, using different initial concentrations of substrate (S0 ). Experiments were performed in duplicate and the results represent means ± standard deviations
of the two independent experiments.

Run S0 (g/L) Fermentation time (h) IE24 (%)

Soybean oil Kerosene Gasoline

1 12.71 ± 0.05 24 0.0 25 ± 0.0 0.0

2 48.96 ± 0.09 48 58 ± 0.12 67 ± 0.0 37 ± 0.02
3 65.04 ± 0.29 72 53 ± 0.06 66 ± 0.1 26 ± 0.19
4 96.10 ± 1.42 72 64 ± 0.00 67 ± 0.0 0.0

produced by B. subtilis LAMI005 showed an emulsification activ- time for the different experimental conditions evaluated. It can be
ity higher than 1.0 U, except when S0 = 12.71 g/L, was used. It is observed that, in all assays (runs 1–4) B. subitilis LAMI005 produced
worthnotice that in this condition, low amounts (concentration) biosurfactant, since surface tension vales below 35 dyne/cm were
of biosurfactant was produced. Higher emulsifying capacity is also detected.
observed on the sample collected after 48 h of inoculation. The It is possible to observe in Fig. 6 that as surfactant concentra-
emulsion stability is always very high and independent of the initial tion increases in the culture medium (see Fig. 3B), lower values of
substrate concentration on the culture medium. Results obtained in surface tension are achieved up to a minimum value (30 dyne/cm).
this work are similar to those described for the synthetic commer- In some cases, when using 65.04 and 96.1 g/L of initial substrate
cial surfactants tested by Amaral et al. [39]. They were also similar concentration, the surface tension remained almost unaltered from
to those described to other biosurfactant from Yarrowia lipolytica 8/24 h until the end of cultivation. After this time of cultivation,
[39], C. lipolytica IA [40] and Nocardia sp. L-417 [41]. although surfactin concentration continued to grow, no further
reduction was observed in the surface tension of the free-cell broth.
3.5. Minimum surface tension and critical micelle concentration Similar behavior was observed by other authors [44] and they sug-
considering the effect of carbon source concentration gested that the biosurfactant concentration in the broth probably
reached or exceeded its CMC at this cultivation time. According to
It is important to distinguish between an effective biosurfac- the literature [18], as surfactant concentration increases the sur-
tant and an efficient biosurfactant. Effectiveness is measured by face tension of the surfactant solution decreases up to a certain
the minimum value to which the surface tension can be reduced, value and then becomes almost constant due to the interface sat-
whereas efficiency is measured by the biosurfactant concentration uration with the surfactant molecules. Barros et al. [23], working
required to produce a significant reduction in the surface tension with B. subtilis LB5, grown in cassava wastewater, report that the
of water. The latter can be determined from the CMC of the bio- biosurfactant tested exhibited excellent surface activity, reducing
surfactant [18]. Moreover, the ability to reduce surface tension the tension of water from 72.31 to 27.01 dyne/cm. Queiroga et al.
below 35 dyne/cm is one of the criteria used to select biosurfactant- [45] using B. subtilis for the production of biosurfactant in the pres-
producing microorganisms [1]. Several factors influence the type ence of oil, reported a reduction in surface tension of the fermented
and amount of surfactant, among which we highlight the grow- broth from 53 dyne/cm to 25.7 dyne/cm. Gouveia et al. [46], work-
ing conditions and nutrient availability in the medium [42,43]. In ing with 13 biosurfactant-producing strains, and using glycerol and
this work, the effect of carbon source concentration on biosurfac- glucose as carbon source, observed a reduction in surface tension
tant effectiveness and efficiency was investigated. Fig. 6 shows of the medium from 58 dyne/cm to 30 dyne/cm. In this work, the
the results of surface tension of the cell-free supernatant along produced surfactin showed to be a highly effective biosurfactant
since its crude solution could lower the surface tension of the fer-
mented broth to 30 dyne/cm. However, it was documented that
surfactin can lower the surface tension to even lower values, reach-
ing 27 dyne/cm [1]. This contradiction may be attributed to the low
purity of the surfactin preparation, since it was not extracted from
the culture medium.
By definition, the CMC is the surfactant concentration at which
an abrupt increase in surface tension is observed. Regardless of the
surfactant concentration, a further decrease in the surface tension
will not be observed once the CMC has been reached [22]. Zhang
and Miller [47] reported that the concentration of biosurfactant
required to reach the CMC is typically between 1 and 200 mg/L,
while the interfacial tension (oil/water) is around 1 and 30 dyne/cm
[48]. Table 4 shows the critical micelle concentration (CMC) of sur-
factin produced by B. subtilis LAMI005 at this work, as well as SDS
and several biosurfactant isolated from different B. subtilis strains,
including a standard sample (surfactin from Sigma–Aldrich).
It can be observed in Table 4 that surface active com-
pounds can reduce the surface tension of water to values around
27–37 dyne/cm and their CMCs range from 15 to 180 mg/L. Puri-
fied surfactin (standard) is even more efficient since its CMC could
Fig. 6. Surface tension of the fermented broth free of cells after 72 h of fermen- reach 7.8 mg/L, see Table 4. Variations in the values of CMC (13, 22,
tation of clarified cashew apple juice (CCAJ) + ammonium sulfate (1.0 g/L) with and 17 mg/L) for surfactin have been described by other authors
different initial substrate concentrations: () 12.71 g/L, () 48.96 g/L, () 65.04 g/L,
[51–53]. The literature [4,52] reports that these variations are
() 96.10 g/L (undiluted juice). Error bars illustrate experimental errors (standard
deviations), calculated from two independent experiments. observed depending on the nature of the solvent used to dissolve
42 D.W. Freitas de Oliveira et al. / Colloids and Surfaces B: Biointerfaces 101 (2013) 34–43

Table 4
Minimal surface tensions and critical micelle concentration (CMC) obtained for several surface active compounds. Experiments were performed in duplicate and the results
represent means ± standard deviations of the two independent experiments.

Surfactant Surface tension (dyne/cm) CMC (mg/L) Reference

Run 1 – S0 = 12.71 g/L 34.7 ± 0.0 10.24 This work

Run 2 – S0 = 48.96 g/L 30.0 ± 0.0 16.90 This work
Run 3 – S0 = 65.04 g/L 30.6 ± 0.5 63.00 This work
Run 4 – S0 = 96.10 g/L 30.0 ± 0.0 21.41 This work
SDS (synthetic) 37.0 2.888 [49]
Surfactin from Bacillus subtilis isolate bs5 42.5 15.6 [18]
Surfactin from Bacillus subtilis 27 25.0 [32]
Standard surfactin (Sigma–Aldrich) 27 7.8–20.7a [50]
a
CMC = 7.5–20 ␮mol/L and molecular weight = 1036.34 g mol [50].

industry, supplemented with ammonium sulfate. Since economic

considerations are one of the main problems concerning the expan-
sion of the biosurfactant market, CCAJ appears as an alternative
water soluble low cost medium. Based on the literature [7], this
result is interesting once water-soluble substrates are cheaper than
hydrocarbons and are preferred because single-phase fermenta-
tion is simpler than biphasic fermentation. Analyses of the culture
supernatants along cultivation time showed that surfactin produc-
tion was influenced by the amount of CCAJ used to prepare the
culture medium, reaching a maximum concentration of 319.3 mg/L.
Furthermore the emulsification index (IE24 ) value of the biosurfac-
tant was found to be 65% demonstrating its capacity to emulsify
kerosene and soybean oil. The biochemical analysis of the semi-
purified biosurfactant indicates that surfactin was produced by B.
subtilis LAMI005 under the conditions assayed in this work.

Acknowledgments

The authors kindly acknowledge the financial aid and research

Fig. 7. A comparison of vibrational spectroscopy in the infrared region – FT-IR, scholarships given by Conselho Nacional de desenvolvimento
spectrograms of standard surfactin (dashed line) and semi-purified extracellular
Científico e Tecnológico (CNPq), Fundação Cearense de Apoio
biosurfactant (solid line) produced by B. subitilis LAMI005 grown in clarified cashew
apple juice (CCAJ) supplemented with ammonium sulfate (1.0 g/L) at 30 ◦ C, 180 rpm ao Desenvolvimento Científico e Tecnológico (FUNCAP) and
and S0 = 65.04 g/L. Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
(CAPES).
surfactin as well as the purity of surfactin preparation. By com-
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