International Review of Chemical Engineering (I.RE.CH.E.), Vol. 5, N.

4
ISSN 2035-1755 July 2013
Manuscript received and revised June 2013, accepted July 2013 Copyright 2013 Praise Worthy Prize S.r.l. - All rights reserved
310
Biosurfactant Production from Industrial Residues
Using Microorganisms Isolated from Oil Wells


Ana Katerine de C. Lima Lobato
1
, Andra F. Almeida
2
, Mrcio S. Bezerra
3
,
Laerte M. Barros J nior
4
, Luiz Carlos L. Santos
5
, Gorete R. Macedo
6


Abstract Biosurfactants can be used in several industries, such as the petrochemical industry.
It is therefore of great interest to improve biosurfactant production processes to make
biosurfactants economically competitive compared to synthetic surfactants. To achieve economic
competitiveness, it is important to select microorganisms that can produce biosurfactants, to
establish an appropriate and economical media and optimized cultivation conditions for the
biosurfactant synthesis. In this work, the production of biosurfactant by microorganisms isolated
from oil wells was studied in a medium composed of sugar cane molasses and petroleum as
carbon sources. The kinetics of microbial growth were evaluated by monitoring the cell
concentration, substrate consumption and surface tension variation, which could indicate
biosurfactant production. A reduced cultivation time of 8 hours with a significant surface tension
reduction (30.77%) was achieved with molasses as a low-cost substrate in the presence of
petroleum. The obtained surface tension reduction was similar to that obtained by the previous
literature. However, to the best of our knowledge, no report has yet been published with a reduced
cultivation time of 8 hours and a significant surface tension reduction using molasses as a low-
cost substrate in the presence of petroleum. Copyright 2013 Praise Worthy Prize S.r.l. - All
rights reserved.

Keywords: Biosurfactant, Microorganism, Molasses, Petroleum, Surface Tension


I. Introduction
Biosurfactants are amphoteric substances produced
from different sources of carbon by microorganisms such
as bacteria, yeasts and filamentous fungi. Little is known
about what leads microorganisms to produce these
metabolites, but one reason may be the need to solubilize
insoluble substrates in media [1].
These molecules act preferentially on the interface
between fluid phases with different polarities, and for the
case of an oil reservoir, would link the oil/water or
water/oil interfaces and reduce the surface and interface
tensions between these phases [2], [3], [4].
This action strongly affects the rheology of the
medium and improves the oil recovery factor [5]. The
main classes of biosurfactants are glycolipids,
lipopeptides, lipoproteins, phospholipids, fatty acids, and
polymeric and particulate surfactants [6]. Some
researchers prefer to group biosurfactants by their
molecular weights [7], [8].
For example, low-molecular-weight biosurfactants are
generally glycolipids, and rhamnolipids are the main
example of this class [9].
This type of biosurfactant is produced by different
species of Pseudomonas [10].
Biosurfactants compete with synthetic surfactants in
three main aspects: cost, functionality and production
volume (productivity) [11].
Compared to synthetic surfactants, biosurfactants
perform better under extreme temperatures and pH and at
high salinity [12], [13]. In addition, biosurfactants have
low toxicity and are biodegradable [3], [14], [15], [16].
In spite of these advantages, the economical
production of biosurfactants has several barriers that
must be overcome, such as low yield, high cost for large-
scale production, the need for sterilization, and process
control problems including foam formation, product
recovery and purification problems and the difficulty in
conducting a chemical analysis of the products, given the
complex nature of the produced metabolite [17].
According to Kosaric [6], the greatest use of
biosurfactants in the oil industry is for MEOR (Microbial
Enhanced Oil Recovery).
This tertiary oil recovery technique consists of using
microorganisms or some of their metabolic products to
recover residual oil. Polymers and surfactants produced
by these microorganisms could reduce the oil-rock
surface tension; this reduction mitigates capillary forces
and improves oil flow through the rock pores [11], [14],
[18].
For an economical biosurfactant production process, it
is important to identify the microorganisms that produce
biosurfactant and to optimize the cultivation medium and
the fermentation process itself [16]. It is estimated that
raw materials account for 10 to 30% of the overall
production cost of biosurfactants [19], [20].

Ana Katerine de C. Lima Lobato et al.

Copyright 2013 Praise Worthy Prize S.r.l. - All rights reserved International Review of Chemical Engineering, Vol. 5, N. 4
311
Several renewable substrates, especially those from
residue-generating industries, have been used for
microorganism cultivation and biosurfactant production
on an experimental scale in an attempt to make the
produced metabolite more competitive with its synthetic
counterpart [4], [16], [21], [22], [23], [24]. The main
drawback of using alternative substrates as a culture
medium is finding a residue with high nutrient value that
allows cell growth and product accumulation [19].
Olive Oil Mill Effluent (OOME), animal fat, frying
oil, soapstock, molasses, milk serum and other starch-
rich residues [3], [25] have been used as substrates for
biosurfactant production. Manioc liquid residue, soy
seed, beet, sweet potato and potato sugars have also been
used. Other residues include wheat bran, rice and wheat
stalks; soy, corn and rice husks; sugar cane and manioc
bagasse; coffee processing residues such as coffee pulp;
fruit processing residues such as apple and grape pulp;
pineapple and carrot processing wastes, banana wastes;
and oil mill wastes, such as coconut, soy, peanut and
canola [19], [26].
These metabolites produced from inexpensive
renewable substrates, obtained through high-yield lead to
economically viable processes and make biosurfactants a
promising product.
In this work, Pseudomonas aeruginosa sp. isolated
from oil wells was studied for biosurfactant production.
It was cultivated in Erlenmeyer flasks under agitation to
identify, through kinetic variables, a better culture
medium with a substrate of sugar cane molasses and
petroleum.
II. Materials and Methods
II.1. Microorganism
The Pseudomonas aeruginosa sp. strain isolated from
PETROBRAS oil wells in the AU100 zone, Mossor,
Brazil, was selected as a promising microorganism. The
strain belongs to UN-RNCE (Rio Grande do Norte/Cear
Business Unit) and is maintained by the Department of
Antibiotics of the Federal University of Pernambuco.
II.2. Cultivation Media
Cultivation media was prepared from yeast extract,
agar, sucrose, molasses, petroleum (from AU100 zone
PETROBRAS - Mossor/Brazil) and medium A, which
was prepared from 100 mM phosphate buffer (pH 7) with
1 g/l of (NH
4
)
2
SO
4
, 0.25 g/l of MgSO
4
.7H
2
O and 1 ml/l
of a stock salts solution containing (g/100 ml) 0.01 of
EDTA, 0.30 of MnSO
4
.H
2
O, 0.01 of FeSO
4
.7H
2
O, 0.01
of CaCl
2
, 0.01 of CoCl
2
.6H
2
O and 0.01 of ZnSO
4
.7H
2
O.
II.3. Culture Maintenance
The culture was maintained in solid medium with
PCA and 25% (w/v) of agar. Cell renewal was performed
by transferring the cells from the master culture to the
sub-master culture and incubating the cells at 38C for 48
hours. After incubation, the sub-master culture was
stored at 5C.
II.4. Cell Propagation
Erlenmeyer flasks with capacities of 500 mL and
containing 150 mL of culture medium were inoculated
with cells from the sub-master culture. The flasks were
placed in a shaker (New Brunswick) at 38C for 24
hours, under agitation at 250 rpm. This culture was used
as an inoculum for the biosurfactant production. A period
of 24 hours was established (in previous work) because
at this time, the cells are in the exponential phase.
II.5. Cultivation Procedures
Erlenmeyer flasks with aeration rates of 0.4
(V
medium
/V
flask
) were inoculated with 7% (v/v) of
inoculum. After inoculation, the flasks were placed in the
shaker and were agitated at 250 rpm and 38C for 96
hours. Samples were taken every 4 hours for the first 12
hours and every 12 hours afterwards to determine
biomass concentration, substrate concentration and
surface tension variation.
II.6. Growth Kinetics
To study the growth kinetics of the microorganism,
several experiments were conducted to accompany
culture growth in different carbon sources, such as
sucrose, molasses and petroleum, and the conditions of
the inoculum and the medium were varied (see Table I).

TABLE I
DESCRIPTION OF THE EXPERIMENTS PERFORMED
Experiment Inoculum medium Culture medium
1 Medium A, 1%S Medium A, 1%S
2 Medium A, 1%S Medium A, 1%S, 1%P
3 Medium A, 1%M, 1%YE Medium A, 1%M
4 Medium A, 1%M, 1%YE Medium A, 1%M, 2%P
*S Sucrose; M Molasses; P Petroleum; YE Yeast Extract
II.7. Cell Concentration
The cell concentration was obtained with the dry
weight method [27]. Samples (2 ml) taken from the
shaker were centrifuged (16.000 g for 15 min) and were
washed with distilled water. After a second washing, the
samples were placed in a furnace at 105C for 24 hours
and weighed until a constant weight (dry weight) was
obtained. Therefore, the cell concentration (g/l) was
obtained by dividing the dry weight by the sample
volume.
II.8. Substrate Concentration
The DNS method is based on the reduction of 3,5
dinitrosalicylic acid to 3-amino-5-nitrosalicylic acid with
the simultaneous oxidation of the aldehyde group of the

Ana Katerine de C. Lima Lobato et al.

Copyright 2013 Praise Worthy Prize S.r.l. - All rights reserved International Review of Chemical Engineering, Vol. 5, N. 4
312
sugar to a carboxylic group; a red color develops and is
measured spectrophotometrically at 600 nm [28]. The
original procedure determines glucose and fructose.
Because our sample contains sucrose, a preliminary
hydrolysis is needed. The hydrolysis was performed in a
50 mL volumetric balloon by the addition of 1 mL of the
sample, 0.5 mL of HCl and 6 mL of water. The final
solution was heated at 70C for 10 minutes and then
cooled to ambient temperature and neutralized with 4N
NaOH solution. The flask was filled to the mark with
distilled water.
II.9. Surface Tension
The surface tension was measured in a Du Noy
tensiometer through the ring method [29]. The analyses
were conducted at 30C on the supernatant obtained after
centrifugation of the raw sample.
The surface tension reduction provides an indirect
measurement of biosurfactant production.
II.10. Emulsification Index
The emulsification activity was determined by
measuring the emulsification index (E24) at 25C [24].
In general, 4 ml of crude oil was poured into a test
tube containing 6 ml of biosurfactant solution or
commercial surfactant. After being vigorously vortexed
for 2 min, the test tube was kept still for 24 h and the
heights of the emulsion, oil and aqueous zones were
measured. The emulsion index (E24) was then calculated
from the ratio of the height of the emulsion zone to the
total of the heights of the oil, emulsion, and aqueous
zones.
II.11. Kinetic Parameters
The maximum specific growth rate was estimated
with the software Lissage [30] based on linearization of
the exponential phase of cell growth. The factor for the
conversion of substrate to cell was calculated by dividing
the change in biomass by the substrate concentration.
III. Results and Discussion
The evaluation of biosurfactant production processes
using low-cost substrate and real environmental
conditions is important from an economic and
application perspective [31]. To this, the study explored a
set of variations in medium composition, from the
presence of simple substrates, such as pure sucrose, to a
potentially more economical carbon source easily
assimilated by the cells (sugar cane molasses) and a
combination of those substrates with crude oil.
The use of crude oil in the medium is expected
because the working strain has been isolated from oil
wells and also because of the possibility for using the
produced biosurfactant in oil industry activities, such as
MEOR (Microbial Enhanced Oil Recovery) and
bioremediation of oil spills. The carbon source, medium
composition, cultivation time and knowledge of the
kinetics are essential variables in the overall assessment
of the process [32], [33].
The kinetic study of the cell growth (experiments 1 to
4) is shown in Fig. 1.
Fig. 1 shows that the exponential growth phase occurs
in the first 24 hours of cultivation, with the exception of
experiment 2. In experiment 2, no yeast extract was used
in the medium during preparation of the inoculum, and
oil was added to the cultivation medium. In this case, the
existence of a lag phase was observed; this phase
indicated the reaction of the microorganism to the
medium, which is now more complex.
However, this was not observed in the experiment 4,
in which the medium contained petroleum and the
inoculum preparation medium contained yeast extract.
The inclusion of this source of essential amino acids
and proteins, which are responsible for cell growth, was
strategic for the elimination a lag phase in a more
complex medium. For most of the conditions studied,
after 36 hours of cultivation, the cell concentration
begins to decline phase.
This decline is attributed to cell death as a function of
the substrate limitation or to biosurfactant action on the
cell wall. The adherence of biosurfactants to cell surfaces
caused deterioration in the integrity of the cell membrane
and also cause breakdown in the nutrition cycle [34],
[35].
Experiments 1 to 4 show that in most of the cases, the
cells developed adequately under the conditions studied.
The substrate was exhausted, and the biomass
concentration reached a plateau after 48 hours of
cultivation (Fig. 2).
The reduced surface tension coincides with the
exponential growth phase between 0 and 12 hours (Fig.
3).

0 4 8 12 24 36 48 60 72 84 96 108
2
4
6
8
10
12
14
16
18
B
i
o
m
a
s
s

(
1
0
-
3
g
/
L
)
Time (h)
Experiment 1 Experiment 2
Experiment 3 Experiment 4


Fig. 1. Biomass production curves for experiments 1 to 4 at 250 rpm
and 38C. Experiment 1 (Medium A, 1% S); Experiment 2 (Medium A,
1% S, 1% P); Experiment 3 (Medium A, 1% M); Experiment 4
(Medium A, 1% M, 2% P)

Ana Katerine de C. Lima Lobato et al.

Copyright 2013 Praise Worthy Prize S.r.l. - All rights reserved International Review of Chemical Engineering, Vol. 5, N. 4
313
0 4 8 12 24 36 48 60 72 84 96 108
0,0
0,8
1,6
2,4
3,2
4,0
4,8
6
8
10
12
14
S
u
b
s
t
r
a
t
e

(
g
/
L
)
Time (h)
Experiment 1 Experiment 2
Experiment 3 Experiment 4


Fig. 2. Substrate consumption curves for experiments 1 to 4 at 250 rpm
and 38C. Experiment 1 (Medium A, 1% S); Experiment 2
(Medium A, 1% S, 1% P); Experiment 3 (Medium A, 1% M);
Experiment 4 (Medium A, 1% M, 2% P)
0 4 8 12 24 36 48 60 72 84 96 108
30
40
50
60
70
S
u
r
f
a
c
e

t
e
n
s
i
o
n

(
d
y
n
a
/
c
m
)
Time (h)
Experiment 1 Experiment 2
Experiment 3 Experiment 4


Fig. 3. Surface tension variation curves for experiments 1 to 4 at 250
rpm and 38C. Experiment 1 (Medium A, 1% S); Experiment 2
(Medium A, 1% S, 1% P); Experiment 3 (Medium A, 1% M);
Experiment 4 (Medium A, 1% M, 2% P)

This relation suggests that the biosurfactant synthesis
is associated with the growth because parallel relations
could be observed between biomass production, substrate
consumption, and the decrease in the surface tension of
the culture broth.
Experiment 1 shows only a small reduction in surface
tension (Fig. 3) when only sucrose is used as a carbon
source. This result is most likely because of the easy
assimilation of sucrose, which resulted in the absence of
biosurfactant synthesis.
Some cultivation parameters, such as the maximum
specific growth rate (
Xmax
), the substrate-to-cell
conversion factor (Y
X/S
), the surface tension reduction
percentage (STR %) and the relation between the
minimum surface tension and time (ST
min
/t), were
calculated, and they are shown in Table II.
As shown in Table II, the highest specific cell growth
rate occurred in experiment 4 (
Xmax
= 0.0885). In
addition, experiment 4 presented a surface tension
reduction percentage (STR) of 30.77% for the medium
containing molasses and petroleum. This result indicated
that the microorganism was well adapted to the proposed
medium.
Experiments 1 and 3, which substitutes sucrose for
molasses, show reductions in the surface tension of
12.9% and 44.44%, respectively.
This result suggests a substantial increase in
biosurfactant and also indicates the potential for
molasses as substrate in this process. Ghurye et al. [36]
produced biosurfactant from a mixed culture with a
carbon source of molasses, and they obtained a surface
tension reduction of approximately 30% after 48 hours of
cultivation.
Considering molasses as substrate, Makkar and
Cameotra [23] showed a surface tension reduction in the
medium of 50% after 24 hours. Kashkouli et al. [37]
applied an artificial neural network to model the
fermentation parameters for biosurfactant production by
Bacillus subtilis ATCC 6633 from sugar cane molasses.
In this bioconversion process, the highest reduction of
surface tension (45%) was noticeable at 48 hours.
Experiment 3 achieved a tension reduction (44.44% in 24
hours) comparable to the result obtained by these
authors.
Several studies have shown significant reductions in
surface tension when they adopted low-cost substrates
for biosurfactant production; Fox and Bala [22] managed
to reduce surface tension by 60% in 72 hours by
cultivating Bacillus subtilis with potato.
By cultivating Pseudomonas aeruginosa, Santa Anna
et al. [38] observed that the use of babassu oil resulted in
a surface tension reduction of 31% in 168 hours whereas
with glycerol, a reduction of 48.2% was observed in the
same time period.
Nitschke and Pastore [39] evaluated a cassava flour
processing effluent as a substrate for surfactant
production by two Bacillus subtilis strains; a surface
tension reduction in the medium of 44.9% was obtained
in 24 hours with B. Subtilis ATCC 21332. J ayachandran
and George [40] analyzed rhamnolipid biosurfactants
produced by submerged fermentation with orange fruit
peelings as the sole carbon source, and they found a
surface tension reduction of approximately 45% after 168
hours of fermentation.
Rocha et al. [41] evaluated cashew apple juice for
production of biosurfactants by Bacillus subtilis
LAMI008 and achieved a surface tension reduction of
21.37% in 24 hours.

TABLE II
CULTIVATION PARAMETERS FOR EXPERIMENTS 1 TO 7
Experiment

Xmax

(h
-1
)
Y
X/S
STR
(%)
ST
min
(dyn.cm
-1
)/t(h)
1 0.0223 0.0009 12.90 27/36
2 0.0429 0.0017 47.69 34/48
3 0.0467 0.0028 44.44 30/24
4 0.0885 0.0013 30.77 36/8


Ana Katerine de C. Lima Lobato et al.

Copyright 2013 Praise Worthy Prize S.r.l. - All rights reserved International Review of Chemical Engineering, Vol. 5, N. 4
314
With respect to the addition of petroleum to the
medium, experiment 2 (1% sucrose and 1% petroleum)
showed the greatest reduction in surface tension
(47.69%) followed by experiment 4 (30.77%), where the
medium contained 1% molasses and 2% petroleum.
Some studies were reported in the literature on surface
tension reduction when more complex substrates are used
for the growth of microorganism producers of
biosurfactants; Bento et al. [42] isolated biosurfactant-
producing microorganisms from soil contaminated with
diesel oil and evaluated the capacity for reduction of the
surface tension of the medium when cultivated in a
medium containing diesel oil as the only carbon source.
Among the microorganisms, Acinetobacter junii
showed the highest surface tension reduction (37.33%)
after 168 hours of cultivation. Samadi et al. [43]
cultivated Brevibacterium sp. S-34 in different carbon
sources (glucose, glycerol, molasses, gasoline, canola oil
and residual oil) and found that the greatest reduction in
the surface tension of the medium was obtained when
glycerol was the only carbon source (56.52%) after 72
hours of cultivation.
The highest substrate-to-cell conversion factor was
obtained in experiment 3 (Y
X/S
=0.0028). In this case,
only molasses was used in the medium; this result
indicates that the absence of petroleum favors the
conversion of sugar into cells.
By analyzing the ratio of the surface tension reduction
to the cultivation time (ST
min
/t), it was found that for
experiment 2, with sucrose and petroleum in the medium,
a STR of 47.69% was obtained, and the lowest point of
the surface tension was at 48 hours of cultivation.
The experiment that contains only molasses as a
carbon source (experiment 3), had a STR of 44.44%, and
the lowest surface tension was recorded at 24 hours of
cultivation. However, in experiment 4, with molasses and
petroleum in the medium, a STR of 30.77% was reached
at only 8 hours of fermentation. Considering petroleum
as part of the substrate, Silva et al. [44] studied the
biosurfactant production potential of the Pseudomonas
fluorescens strain and found a surface tension reduction
of 57.14% after 60 hours of cultivation.
Notably, although the highest surface tension
reduction occurred in experiment 2 (47.69%), which uses
sucrose and petroleum as the carbon source, this
reduction only occurred after 48 hours of cultivation. By
using only molasses as the carbon source (experiment 3),
the surface tension reduction was practically the same as
in experiment 2 (44.44%) but in half the time (24 hours)
of cultivation.
Experiment 4, which uses molasses and petroleum in
the medium, also showed a significant surface tension
reduction (30.77%) after only 8 hours of cultivation. The
latter experiment is of greatest interest to this study
because a satisfactory surface tension reduction was
attained in a lower time and because molasses, which is a
non-conventional substrate, and petroleum were used in
the medium. The emulsification activity of the
biosurfactant produced in the experiment 4 with molasses
and petroleum in the medium (Medium A, 1% M, 2% P)
was 100%. The ability to form emulsions with oil
suggests that the biosurfactant produced by fermentation
with Pseudomonas aeruginosa sp. is a good candidate for
a cleaning and emulsifying agent and that the
biosurfactant has potential applications in microbial
enhanced oil recovery (MEOR), environment
preservation [12], medicine and the cosmetics industry.
Although some investigators have managed to obtain
similar surface tension reductions, the cultivation time
required was far greater than that observed in the present
study; this result indicates the potential of the
Pseudomonas aeruginosa sp. strain and the conditions
adopted here.
IV. Conclusion
The Pseudomonas aeruginosa sp. strain proved to be a
promising producer of biosurfactants from sugar cane
molasses. The highest STR of the medium coincides with
the exponential growth phase; thus, biosurfactant
synthesis is associated with cell growth. The optimum
cultivation conditions for biosurfactant synthesis were
found to use inoculum with 1% molasses and 1% yeast
extract in medium A, and a cultivation medium with the
same composition and 2% petroleum; under these
conditions, a STR of 30.77% was reached after only 8
hours of cultivation.
Compared to the current literature, the surface tension
reduction was similar to that obtained by other
researchers that used an alternative substrate for
biosurfactant production. However, minimization of the
cultivation time is another important process parameter
for reducing the production cost of biosurfactant. To the
best of our knowledge, no report has yet been published
with a reduced cultivation time of 8 hours and a surface
tension reduction of approximately 30% with a low-cost
substrate in the presence of petroleum.
Acknowledgements
The authors thank FINEP, CTPETRO and
PETROBRAS for financial support.
References
[1] F. Sineriz, R. K. Hommel, H. P. Kleber, Production of
biosurfactants. In: Encyclopedia of Life Support Systems (Eolls
Publishers, 2001).
[2] G. Bognolo, Biosurfactants as emulsifying agents for
hydrocarbons, Colloids Surf. A. 152 (1999) 41-52.
[3] J . D. Desai, I. M. Banat, Microbial production of surfactants and
their commercial potential, Microbiol. Mol. Biol. Rev. 61 (1997)
47-64.
[4] R. S. Makkar, S. S. Cameotra, An update on the use of
unconventional substrates for biosurfactant production and their
new applications, Appl. Microbiol. Biotechnol. 50 (1998) 520-
529.
[5] M. Benincasa, A. Abalos, I. Oliveira, A. Manresa, Chemical
structure and biological activities of the biosurfactant produced
by Pseudomonas aeruginosa LBI from soapstock, Antonie Van
Leeuwenhoek. 85 (1) (2004) 1-8.

Ana Katerine de C. Lima Lobato et al.

Copyright 2013 Praise Worthy Prize S.r.l. - All rights reserved International Review of Chemical Engineering, Vol. 5, N. 4
315
[6] N. Kosaric, Biosurfactants in Industry, Pure Appl. Chem. 64 (11)
(1992) 1731-1737.
[7] H. Bach, Y. Berdichevsky, D. Gutnick, An exocellular protein
from the oildegrading microbe Acinetobacter venetianus RAG-1
enhances the emulsifying activity of the polymeric bioemulsifier
Emulsan, Appl. Environ. Microbiol. 69 (5) (2003) 2608-2615.
[8] E. Z. Ron, E. Rosenberg, Biosurfactants and oil bioremediation,
Curr. Opin. Biotechnol. 13 (2002) 249-252.
[9] N. Christofi, I. B. Ivshina, Microbial surfactants and their use in
field studies of soil remediation, J. Appl. Microbiol. 93 (2002)
915-929.
[10] G. Sobern-Chavz, F. Lpine, E. Dziel, Production of
rhamnolipids by Pseudomonas aeruginosa, Appl Microbiol
Biotechnol 68 (2005) 718725.
[11] I. M. Banat, R. S. Makkar, S. S. Cameotra, Potencial commercial
application of microbial surfactants, Appl. Microbiol. Biotechnol.
53(2000) 495-508.
[12] F. A. Kronemberger, C. P. Borges. D. M. G. Freire, Fed-batch
biosurfactant production in a bioreactor, I.RE.CH.E 2(4) (2010)
513-518.
[13] R. S. Makkar, S. S. Cameotra, I. M. Banat, Advances in
utilization of renewable substrates for biosurfactant production,
AMB Express 1(5) (2011) 1-19.
[14] M. Nitschke, G. M. Pastore, Biossurfactantes: propriedades e
aplicaes, Quim. Nova 25 (5) (2002) 772-776.
[15] S. S. Cameotra, R. S. Makkar, J . Kaur, S. K. Mehta, Adv. Exp.
Med. Biol., 672 (2010) 261-280.
[16] D. F. Coelho, S. A. Barbosa. E. Silveira, R. R. Souza, E. B.
Tambourgi, Biosurfactant production from unconventional
resources: a short overview, (2012) International Rewiew of
Chemical Engineering (IRECHE) 4(2), pp. 175-183.
[17] M. G. Healy, C. M. Devine, R. Murphy, Microbial production of
biosurfactants, Resour. Conservat. Recycl. 18(1996) 41-57.
[18] A. Perfumo, I. Rancich, I.M. Banat, Possibilities and challenges
for biosurfactants use in petroleum industry, Adv. Exp. Med. Biol.,
672 (2010) 135-145.
[19] R. S. Makkar, S. S. Cameotra, An update on the use of
unconventional substrates for biosurfactant production and their
new applications, Appl. Microbiol. Biotechnol. 58 (2002) 428-
434.
[20] S. N. Al-Bahry, Y. M. Al-Wahaibi, A. E. Elshafie, A. S. Al-
Bemani, S. J . J oshi, H. S. Al-Makhmari, H.S. Al-Sulaimani,
Biosurfactant production by Bacillus subtilis B20 using date
molasses and its possible application in enhanced oil recovery,
Int. Biodeterior. Biodegrad. 81 (2013) 141146.
[21] P. S. Babu, A. N. Vaidya, A. S. Bal, Kinetics of biosurfactant
production by Pseudomonas aeruginosa strain BS2 from
industrial wastes, Biotechnol. Lett. 18 (3) (1996) 263-268.
[22] S. Fox, G. A. Bala, Production of surfactant from bacillus ATCC
21332 using potato substrates, Bioresour. Technol. 75 (2000)
235-240.
[23] R. S. Makkar, S. S. Cameotra, Utilization of molasses for
biosurfactant production by two bacillus strains at thermophilic
conditions, J Am Oil Chem Soc. 74(1997) 887-889.
[24] R. M. Patel, A. J . Desai, Biosurfactant production by
Pseudomonas aeruginosa GS3 from molasses, Lett. Appl.
Microbiol. 25(1997) 91-94.
[25] S. Maneerat, Production of biosurfactants using substrates from
renewable-resources, Songklanakarin. J. Sci. Technol. 27 (3)
(2005) 675-683.
[26] S. Mukherjee, P. Das, R. Sem, Towards commercial production
of microbial surfactants, Trends Biotechnol. 24 (11) (2006) 509-
515.
[27] W. Borzani, W. Schmidell, U. A. Lima, E. Aquarone,
Biotecnologia Industrial Fundamentos (Edgard Blucher, 2001).
[28] G. L. Miller, Use of dinitrosalicylic acid reagent for determination
of reducing sugar, Anal. Chem. 31 (1959) 426428.
[29] P. L. Du Noy, An interfacial tensiometer for universal use, J.
Gen. Physiol. 7 (1925) 625-633.
[30] D. A. Simes, G. Arroyo, J . L. Uribelarrea, Logiciel de Lissage
(1994). Software.
[31] B. Hahn-Hgerdal, K. Karhumaa, C.U. Larsson, M. Gorwa-
Grauslund, J . Grgens, W. H. Van Zyl, Role of cultivation media
in the development of yeast strains for large scale industrial use,
Microb. Cell Fact. 4 (2005) 1-16.
[32] W. Schmidell, U. A. Lima, E. Aquarone, W. Borzani, Engenharia
Bioqumica - Biotecnologia Industrial (Edgard Blcher, 2001).
[33] M. H. Pinto, R. G. Martins, J . A. V. Costa, Avaliao cintica da
produo de biossurfactantes bacterianos, Quim. Nova 32 (2009)
2104-2108.
[34] S. T. Hingley, A. T. Hastie, F. Kueppers, Effect of ciliostatic
factors from Pseudomonas aeruginosa on rabbit respiratory cilia,
Infect. Immun. 51 (1986) 254-258.
[35] E. Z. Gomaa, Antimicrobial activity of a biosurfactant produced
by Bacillus licheniformis strain M104 grown on whey, African
Afr J Microbiol Res 6(20) (2012) 4396-4403.
[36] G. L. Ghurye, C. Vipulanandan, R. C. Willson, A practical
approach to biosurfactant production using nonaseptic
fermentation of mixed cultures, Biotechnol. Bioeng. 44 (1994)
661-666.
[37] Y. R. Kashkouli, A. Mogharei, S. Mousavian, F. Vahabzadeh,
Performance of Artificial Neural Network for predicting
fermentation characteristics in biosurfactant production by
Bacillus subtilis ATCC 6633 using sugar cane molasses, Int J
Food Eng 7(6) (2011) 1-19.
[38] L. M. Santa Anna, G. V. Sebastian, E. P. Menezes, T. L. M.
Alves, E. P. Menezes, D. M. G. Freire, Production of
biosurfactants from Pseudomonas aeruginosa PA1 isolated in oil
environments, Braz. J. Chem. Eng 19 (2002) 159-166.
[39] M. Nitschke, G. M. Pastore, Biossurfactantes: propriedades e
aplicaes, Appl. Biochem. Biotechnol. 112 (2004) 163-172.
[40] S. George, K. J ayachandran, Analysis of rhamnolipid
biosurfactants produced through submerged fermentation using
orange fruit peelings as sole carbon source, Appl. Biochem.
Biotechnol. 158 (2009) 694705.
[41] M. V. P. Rocha, R. V. Barreto, V. M. M. Melo, L. R. B.
Gonalves, Evaluation of cashew apple juice for surfactin
production by Bacillus subtilis LAMI008, Appl. Biochem.
Biotechnol. 155 (2009) 366378.
[42] F. M. Bento, F. A. O. Camargo, B. C. Okeke, W. T.
Frankenberger J r, Diversity of biosurfactant producing
microorganisms isolated from soils contaminated with diesel oil,
Microbiol. Res. 160 (2005) 249-255.
[43] N. Samadi, N. Abadian, A. Akhavan, M. R. Fazeli, A. Tahzibi, H.
J amalifar, Biosurfactant production by the strain isolated from
contaminated soil, J Biol Sci. 7 (2007) 1266-1269.
[44] T. A. L. Silva, H. W. C. Arajo, E. B. Tambourgi, C. A. A. Silva,
G. M. C. Takaki, Potencial biotecnolgico de uma nova linhagem
de Pseudomonas fluorescens na produo de biossurfactante
utilizando petrleo como substrato, Exacta 7 (2009) 31-37.
Authors information
1
Federal University of Bahia Postgraduate Program of Chemical
Engineering - R. Prof. Aristides Novis, 2, 2 andar, Federao, CEP
40210-630, Salvador/BA, Brazil. Email: katycarvalho@hotmail.com

2
Federal University of Paraba Department of Biotechnology -
Campus I, Cidade Universitria, Castelo Branco, CEP 58.051-900 J oo
Pessoa/PB, Brazil.

3
Federal Institute of Education, Science and Technology of Rio Grande
do Norte - R. Manoel Lopes Filho, 773, Valfredo Galvo, CEP: 59380-
000, Currais Novos/RN, Brazil.

4
Petrobras E&P Business Unit for Rio Grande do Norte & Cear - R.
Euzbio Rocha,1000, Cidade da Esperana, CEP 59164-100, Natal/RN,
Brazil.

5
Federal University of Bahia - Department of Materials Science and
Technology - R. Prof. Aristides Novis, 2, 3 andar, Federao, CEP
40210-630, Salvador/BA, Brazil.

6
Federal University of Rio Grande do Norte Department of Chemical
Engineering - Campus Universitrio, CEP 59.072-970 Natal/RN,
Brazil.

Ana Katerine de C. Lima Lobato et al.

Copyright 2013 Praise Worthy Prize S.r.l. - All rights reserved International Review of Chemical Engineering, Vol. 5, N. 4
316
Ana Katerine de Carvalho Lima Lobato
(Natal, 1973) has a bachelors degree in
Chemical Engineering from the Federal
University of Rio Grande do Norte, Natal, Brazil
(1999), a master's in Chemical Engineering from
the Federal University of Rio Grande do Norte,
Natal, Brazil (2003) and a PhD in Chemical
Engineering from the Federal University of Rio
Grande do Norte, Natal, Brazil (2010) with a sandwich period at the
University of Manchester, United Kingdom. Currently, she is a
researcher and an assistant professor in the Post-Graduate Program in
Chemical Engineering at the Federal University of Bahia.
She has experience in Chemical Engineering with an emphasis in
Biochemical Engineering. Her research interests cover the following
subjects: petroleum, biofuels, biosurfactant production, antibiotic
production and metabolic flux analysis.
Prof. Lobato is a member of the technical chamber of biological
products at the general management of medicines national agency of
sanitary surveillance.

Andrea Farias de Almeida has degree in
Chemical Engineering from the Federal
University of Rio Grande do Norte (2003), a
masters in Chemical Engineering from the
Federal University of Rio Grande do Norte
(2005) and a Ph.D. in Chemical Engineering
from the Federal University of Rio Grande do
Norte (2010). She is currently a researcher and
assistant professor in the Department of Biotechnology at the
Biotechnology Center of the Federal University of Paraba.
She has experience in Chemical Engineering with an emphasis on
biochemical processes in the following subjects: fermentative
processes, production of animal cells, in vitro production of
baculoviruses, AgMNPV and SfMNPV, and biopesticides.

Mrcio Silva Bezerra (So Paulo, 1979) has a
bachelors degree in Chemical Engineering from
the Federal University of Rio Grande do Norte
(2004), a masters in Chemical Engineering
from the Federal University of Rio Grande do
Norte (2006) and a Ph.D. in Chemical
Engineering from the Federal University of Rio
Grande do Norte (2012). He is currently a
professor at the Federal Institute of Education, Science and Technology
of Rio Grande do Norte (IFRN). He has experience working on the
following topics: biosurfactants, agroindustrial residues and
fermentation in submerged cultures.

Laerte Medeiros Barros Jnior has a
bachelors degreein Chemical Engineering from
the Federal University of Rio Grande do Norte
(1999), a masters in Chemical Engineering
from the Federal University of Rio Grande do
Norte (2001) and a Ph.D. in Chemical
Engineering from the Federal University of Rio
Grande do Norte (2008). He is currently a
process engineer at PETROBRAS.
He has experience in Chemical Engineering with an emphasis in
Environmental Engineering in the following topics: utilities,
wastewater treatment and the manufacturing process of urea.
Luiz Carlos L. Santos (Rio de J aneiro, 1976)
has a bachelors degree in Chemical
Engineering from the Federal University of Rio
Grande do Norte, Natal, Brazil (1999), a
master's in Chemical Engineering from the
Federal University of Rio Grande do Norte,
Natal, Brazil (2002) and a PhD in Chemical
Engineering from the University of Manchester,
United Kingdom (2007). He has experience in Chemical Engineering
with an emphasis in Oil and Gas. His research interests cover the
following subjects: petroleum, biofuels, surfactants, microemulsions,
adsorption, catalysis and ceramic membranes. Dr. Santos is a member
of the Society of Petroleum Engineering.

Gorete Ribeiro de Macedo has a bachelors
degree in Chemical Engineering from the
Federal University of Rio Grande do Norte
(1978), a master's in Chemical Engineering from
the University of So Paulo (1982) and a Ph.D.
in Chemical Engineering from the University of
So Paulo (1998). She is currently professor at
the Federal University of Rio Grande do Norte.
She has experience in Chemical Engineering with an emphasis in
Biochemical Processes in the following subjects: enzyme production,
bioethanol, biosurfactants, obtaining oligosaccharides by fermentation,
cultivation of recombinant microorganisms, biological treatment of
liquid and solid waste. Prof. Macedo was the coordinator of the
Graduate Program in Chemical Engineering Federal University of Rio
Grande do Norte (2001-2005) and of the Ph.D in Biotechnology of the
Northeast Biotechnology Network (2008-2012).