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World J Microbiol Biotechnol (2008) 24:1901–1907

DOI 10.1007/s11274-008-9691-9

ORIGINAL PAPER

Biosurfactants, an help in the biodegradation of hexadecane?


The case of Rhodococcus and Pseudomonas strains
Murielle Bouchez-Naı̈tali Æ Jean-Paul Vandecasteele

Received: 14 December 2007 / Accepted: 6 February 2008 / Published online: 20 February 2008
 Springer Science+Business Media B.V. 2008

Abstract The roles of the extracellular biosurfactants bacterium. The roles of biosurfactants thus differ widely
produced by two bacterial strains, Pseudomonas aerugin- among bacteria degrading hydrophobic compounds.
osa GL1 and Rhodococcus equi Ou2, in hexadecane uptake
and biodegradation were compared. For this purpose, cell Keywords Alkane biodegradation  Bioremediation 
hydrophobicity and production of glycolipidic biosurfac- Cell hydrophobicity  Emulsification  Insoluble substrate 
tants were evaluated during bacterial growth on Pseudosolubilization
hexadecane, as well the effects of these biosurfactants on
culture supernatants properties i.e., surface and interfacial
tensions, and emulsification and pseudosolubilization Introduction
capacities. The results showed that the role of biosurfac-
tants was different in these two strains and was directly Hydrocarbons are the most widespread organic pollutants
related to the hydrophobicity of the bacterial cells con- in the environment because of their massive utilization and
cerned. Extracellular biosurfactants produced by strain their biodegradation is currently studied in detail. The
R. equi Ou2 had only a minor role in hexadecane degra- microorganisms which degrade these hydrocarbons are
dation. Direct interfacial accession appeared to be the main numerous and possess original characteristics due in par-
mechanism for hexadecane uptake by the hydrophobic ticular to the low solubility of their substrate, that requires
cells of strain R. equi Ou2. On the contrary, the biosurf- specific mechanisms of transfer to microbial cells. The
actants produced by P. aeruginosa GL1 were required for uptake mechanisms of hydrophobic compounds have been
growth on hexadecane, and their pseudosolubilization described (Haferburg et al. 1986; Hommel 1994) and only
capacity rather than their emulsification capacity was two modes of uptake are relevant to long-chain alkanes
involved in substrate degradation, allowing uptake from because of their very low solubility (9  10-4 mg l-1 at
hexadecane micelles by the hydrophilic cells of this 25C for hexadecane): direct contact of alkane droplets
with the bacterial cell (interfacial accession) and biosur-
factant-mediated uptake. Studies have been performed to
J.-P. Vandecasteele—in retirement
distinguish between these modes (Goswami and Sing 1991;
M. Bouchez-Naı̈tali (&) Song et al. 2006) but the role of biosurfactant needs further
AgroParisTech, UMR763 Bioadhésion et Hygiène des investigations.
Matériaux, 25 avenue de la République, 91300 Massy Cedex, The production of biosurfactants by hexadecane-
France
degrading strains is a frequent phenomenon (Bouchez-
e-mail: murielle.naitali@agroparistech.fr
Naı̈tali et al. 1999; Puntus et al. 2005) which occurs both
M. Bouchez-Naı̈tali in microorganisms possessing hydrophobic or hydrophilic
INRA, UMR763 Bioadhésion et Hygiène des Matériaux, 25 cell surfaces (Neu and Poralla 1990; Bouchez-Naı̈tali et al.
avenue de la République, 91300 Massy Cedex, France
1999). In a previous study, we suggested different roles for
J.-P. Vandecasteele the biosurfactants produced in relation with strain hydro-
1 allée des Ormes, 78112 Fourqueux, France phobicity: (i) Pseudosolubilization i.e., formation of

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1902 World J Microbiol Biotechnol (2008) 24:1901–1907

micelles, a mechanism well suited for hydrocarbon transfer hexadecane (conducted as previously described) was added
to hydrophilic strains since the hydrophobic compounds to the culture medium. Quadruplicates were performed for
contained in the micelles are surrounded by the hydrophilic each condition of supplementation.
outer layer formed by the biosurfactant; (ii) emulsification
which increases the surface area of the hydrocarbon phase
Analyses
and, as a consequence, the biodegradation rates in hydro-
phobic strains (Bouchez-Naı̈tali et al. 1999). In addition,
Growth analysis
biosurfactants may also bind to the cell surface and make it
more hydrophobic (Al-Tahhan et al. 2000; Hua et al. 2003;
Because growth was heterogeneous for some strains
Mulligan 2005), thus increasing interaction with hydro-
growing on hydrophobic substrates, it was indirectly
phobic compounds. In the present work, we characterized
monitored by nitrate depletion. For homogenous GL1
the pseudosolubilization and emulsification capacities of
cultures, growth was also evaluated by OD600 measurement
biosurfactants produced by two strains selected for their
after removal of culture supernatant and suspension in the
different cell hydrophobicity after growth on hexadecane,
same volume of physiological water. In the latter case, two
Pseudomonas aeruginosa GL1 and Rhodococcus equi Ou
parameters were determined, the lag phase (k) and the
2. In the case of P. aeruginosa GL1, we also tested the
specific growth rate (l). The lag phase was defined as the
effect of supplementation with biosurfactants on the
time required to reach an OD600 = 0.1. The specific
growth on hexadecane.
growth rate was the slope of the straight line obtained when
plotting OD600 against time on a semi-logarithmic scale.
Materials and methods
Nitrate determination
Strains
Residual nitrate concentrations of culture supernatants
Pseudomonas aeruginosa GL1 and Rhodococcus equi Ou2, were determined with nitrate reductase using commercial
two strains using hexadecane as sole source of carbon and kits (Boehringer Mannheim, Mannheim, Germany). Each
energy, were isolated from polluted soils (Bouchez-Naı̈tali result was the mean of three measurements conducted on
et al. 1999). They were stored at -80C in 50% v/v independently grown cultures.
glycerol/physiological salt solution.
Glycoside determination
Cultures
Glycoside concentrations were measured in supernatants of
Cultures were carried out in series of 250 ml flasks con- cultures by the colorimetric method of Dubois et al.
taining 100 ml of mineral salt medium MMS4 (Bouchez- (1956). Glucose was used as a standard and results were
Naı̈tali et al. 1999) and hexadecane (12 g l-1 of carbon) as expressed in glucose equivalents. For a given sample, the
sole source of carbon and energy and inoculated with 3% v/ measurements were made three times in separate tubes and
v of cultures grown on the same medium. They were the standard deviation was within 5%. Each result was the
incubated on a rotary shaker (160 rpm) at 30C. Kinetic mean of nine measurements (three measurements on three
analysis of hexadecane biodegradation was performed by independently grown cultures).
harvesting (centrifugation, 15 min, 4,000 rpm), at various
times, the cells of three flasks and filtering the supernatants
(0.22 lm Analypore filters, OSI, Elancourt, France) before Interfacial tension and surface tension measurements
analysis.
Besides being cultivated in the conditions just described, Surface tensions (cs) and interfacial tensions against
strain GL1 was also grown using another mode of inocu- hexadecane (ci) of filtered supernatants were determined at
lation and with a supplementation in biosurfactants. The 30C by the Wilhelmy plate method, using a K-12 tensi-
flasks were inoculated with 3 ml of a suspension in phys- ometer (Krüss, Hamburg, Germany). Stabilization was
iological water (optical density at 600 nm, OD600 = 1) allowed to take place until the standard deviation of 10
prepared with cells grown on trypticase soy agar (bio- successive measurements was below 0.4 mN m-1. Surface
Mérieux, Marcy l’Etoile, France) and washed twice to tensions and interfacial tensions values of a given sample
prevent transfer of extracellular biosurfactant with the were the average of 10 determinations after stabilization
inoculum. For supplementation with biosurfactant, a and each result was the mean of three values obtained on
known volume of a filtered supernatant of a GL1 culture on independently grown cultures.

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World J Microbiol Biotechnol (2008) 24:1901–1907 1903

Measurement of the capacity of pseudosolubilization Results and discussion

The capacity of the culture supernatants to pseudosolubi- Kinetic studies of the effects of biosurfactant
lize hexadecane was evaluated by a method adapted from production in Rhodococcus equi Ou2
that described by Reddy et al. (1983). Hexadecane (2 ml)
was mixed with 20 ml of supernatant on an orbital shaker During all the growth phase of cultures on hexadecane,
(160 rpm) at 30C during 24 h. Then, the excess of indirectly monitored by nitrate depletion, strain Ou2 was
hydrocarbon was separated from the aqueous phase by found to be hydrophobic (above 70% of adhesion to
centrifugation (2 h, 40,000 g) and filtration (0.22 lm hexadecane, Fig. 1a). This property was probably due to
Analypore filters). Several successive filtrations were cell-associated glycolipids (Lang and Philp 1998). Strain
sometimes necessary in order to obtain a limpid aqueous Ou2 also released biosurfactants as illustrated by the
phase. The hexadecane pseudosolubilized was analysed by decrease of interfacial tension of supernatants and by the
gas phase chromatography (ionisation flame detector) after presence of extracellular glycosides (Fig. 1a). The glyco-
extraction with cyclohexane (25% v/v, 24 h). Isothermic sides produced were shown by migration on thin layer
separation (160C) was performed using an HP5 column chromatography to be glycolipids, a group of biosurfac-
(30 m 9 0.32 mm 9 0.25 lm, Hewlett Packard, USA). tants frequently produced by Rhodococcus strains, as for
For a given sample, three independent extractions and example trehalose mycolates in Rhodococcus erythropolis
measurements were made and the standard deviation was (Lang and Philp 1998). The evolution in time of surface
within 10%. Each result presented was the mean of nine tension of supernatants was similar to that of interfacial
measurements (three measurements on three independently
grown cultures). a
γ i (mN/m), glycosides 25 100
and nitrates (x 10 g/l)
80

Hydrophobicity (%)
Measurement of the capacity of emulsification 20

15 60
The capacity of the supernatants to emulsify hexadecane
was evaluated according to an adaptation of the method
10 40
described by Reddy et al. (1983). One ml of supernatant
was vortex-shaken (2 min) with 200 ll of hexadecane. The
5 20
OD600 was then read directly and after 2 and 24 h of set-
tling. For a given sample, the measurements were made
0 0
three times in separate tubes and the standard deviation was 0 25 50 75 100 125 150 175 200
within 10%. Each result presented was the mean of nine Time (h)
measurements (three measurements on three independently
grown cultures). b 250 3.5

3.0
C16 pseudosolubilized

Emulsification (OD 600)


200
Cell hydrophobicity 2.5
150
(mg/l)

2.0
Cell hydrophobicity was measured by bacterial adhesion to
hexadecane according to the BATH method (Rosenberg 100 1.5

1984) in the experimental conditions previously described 1.0


(Bouchez-Naı̈tali et al. 1999). For a given sample, the 50
0.5
measurements were made three times in separate tubes and
the standard deviation was within 5%. As above, each 0 0.0
0 25 50 75 100 125 150 175 200
result was the mean of nine measurements.
Time (h)

Fig. 1 Culture characteristics during hexadecane degradation by


Statistical analyses Rhodococcus equi Ou2. (a) Cell hydrophobicity (¤), interfacial
tensions of culture supernatants (9), glycosides produced (D) and
The standardized format for variance analysis, ANOVA, residual nitrate in culture supernatants (h); (b) capacity of the
and the determination of the critical probability associated supernatant to pseudosolubilize hexadecane (j) and to emulsify
hexadecane, OD600 being measured after 30 s (e), 2 h (s) and 24 h
with the F-test (P value), were performed using Stat- (D) of settling. Mean ± standard deviation. Small (non-visible)
graphics (ManugisticsTM, Maryland, USA). standard deviations are within the symbols

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tension and its value decreased from 66 to 27 mN m-1 a 25 100


(data not shown).

γ i (mN/m), glycosides
The surfactants produced by strain Ou2 were able to 20 80

and nitrate (x 10 g/l)

Hydrophobicity (%)
pseudosolubilize and emulsify hexadecane (Fig. 1b) but
these properties were observed only at a late stage (after 15 60
about 50%) of growth, indicating that the biosurfactants
produced were not necessary to initiate degradation of 10 40
hexadecane. These results clearly point to direct interfacial
accession as the main mechanism of hexadecane uptake for 5 20
strain Ou2. They are in agreement with those of other
authors and indicate, according to Lang and Philp (1998), 0 0
0 25 50 75 100 125 150 175 200
that the major mode of alkane uptake by rhodococci is
Time (h)
likely to be direct contact with large oil droplets, although a
contribution can also be made by emulsification. In addi- b 250 3.5
tion, it can be noted that, in several biosurfactant-producing

C16 pseudosolubilized
3

Emulsification (OD 600)


strains including Rhodococcus erythropolis, Noordman and 200
Janssen (2002) observed that addition of their own bio- 2.5
surfactant did not stimulate hexadecane degradation. The 150

(mg/l)
2
production of extracellular glycolipids may be, in fact, a
1.5
consequence of the synthesis of cell wall-bound hydro- 100
phobic glycolipidic constituents which contribute to cell 1
hydrophobicity as stated above, and constitute in this way a 50
0.5
physiological response favouring growth on insoluble
hydrophobic compounds. In agreement with this interpre- 0 0
tation, we showed that cells of strain Ou2 were hydrophilic 0 25 50 75 100 125 150 175 200
and did not produce surfactants during growth on glycerol Time (h)
(Bouchez-Naı̈tali et al. 1999). However, extracellular bio- Fig. 2 Culture characteristics during hexadecane degradation by
surfactants may also contribute to hydrocarbon uptake in a Pseudomonas aeruginosa GL1. Symbols as in Fig. 1
different way by limiting cell flocculation at high cell
densities. Only little flocculation was observed in strain
Ou2 cultures whereas we demonstrated that several Rho- of strain GL1, the cells of biosurfactant-producing cultures
dococcus strains that did not produce any extracellular of microorganisms were more generally reported to be
biosurfactant formed flocs during growth on hexadecane, hydrophobic (Pruthi and Cameotra 1997; Al-Tahhan et al.
resulting in a limitation in substrate uptake (Bouchez- 2000; Hua et al. 2003). According to Al-Tahhan et al.
Naı̈tali et al. 2001). (2000), the high hydrophobicity of a biosurfactant-pro-
ducing strain of P. aeruginosa could be explained by the
Kinetic studies of the effects of biosurfactant removal of lipopolysaccharides from the outer membrane
production by Pseudomonas aeruginosa GL1 induced by these biosurfactants.
Concerning the rhamnolipids produced by P. aerugin-
The characteristics of cultures of strain GL1 were found osa GL1, kinetic monitoring showed that both interfacial
very different from those of strain Ou2. As shown in and surface tensions remained low (below 7 and 37 mN
Fig. 2a, during all the growth period on hexadecane of m-1, respectively) from the beginning of the culture
strain GL1, the cells remained hydrophilic (below 20% of (Fig. 2a) when the inocula came from hexadecane-grown
adhesion to hexadecane). The strain produced extracellular cultures and thus contained biosurfactants. Only interfacial
glycosides in concentrations which increased with time tensions are illustrated on Fig. 2a. The capacity of the
from 0.1 to 1.1 g l-1 of glucose equivalents (Fig. 2a). They biosurfactants produced in the culture to pseudosolubilize
were shown to be glycolipids and were identified as and emulsify hexadecane varied with time (Fig. 2b). Dur-
rhamnolipids (Arino et al. 1996), which are frequently ing the active growth phase (the 44 first hours that
produced by Pseudomonas aeruginosa strains (Mulligan corresponded to 70% of total growth), the pseudosolubili-
2005; Van Hamme et al. 2006). Although hydrophilic cell- zation capacity of the supernatant increased very quickly
surface properties have been reported in the literature for up to a value of 170 mg l-1, suggesting that it was
emulsifier- or biosurfactant-producing cultures (Neu and involved in hexadecane degradation. A similar situation
Poralla 1990; Bouchez-Naı̈tali et al. 1999) like in the case was observed in P. aeruginosa Au1 (data not shown) and

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World J Microbiol Biotechnol (2008) 24:1901–1907 1905

also described for Pseudomonas N1 (Goswami and Singh 7


1991). An emulsification capacity was also observed but
6
later in time. As illustrated by the OD decrease with
increasing time of settling, the emulsions formed were 5

Growth (OD600)
unstable (Fig. 2b). Moreover, the emulsification property
appeared at the end of the active growth phase (after 70% 4
of nitrate consumption), a point making unlikely its
3
involvement in hexadecane degradation. All these results
clearly indicate that pseudosolubilization, and not emulsi- 2
fication, is the key property of rhamnolipids involved in
1
hexadecane degradation. It should allow direct interaction
between hydrophilic cells of strain GL1 and hydrophilic 0
micelles containing hexadecane, as previously supposed 0 50 100 150 200 250
(Bouchez-Naı̈tali et al. 1999). In fact, although emulsifi- Time (h)
cation and pseudosolubilization are generally considered as
Fig. 3 Kinetics of growth of Pseudomonas aeruginosa GL1 on
acting together in alkane uptake, the importance of their
MMS4 + hexadecane unsupplemented (9) or supplemented with 3%
respective roles is probably dependent on the hydropho- (open symbols) or 10% (closed symbols) of a filtered supernatant of a
bicity of the cells concerned. culture grown during 44 h (square) or 188 h (circle), respectively
After reaching a maximum, the pseudosolubilization
and emulsification capacities of the supernatants decreased capacity (Fig. 2b). When referring to Fig. 2a, these addi-
although the concentration in glycolipids still increased. tions corresponded respectively to amounts of glycosides
These unexpected observations, have also been observed (expressed in glucose equivalents in the culture medium) of
by other authors (Goswami and Singh 1991; Prabhu and 5 and 17 mg l-1 for the 44 h-culture and of 34 and
Phale 2003). In the present case, they may result from 113 mg l-1 for the 188 h-culture. Biosurfactant addition
changes in the composition of the excreted products since also increased the specific growth rates. When plotting
strain GL1 is known to produce several types of extracel- OD600 of the cultures versus time on a semi-logarithmic
lular rhamnolipids and also to excrete a significant amount scale, a portion of straight line was observed in all cases in a
of proteins (Arino et al. 1996). range of OD600 values between 0.1 and 1, with a good
correlation coefficient (0.982 \ r2 \ 0.997). The values of
Influence of biosurfactant supplementation on growth l observed (Table 1) were quite different when the super-
of P. aeruginosa GL1 natants came from cultures of various ages. The
supernatants from a 188 h-old culture led to higher specific
During our experiments, we observed that cultures on growth rates than those from a 44 h-old culture in spite of
hexadecane of Pseudomonas GL1—and not of Rhodococ- their lower pseudosolubilization capacity (Fig. 2b). Thus, it
cus OU2—presented important growth delays (more than clearly appears that supplementation with its own biosur-
1 week) when inoculated with cells cultivated on a solid factant favoured hexadecane degradation by strain GL1, a
rich medium (Fig. 3), whereas cultures inoculated with an
aliquot of a culture grown on hexadecane in liquid medium Table 1 Influence of the initial biosurfactant supplementation on the
did not present such lag phases (Fig. 2a). As discussed growth on hexadecane of Pseudomonas aeruginosa GL1
above, biosurfactants were brought into the cultures by Supernatanta Lag phase (h)b Specific
liquid GL1 inocula grown on hexadecane, facilitating the growth rate (h-1)b
beginning of the degradation. In order to study this point, No [200 n.d.
various amounts (3 and 10%) of filtered supernatants 44 h, 3% 77 ± 4 0.031 ± 0.006
obtained from hexadecane-grown cultures of Pseudomonas 44 h, 10% 13 ± 1* 0.041 ± 0.007
GL1 (44- and 188-h-old) were used to supplement the 188 h, 3% 11 ± 2* 0.094 ± 0.005
MMS4/hexadecane medium. The inocula were then pre-
188 h, 10% \2 0.081 ± 0.005
pared from washed cells grown on a solid medium in order
a
to remove extracellular biosurfactants from bacterial The first number indicates the age (h) of the culture from which the
supernatant originated. The second number indicates the amount (in
inocula. % v/v) of the supernatant added in the new culture
As illustrated in Fig. 3 and Table 1, the growth lag (k) b
Mean ± standard deviation, determined as described in Materials
was strongly shortened by the addition of biosurfactants and and Methods on four independent cultures. All the values are statis-
the effect increased with the amount of biosurfactants added tically different (P \ 0.05) except those with an asterisk (*)
(Fig. 2a), but not always with their pseudosolubilization n.d., Not determined

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result also observed for P. aeruginosa UG2 (Noordman and Bouchez-Naı̈tali M, Rakatozafy H, Marchal R, Leveau JY, Vandec-
Janssen 2002). Furthermore, the data suggest an interesting asteele JP (1999) Diversity of bacterial strains degrading
hexadecane in relation to the mode of substrate uptake. J Appl
point in the case of strain GL1, namely that the favourable Microbiol 86:421–428
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pseudosolubilization capacity. Rhodococcus equi: the importance of cell flocculation. Micro-
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thank Daniel Ballerini for having access to the tensiometer equipment Noordman WH, Janssen DB (2002) Rhamnolipid stimulates uptake of
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