Journal of Applied Microbiology 1999, 86, 421428

Diversity of bacterial strains degrading hexadecane in

relation to the mode of substrate uptake
M. Bouchez-Natali1, H. Rakatozafy1*, R. Marchal2, J.-Y. Leveau1 and J.-P. Vandecasteele2
1

Ecole Nationale Superieure des Industries Agro-alimentaires, Laboratoire de Microbiologie Industrielle, Massy
cedex, and 2Division Chimie et Physico-chimie Appliquees, Institut Francais du Petrole, Rueil Malmaison
cedex, France
6783/06/98: received 29 June 1998, revised 5 October 1998 and accepted 7 October 1998
M . B OU C HE Z- N AI TA L I, H. R AK AT O ZA FY , R. MA R CH AL , J. -Y . LE VE A U A ND J .- P.
V AN DE C AS TE E LE . 1999. The relative distribution of the modes of hydrocarbon uptake, used
by bacteria of the environment for the degradation of long-chain alkanes, has been
evaluated. The first mode of uptake, direct interfacial accession, involves contact
of cells with hydrocarbon droplets. In the second mode, biosurfactant-mediated transfer,
cell contact takes place with hydrocarbons emulsified or solubilized by
biosurfactants. Sixty-one strains growing on hexadecane were isolated from polluted
and non-polluted soils and identified. The majority (61%) belonged to the CorynebacteriumMycobacterium-Nocardia group. Criteria selected for characterizing hexadecane uptake
were cell hydrophobicity, interfacial and surface tensions and production of glycolipidic
extracellular biosurfactants. These properties were determined in flask cultures on an
insoluble (hexadecane) and on a soluble (glycerol or succinate) carbon source for
a subset of 23 representative strains. Exclusive direct interfacial uptake was utilized by
47% of studied strains. A large proportion of strains (53%) produced
biosurfactants. The data on cellular hydrophobicity suggested the existence of two
distinct alkane transfer mechanisms in this group. Accordingly, tentative
assignments of biosurfactant-mediated micellar transfer were made for 11% of the
isolated strains, and of biosurfactant-enhanced interfacial uptake for 42%.

INTRODUCTION

Present interest in hydrocarbon biodegradation is motivated

for a large part by their presence as environmental pollutants
(Atlas 1984; Leahy and Colwell 1990). Crude oil is a major
sea pollutant and petroleum products, such as gasoline or
diesel and fuel oils, are the most frequent organic pollutants of
soils and ground-waters. The biodegradation of hydrocarbons
has a high ecological significance as it constitutes the major
process for remediation of the contaminated areas. Research
aims at a better understanding of the fate of polluting hydrocarbons (natural attenuation) and of the prospects for bioCorrespondence to: Dr M. Bouchez-Natali, Ecole Nationale Superieure des
Industries Agro-alimentaires, Laboratoire de Microbiologie Industrielle,
1, avenue des Olympiades, 91744 Massy cedex, France (e-mail:
naitali@ensia.inra.fr).
* Present address: Laboratoire de Matrise des Technologies AgroIndustrielles, Universite de la Rochelle, Avenue Marillac, 17042 La
Rochelle cedex 01, France.
1999 The Society for Applied Microbiology

technological improvement of biodegradation processes

(engineered bioremediation). A key point, which is only partially understood, is the mechanism of uptake by microorganisms of these strongly hydrophobic compounds. For
long-chain alkanes, which are practically non-water soluble,
two uptake modes are generally considered (Boulton and
Ratledge 1984; Singer and Finnerty 1984; Haferburg et al.
1986; Hommel 1994): interfacial accession (direct contact of
cells with hydrocarbon droplets) and biosurfactant-mediated
hydrocarbon uptake (cell contact with so-called accommodated or with solubilized hydrocarbons). Such mechanisms are also involved in the biodegradation of poorly
soluble, polycyclic aromatic hydrocarbons (Deziel et al. 1996;
Bouchez et al. 1997). A question often raised concerning
hydrocarbon biodegradation in the environment is the
importance of the contribution of biosurfactants to the
process. However, this complex question remains a matter of
debate, as conflicting results have been reported regarding

422 M . B OU C HE Z- N AI TA L I E T A L .

the efficiency of biosurfactants in promoting hydrocarbon

degradation in various situations (Haferburg et al. 1986).
Another approach was followed in the present work. A series
of bacterial strains representative of the microflora degrading
long-chain alkanes was isolated and identified. Physiological
properties useful as criteria for diagnosing mechanisms of
alkane uptake were selected and their determination was
applied to the strain collection. The approach aimed at widening the basis of observations concerning alkane uptake with
respect to bacterial diversity, and at discerning the relative
distribution of either mode of uptake in alkane-degrading
bacterial strains.

Strain cultures

Cultures were incubated in duplicate in 250 ml flasks containing 100 ml MSM4. Glycerol, succinate and hexadecane
(12 g carbon l1) were used individually as sole sources of
carbon and energy. Flasks were inoculated (1% v/v culture
grown on the same medium) and incubated on a rotary shaker
(160 rev min1) at 30 C. Growth was followed by optical
density (O.D.) measurement at 600 nm and by nitrate consumption for cultures grown on soluble substrate and on
hexadecane, respectively. Residual nitrate concentrations of
culture supernatant fluid were determined with nitrate
reductase (Beutler and Wurst 1986) using commercial kits
(Boehringer Mannheim).

MATERIALS AND METHODS

Analyses

Culture media

Trypticase soy agar (TSA) was obtained from Biomerieux.

The vitamin-supplemented mineral salt medium (MSM4)
used contained (g l1): 28 KH2PO4, 167 Na2HPO4.12H2O,
36 KNO3, 01 MgSO4.7H2O, 0001 FeSO4.7H2O, trace
elements and vitamins (Bouchez et al. 1995).

Isolation of strains

A series of bacterial strains was isolated, by selective enrichment, from soils polluted by hydrocarbons (gasoline, diesel
oil, coal tar) or from unpolluted (garden and forest) soils, for
their capacity to use hexadecane as sole source of carbon and
energy. Enrichment was conducted with 1 g soil in 250 ml
flasks containing MSM4 (100 ml) with hexadecane (18 ml) as
carbon source. After 1 week of enrichment (30 C, orbital
shaking at 160 rev min1), cultures were plated on MSM4
agar plates containing hexadecane on the inside wall of the
lid. Morphologically different colonies were then purified on
TSA. The degradative capacity of purified strains was verified
by growth on hexadecane-MSM4 agar plates. Strains from
other origins, using hexadecane as sole source of carbon and
energy, were also used; strain GL1 was isolated from a soil
of a manufactured gas plant (Arino et al. 1996), and strains
PyrGe1, Mu1.4 and NapRu1 were isolated for their capacity
to use pyrene, anthracene and naphthalene, respectively, as
growth substrate. Strains were stored at 80 C in 50% v/v
glycerol/physiological salt solution.

Identification of strains

Identification was performed by numerical taxonomy with

API kits (Biomerieux). Gram-positive rods and Gram-negative, oxidase-negative rods were identified on API Coryne
and API 20 NE kits, respectively. Gram-positive, catalasepositive cocci were identified on API Staph kits.

Glycoside and glycolipid determination. Glycosides were

evaluated on supernatant fluids of cultures by the colorimetric
method of Dubois et al. (1956). Glucose was used as a standard and results were expressed in glucose equivalents. Each
result was the average of three determinations, and standard
deviation was within 5%. Glycolipids were recovered as
follows. Supernatant fluid of cultures was heated at 100 C
for 10 min, acidified to pH 2 after cooling and centrifuged
in order to eliminate extracellular proteins. Subsequently,
deproteinized supernatant fluid was extracted three times
with ethyl acetate (1/1 v/v); finally, after solvent evaporation,
extracted glycolipids were taken up in methanol. The different glycolipidic types were separated by analytical thin
layer chromatography (TLC) carried out on silica gel plates
60 F-254 (Merck) using the solvent system CHCl3/CH3OH/
H2O (65/25/4 in volume). Detection was performed with the
Molish reagent (Arino et al. 1996).
Interfacial tension and surface tension measurement. Interfacial tension against hexadecane (gi) and surface tension (gs)
were determined, at 30 C, on filtered (045 mm Analypore
filters, OSI, Elancourt, France) supernatant fluid of cultures
with, respectively, the De Nouy ring method and the
Wilhelmy plate method, using a K-12 tensiometer (Kruss,
Hamburg, Germany). Stabilization was allowed to occur until
standard deviation of 10 successive measurements was less
than 04 mN m1. Each result was the average of 10 determinations after stabilization.
Cell hydrophobicity. Cell hydrophobicity was measured by
bacterial adherence to hydrocarbons (BATH) according to a
method similar to that described by Rosenberg et al. (1980).
The cells were washed twice and resuspended in a buffer salt
solution (169 g l1 K2HPO4, 73 g l1 KH2PO4) to give an
O.D. at 600 nm of 05. The cell suspension (2 ml) with 100 ml

1999 The Society for Applied Microbiology, Journal of Applied Microbiology 86, 421428

H EX AD E CA NE U PT AK E IN BA C TE RI A 423

hexadecane added was vortex-shaken for 3 min in haemolysis

tubes (10 100 mm). After shaking, hexadecane and aqueous
phases were allowed to separate for 1 h. The O.D. of the
aqueous phase was then measured at 600 nm. Hydrophobicity
is expressed as the percentage of adherence to hexadecane
calculated as follows: 100 (1O.D. of the aqueous
phase/OD of the initial cell suspension). For a given sample,
three independent determinations were made and the standard deviation was within 5%.
RESULTS
Strain isolation and identification

Sixty-one different bacterial strains were selected for their

capacities to use hexadecane as sole source of carbon and
energy, 57 of them having being isolated on this compound.
Phenotypic identification showed that strains belonged to
various genera (Fig. 1), with the Corynebacterium/Mycobacterium/Nocardia (CMN) group (Barksdale 1970) predominating.
Thirty-four categories of strains were defined, each one
regrouping strains with the same phenotypic characteristics
on API kits and identical colonial morphology when grown
on TSA and on MSM4-hexadecane plates (data not shown).
For further studies, 23 strains (presented in Tables 1, 2 and
3) were chosen. They belonged to 19 different categories of
strains representing 735% of the total. For the two largest
categories, several strains were tested in order to study the
homogeneity of substrate uptake characteristics.
Characterization of the cultures in relation to the
mode of substrate uptake

Several types of determinations were selected for diagnosing

the modes of substrate uptake used by the strains in alkane-

Percentage of total strain set

45
40
35
30
25
20
15
10
5

CMN group

0
Genera of strains

Fig. 1 Distribution in genera of strains degrading hexadecane as

sole source of carbon and energy. ( ), Rhodococcus;
(,), Corynebacterium; ( ), Micrococcus; (), Pseudomonas;
(-), Alcaligenes; (), Staphylococcus

grown cultures. They involved cell hydrophobicity, which

constitutes an important parameter in interfacial uptake, and
surface and interfacial tensions of culture supernatant fluids,
which provide basic information concerning biosurfactantmediated substrate transfer. For further characterization of
biosurfactant production, the production of glycolipids, one
of the main classes of biosurfactants, was also studied. First,
glycoside concentrations in culture supernatant fluid were
determined. Then, the glycosides were tentatively classified
as glycolipidic biosurfactants (S) when they were amphiphilic
(as shown by TLC migration after ethyl acetate extraction) or
as polysaccharides (Ps) when they were not. These properties
were determined at the end of the growth phase but kinetic
studies performed with strains GL1 and NapRu1 showed
that they did not undergo large variations after the initial
growth phase (data not shown).
In order to promote biosurfactant production, cultures
were conducted, with nitrate as nitrogen source, until nitrate
depletion (Guerra-Santos et al. 1984; Syldatk et al. 1985;
Ramsay et al. 1988; Arino et al. 1996; Desai and Banat 1997).
Moreover, in most cases, two carbon sources were tested, one
insoluble (hexadecane) and one soluble (glycerol or succinate), to compare their influence on biosurfactant production.
The results obtained with Gram-negative rods are presented in Table 1. Pseudomonas aeruginosa GL1 and Au1 presented identical characteristics in cultures on hexadecane or
on glycerol, i.e. low cell hydrophobicity and high biosurfactant production resulting in low surface and interfacial
tensions. Cultures of Ps. fluorescens Es1 also presented very
low surface and interfacial tensions, but higher cell hydrophobicity, when grown on hexadecane. During growth of
strain HeB2, the glycosides produced were polysaccharides
which, consequently, reduced surface and interfacial tensions
only moderately. It can be noted that, although no glycolipidic
biosurfactants were produced by Alcaligenes Vi1, interfacial
tension was notably decreased, suggesting the production of
another class of biosurfactants.
Each of the three strains of Micrococcus tested presented
different culture characteristics when grown with hexadecane
as sole source of carbon and energy (Table 2). For Micrococcus
Ou3, high cell hydrophobicity, relatively high surface tension
and intermediate interfacial tension of the culture supernatant
fluid were observed. The high amount of polysaccharides
produced may explain the interfacial tension decrease. The
complex role of extracellular polysaccharides in hydrocarbon
uptake has been well documented in the case of emulsan
produced by Acinetobacter calcoaceticus strains (Rosenberg
and Rosenberg 1981; Sar and Rosenberg 1983; Rosenberg
1993; Marin et al. 1996). Cultures of Micrococcus Fl2 were
different, as the glycosides produced were glycolipids and
the surface and interfacial tensions of the culture supernatant
fluid were low. Micrococcus Cb1 also produced amphiphilic
glycosides which, surprisingly, only moderately decreased

1999 The Society for Applied Microbiology, Journal of Applied Microbiology 86, 421428

424 M . B OU C HE Z- N AI TA L I E T A L .

Table 1 Cultural characteristics of the Gram-negative rods

Relative frequency
Growth
Cell hydrogi
gs
Glycosides
Strain name
Strain identification
in set*
substrate
phobicity (%) (mN m1) (mN m1) (g l1)

GL1
Pseudomonas aeruginosa
66
hexadecane
3
51
326
13 (S)
glycerol
5
36
295
25 (S)
Au1
Pseudomonas aeruginosa
16
hexadecane
2
34
307
28 (S)
glycerol
6
45
311
42 (S)
Es1
Pseudomonas fluorescens
16
hexadecane 37
2
266
05 (S)
glycerol
5
230
561
03 (Ps)
HeB2
unidentified
16
hexadecane 43
230
596
04 (Ps)
glycerol
27
175
498
25 (Ps)
Vi1
Alcaligenes faecalis
16
hexadecane 49
100
429
05 (Ps)
succinate
11
123
447
00

Experiments were run in duplicate as described in Materials and Methods. Analyses were performed after the end of growth phase,
indicated by nitrate depletion. Results were reproducible for cultures in duplicate, within 5% for interfacial and surface tension determinations,
8% for hydrophobicity measurements and 10% for glycoside productions. gi and gs, evaluated on MMS4 medium (blank) were found
equal to 267 mN m1 and 591 mN m1, respectively.
* Frequency (%) of strains of the same category, i.e. presenting the same phenotypic characteristics on API kits and identical visual
aspect when grown on TSA and on agar plate of MSM4 hexadecane.
Gram-negative rod close to Alcaligenes denitrificans subsp. xylosoxydans.
S, biosurfactants, Ps, polysaccharides.

Table 2 Cultural characteristics of the Micrococcaceae

Relative frequency
Cell hydrophobicity
gi
gs
Glycosides
Strain name
Strain identification
in set*
(%)
(mN m1)
(mN m1)
(g l1)

Fl2
Micrococcus sp.
33
77
2
280
05 (S)
Cb1
Micrococcus sp.
16
21
161
500
05 (S)
Ou3
Micrococcus sp.
16
92
156
495
21 (Ps)
Ac7
Staphylococcus caprae
16
83
264
379
02 (Ps)

* See Table 1.
Experiments, analyses and symbols as in the legend for Table 1. The growth substrate was hexadecane.

interfacial and surface tensions of culture supernatant fluids.

It can be supposed that, in this case, concentrations of amphiphilic glycosides were under the critical micellar concentration (CMC). During growth of Staphylococcus caprae
Ac7, compounds active at the air/aqueous phase interface
(medium surface tension), but inactive at the interface of the
aqueous and hydrophobic phases, were produced (Table 2).
The results obtained with the strains of the CMN group
are presented in Table 3. Most of the strains of this group
(Rhodoccocus Fo2 and La2.1, two strains of the same category,
Rhodoccocus PyrGe1 and NapRu1, and Corynebacterium

Mu1.4, Co1 and Ju1.2) presented identical culture characteristics on soluble and on insoluble substrates (when tested
on both), i.e. high values for cell hydrophobicity and for
surface and interfacial tensions. The glycosides produced
were polysaccharides except for strains PyrGe1 grown on
glycerol and Ju1.2 grown on hexadecane where glycolipids
were found. During culture of Coryne. pseudodiphteriticum
Ac2 and of R. equi Ou2 on hexadecane, glycolipids were
produced that decreased surface and interfacial tensions. It is
notable that, for strain Ou2, culture characteristics were very
different depending on whether growth took place on hexa-

1999 The Society for Applied Microbiology, Journal of Applied Microbiology 86, 421428

H EX AD E CA NE U PT AK E IN BA C TE RI A 425

Table 3 Cultural characteristics of the strains belonging to the CMN group

Relative frequency
Growth
Cell hydrogi
gs
Glycosides
Strain name
Strain identification
in set*
substrate
phobicity (%) (mN m1) (mN m1) (g l1)

Ou2
Rhodococcus equi
197
hexadecane 69
31
275
07 (S)
glycerol
21
281
676
07 (Ps)
BB1
Rhodococcus equi
hexadecane 65
24
254
09
SGB1
Rhodococcus equi
hexadecane 64
21
247
12
DuB1
Rhodococcus equi
hexadecane 38
86
323
10
Fo2
Rhodococcus equi
82
hexadecane 83
216
585
02 (Ps)
glycerol
96
240
593
03 (Ps)
La21
Rhodococcus equi
hexadecane 92
236
510
01
HeA1
Rhodococcus equi
49
hexadecane 61
223
439
02 (Ps)
glycerol
28
203
546
09 (Ps)
HdGe1
Rhodococcus equi
33
hexadecane 37
210
514
10 (Ps)
PyrGe1
Rhodococcus equi
33
hexadecane 95
214
507
08 (Ps)
glycerol
77
243
507
02 (S)
NapRu1
Rhodococcus equi
16
hexadecane 86
254
609
01 (Ps)
succinate
97
290
645
03 (Ps)
Ju12
Corynebacterium jeikeium 66
hexadecane 89
228
473
04 (S)
Mu14
Corynebacterium sp.
16
hexadecane 96
254
596
03 (Ps)
glycerol
83
237
556
06 (Ps)
Co1
Corynebacterium sp.
16
hexadecane 86
254
701
01
Ac2
Corynebacterium
16
hexadecane 81
69
286
16 (S)
pseudodiphteriticum
glycerol
92
75
381
01

* See Table 1.
Strain belonging to the same category as Ou2.
Strain belonging to the same category as Fo2.
Corynebacterium group ANF.
Experiments, analyses and symbols as in the legend of Table 1.

decane or on glycerol, a point already observed in the case

of Ps. fluorescens Es1 (Table 1). Properties of cultures on
hexadecane of strains of the same category, BB1, SGB1 and
Ou2, were quite similar although in the same category, cultures of DuB1 presented a lower cell hydrophobicity.
Strain classification by mode of substrate uptake

Strains were classified according to their predominant mode

of substrate uptake. It was considered that the main characteristics of interfacial uptake were a high cellular hydrophobicity to allow cell adherence to the hexadecane phase,
and a high interfacial tension of culture supernatant fluid
proving that there was no biosurfactant production. The
requisites retained for strictly biosurfactant-mediated transfer were a low interfacial tension as well as a low cell hydrophobicity, preventing cell adherence to hydrophobic
interfaces. It is of interest to note that this classification, as
presented in Table 4, points to the existence of strains that
could employ interfacial uptake, but for which biosurfactant

production took place and was thus susceptible to enhanced

hexadecane uptake.

DISCUSSION

Most of the strains isolated belonged to genera known for

their capacity to degrade alkanes (Britton 1984; Goodfellow
1992; Rosenberg 1992). All soil samples tested (polluted as
well as non-polluted) yielded hexadecane-degrading strains,
confirming the widespread distribution in the environment
of such strains. This fact probably reflected the ubiquitous
presence of long-chain alkanes from plants in the environment (Kolattukudy 1976; Berdie et al. 1995).
Broadly speaking, the results concerning hexadecane
uptake were in line with the modes of substrate transfer
previously described in the literature (Singer and Finnerty
1984; Haferburg et al. 1986; Goswami and Singh 1991; Hommel 1994), direct interfacial uptake and biosurfactantmediated uptake, but they gave new physiological and

1999 The Society for Applied Microbiology, Journal of Applied Microbiology 86, 421428

426 M . B OU C HE Z- N AI TA L I E T A L .

Predominant mode of
Hydrophobicity
hexadecane uptake
of cells*
gi
Strain involved

Interfacial uptake
high
high
Rhodococcus equi Fo2
high
high
R. equi La21
high
high
R. equi NapRu1
high
high
R. equi HeA1
high
high
R. equi PyrGe1
high
high
Corynebacterium sp. Co1
high
high
Corynebacterium sp. Mu14
high
high
Coryne. jeikeium Ju12
high
high
Staphylococcus caprae Ac7
medium
high
Gram-negative rod HeB2
medium
high
R. equi HdGe1
Biosurfactant-mediated
uptake

low
low

low
low

Table 4 Strain classification based

on the predominant mode of hexadecane

uptake

Pseudomonas aeruginosa Au1

Ps. aeruginosa GL1

Biosurfactant-enhanced
interfacial uptake

high
low
Micrococcus sp. Fl2
high
low
R. equi Ou2
high
low
R. equi BB1
high
low
R. equi SGB1
high
low
Coryne. pseudodiphteriticum Ac2
high
medium
Micrococcus sp. Ou3
medium
low
Ps. fluorescens Es1
medium
medium
R. equi DuB1
medium
medium
Alcaligenes faecalis Vi1
medium
medium
Micrococcus sp. Cb1

* High 60%; low 10%.

High 18 mN m1; low 7 mN m1.

environmental insights into the diversity of alkane uptake

mechanisms, as detailed below.
Direct interfacial uptake appeared to be the most frequent
mechanism. Assuming, as suggested by the data, that strains
belonging to the same category presented the same characteristics of substrate transfer as the selected strains, an interfacial mechanism was employed by 467% of strains from
the set. A natural habitat for such strains may be one that
maximizes hydrocarbon interfaces such as the phylloplane of
waxy plants. Neu and Poralla (1990) reported the isolation of
bacteria with hydrophobic cell surfaces from such sources.
The effective production of biosurfactants is usually
viewed as the obvious criterion for the existence of biosurfactant-mediated hydrocarbon transfer. In the present
study, both hydrophilic and hydrophobic bacteria were able
to produce biosurfactants, as reported by Neu and Poralla
(1990). Several hydrophobic strains, however, did not produce biosurfactants. These observations do not corroborate a
recently held view associating high cell hydrophobicity with
biosurfactant production capacity (Pruthi and Cameotra
1997). The mode of action of biosurfactants is usually con-

sidered to promote both substrate emulsification and solubilization. In fact, the case where biosurfactants act mainly
by solubilization of the alkane, thus resulting in the formation
of micelles with a hydrophilic outer layer, appears to be a
mode of transfer well suited to hydrophilic cells, allowing
efficient contact with the alkane-degrading bacteria. Among
the strains studied, such a mechanism (micellar transfer)
appears to be appropriate for the two Ps. aeruginosa strains,
although this is not thought to be a general pattern for this
species (Zhang and Miller 1994). It can be noted that the
biosurfactants produced by Ps. aeruginosa GL1 have been
previously characterized as rhamnolipids (Arino et al. 1996)
and that biosurfactants produced by Ps. aeruginosa Au1 were
likely to be rhamnolipids as well, as suggested by their
migration pattern on TLC (migration identical to that of
GL1 surfactants). These observations were in agreement with
the literature (Guerra-Santos et al. 1984; Syldatk et al. 1985;
Parra et al. 1989; Robert et al. 1989). Using the dilution
method (Bosch et al. 1988), we observed (data not shown)
that the low interfacial and surface tensions values corresponded to biosurfactant concentrations above the CMC

1999 The Society for Applied Microbiology, Journal of Applied Microbiology 86, 421428

H EX AD E CA NE U PT AK E IN BA C TE RI A 427

determined (30-fold the CMC for biosurfactants produced

by GL1 at the end of growth on hexadecane). Such conditions
appear favourable to alkane solubilization and indeed, we
observed solubilization of hexadecane (around 15 mg l1) in
culture supernatant fluid of strain GL1.
A different situation was observed for a large group of
bacteria (422% of the set) exhibiting both a high or medium
hydrophobicity and production of biosurfactants. Assuming
that such strains utilized both direct interfacial uptake and
micellar transfer poses a problem as the solubilization of
alkanes implies the existence of hydrophilic micelles. The
data on bacterial cell hydrophobicity suggest that for hydrophobic biosurfactant-producing bacteria, the alkane transfer
mechanism involves, primarily, substrate emulsification
rather than solubilization, emulsified alkane droplets containing hydrophobic regions on their surface. This is in line
with the observation that supernatant fluids of hexadecanegrown cultures of several strains of this group (such as Micrococcus Fl2, Corynebacterium Ac2 and Pseudomonas Es1) were
found to produce emulsions that remained stable after several
months. In fact, the question of the mode of action of biosurfactants in promoting emulsification (Roy et al. 1979;
Rosenberg and Rosenberg 1981; Neu and Poralla 1990) or
solubilization (Roy et al. 1979; Rosenberg and Rosenberg
1981; Zhang and Miller 1995) of substrate, or in modifying
cell hydrophobicity (Zhang and Miller 1994), is long standing. In a marine bacterium, the coexistence of solubilization
and emulsification mechanisms has been proposed (Husain
et al. 1997). The hydrophobicity data presented here indicate
the occurrence of both mechanisms but with one or the other
usually being privileged, depending on the strain concerned.
Biosurfactants were observed in significant amounts for a
large proportion (533%) of the set of strains studied. This
is somewhat high compared with other authors data (Bosch
et al. 1988; Parra et al. 1989; Neu and Poralla 1990; Mercade
et al. 1996). Production of biosurfactants was also observed
during growth on a soluble carbon source, which is in agreement with Haferburg et al. (1986). These observations suggest
a broader role for biosurfactants than just hydrocarbon
uptake. As discussed by Neu (1996), a likely possibility is
the more general participation in adhesion and de-adhesion
interactions between micro-organisms and interfaces. Biosurfactant-enhanced interfacial uptake of alkanes could be
one aspect of this general role.
ACKNOWLEDGEMENTS

The authors thank V. Bardin for strain isolation, H. Bouzrara

for participation in strain characterization and C. Dalmazzone
for helpful discussions.
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