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Microbiology (2001), 147, 2537–2543 Printed in Great Britain

Evidence for interfacial uptake in hexadecane

degradation by Rhodococcus equi : the
importance of cell flocculation
Murielle Bouchez-Naı$ tali,1 Denis Blanchet,2 Ve! ronique Bardin2
and Jean-Paul Vandecasteele2

Author for correspondence : Murielle Bouchez-Naı$ tali. Tel : j33 1 69 93 51 40. Fax : j33 1 69 93 50 84.
e-mail : naitali!ensia.inra.fr

1 Ecole Nationale Supe! rieure The kinetics of hexadecane degradation were studied in four strains of
des Industries Agricoles et Rhodococcus equi that did not produce biosurfactants. The aim was to analyse
Alimentaires, Laboratoire
de Microbiologie the characteristics of alkane uptake and their relevance to a mechanism of
Industrielle, 91 744 Massy interfacial uptake. The kinetic studies involved continuous determination of
Cedex, France degradation by electrolytic respirometry in a diphasic system where the
2 Institut Franc: ais du hydrophobic phase was hexadecane or a solution of hexadecane in a non-toxic,
Pe! trole, Division Chimie et non-biodegradable solvent, either 2,2,4,4,6,8,8-heptamethylnonane or silicone
Physico-chimie Applique! es,
92 852 Rueil-Malmaison oil. The technique allowed large variations in interfacial area between the
Cedex, France aqueous and hydrophobic phases. For the four strains, the kinetics obtained
were reproducible and showed, in almost all cases, an initial short phase of
exponential growth, followed by a long phase of linear growth. Specific
growth rates during exponential growth varied amongst the strains from 011
to 020 hN1 and were independent of interfacial area, in accordance with the
very strong adsorption of bacterial cells at the interface of solvent and
aqueous media. The degradation rates during linear growth did not increase
with interfacial area but increased with efficiency of stirring. These
characteristics can be explained by the formation of cellular flocs due to the
hydrophobicity of the strains. These flocs were observed during growth on
hexadecane in almost all conditions. In one case, with a non-flocculating
culture, a kinetic pattern with a longer exponential phase, closer to that
expected for simple interfacial uptake, was observed. The results show that
strictly interfacial uptake, limited by floc formation (occurring at moderate
and higher cell densities, and controlled by stirring efficiency) is a common
pattern for growth on long-chain alkanes of micro-organisms that do not
produce biosurfactants.

Keywords : alkane degradation, electrolytic respirometry, flocculating strains,

Rhodoccocus sp., uptake kinetics

INTRODUCTION nature of hydrocarbons has required micro-organisms

to develop specific mechanisms of substrate uptake in
Hydrocarbons of natural origin are widespread in the order to degrade them (Boulton & Ratledge, 1984 ;
environment but, because of the massive utilization of Haferburg et al., 1986 ; Hommel, 1994 ; Singer &
petroleum products, they are nowadays strongly Finnerty, 1984). In the case of long-chain alkanes, the
involved in environmental pollution. Biodegradation, micro-organisms sometimes produce biosurfactants,
either naturally occurring (natural attenuation) or by which results in hydrocarbon micelles or small emulsi-
engineered bioremediation, is a key process for the fied hydrocarbon globules. If biosurfactants are not
decontamination of polluted areas. The hydrophobic produced, direct cell contact with hydrocarbon droplets
can take place, involving strictly interfacial hydrocarbon
Abbreviation : HMN, 2,2,4,4,6,8-heptamethylnonane. Whatever the mechanisms involved, a point which is

0002-4636 # 2001 SGM 2537

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still only partially understood at the present time is the produced oxygen supplied to the cultures at constant pressure
kinetics of long-chain alkane biodegradation. Studies allowed quasi-continuous recording of oxygen consumption.
focused on the kinetics of growth of yeasts on n-alkanes The stirring speed being uniform (300 r.p.m.), two types of
were conducted in the seventies and models were stirrers were employed in order to modify agitation efficiency :
proposed to account for the complex kinetic patterns triangular-section bars (14i55 mm) in most cases, and
smaller, round-section bars (6i20 mm) when specified. Cul-
observed (Blanch & Einsele, 1973 ; Erickson et al., 1969, ture flasks containing 237n5 ml MSM3 were inoculated with
1970 ; Erickson & Humphrey, 1969 ; Gutierrez & Erick- 12n5 ml of a preculture on MSM3 supplemented with succinate
son, 1977 ; Moo-Young & Shimizu, 1971 ; Verkooyen & prepared as above. C was added alone or as a solution in a
Rietma, 1980a, b ; Yoshida & Yamane, 1974). However, hydrophobic solvent,"'either 2,2,4,4,6,8,8-heptamethylnonane
with other alkane-degrading micro-organisms, the inter- (HMN) or silicone oil 47V20 (Prolabo). Both solvents were
est was essentially oriented towards the production of non-toxic and non-water-soluble, and were not degraded by
biosurfactants and kinetic studies are scarce. In a recent the strains. As indicated in the results, experiments were run
work, we analysed the relative distribution of the modes several times but individual recordings are presented in the
of substrate uptake in a large series of bacterial strains figures. Oxygen consumption blanks (inoculated flasks con-
degrading hexadecane (C ) (Bouchez-Naı$ tali et al., taining hydrophobic solvent without C and uninoculated
"' we studied in detail the flasks containing C and hydrophobic"' solvent) were run
simultaneously. No"'significant oxygen consumption was
1999). In the present work,
kinetics of degradation of C by four Rhodococcus
strains that had been identified"'as using direct interfacial
detected in these blanks.
uptake on the basis of the absence of biosurfactant Parameters describing the kinetics obtained in different
production. Cell flocculation was found to constitute an conditions were compared using analysis of variance. When
important characteristic of the cultures of these strains considered as significantly different (P 0n05) or very sig-
nificantly different (P 0n01), Student’s t-tests were per-
on C . The kinetic patterns of degradation were
investigated in relation to the various phenomena
involved in C uptake. Analyses. Analyses were performed as described by Bouchez-
"' Naı$ tali et al. (1999). Interfacial tension against C and surface
tension were determined at 30 mC on filtered supernatant fluids
METHODS of cultures by, respectively, the De Nouy ring and Wilhemy
plate methods, using a K-12 tensiometer (Kru$ ss). Each result
Culture media. Vitamin-supplemented mineral salt medium was the mean of ten determinations and standard deviation
MSM4 was as previously described (Bouchez-Naı$ tali et al., was less than 0n4 mN m−". Cell hydrophobicity was measured
1999). Mineral salt medium MSM3 was similar, except that by the bacterial adherence to hydrocarbon test according to a
the nitrogen source was 1n5 g l−" (NH ) SO instead of the method adapted from Rosenberg et al. (1980). Hydrophobicity
%# %
KNO used in MSM4. was measured with respect to the hydrophobic phase used in
the cultures : C when employed alone, or HMN or silicone
Strains. Four Rhodococcus equi strains were employed : "'
oil. For each sample, three independent determinations were
NapRu1, HdGe1, PyrGe1 and Fo2. They were isolated from made and the standard deviation was within 5 %. Residual
soils as previously described (Bouchez-Naı$ tali et al., 1999) and nitrate concentration was determined in culture supernatant
were able to use C as sole source of carbon and energy.
"' fluids using a commercial kit (Boehringer Mannheim). Gly-
Cultures in flasks. Cultures were carried out in 250 ml flasks cosides were evaluated on supernatant fluids of cultures by the
containing 100 ml medium and incubated on a rotary shaker colorimetric method of Dubois et al. (1956) using glucose as
(160 r.p.m.) at 30 mC. They were used as inocula for fer- standard. Each result was the mean of three determinations
mentation work and kinetic studies in the conditions described and the standard deviation was within 5 %.
below. Flask cultures were also used for hydrophobicity
determinations. In this case, the medium was MSM3 and the
carbon source was C (alone or in the hydrophobic solvents RESULTS
studied). Floculating properties of strains degrading
Growth on hexadecane in laboratory fermenters. Batch hexadecane
cultures were performed in 2 litre fermenters (Inceltech), in Four Rhodococcus strains, NapRu1, HdGe1, PyrGe1
1n47 litre MSM4 with 30 ml C as sole source of carbon and
"' and Fo2, were selected as non-producers of bio-
energy. The inoculum was grown in the same medium
containing succinate as carbon source. To remove the residual surfactants (Bouchez-Naı$ tali et al., 1999). Visual and
carbon source from the inoculum, 30 ml of culture was microscopic observations of shake-flask cultures of the
strains grown on C alone or on C dissolved in HMN
centrifuged (10 000 g, 10 min) and the cells were resuspended
or silicone oil were"' made. In all "'cases except one, a
in 30 ml fresh MSM4 before inoculation of the fermenters.
The fermentation conditions were : temperature, 30 mC ; stir- distinct but more or less pronounced tendency to
ring speed, 500 r.p.m. ; aeration, 1 vol. vol.−" min−". The pH produce flocs, exhibiting various sizes and consistencies
was recorded but not regulated. Experiments were run in depending on the strains and on the conditions of C
duplicate. supply, was observed (Table 1). Upon decanting, cell "'
Kinetic studies by respirometry. Time courses of oxygen biomass accumulated as pellets floating above an aque-
consumed during growth on C were recorded using a ous phase. Microscopic examination showed that most
"' bacterial cells were found around oil drops or as
sensitive respirometer (D12-S Sapromat, Voith) consisting of
12 conical flasks (500 ml) placed in a water bath maintained at aggregates in the hydrophobic phase. This can be related
30 mC (Bouchez et al., 1997a). Determination of electrolytically to the high hydrophobicity of the cells (Table 1). A

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Kinetics of interfacial hexadecane uptake

Table 1. Aggregation properties of Rhodococcus strains as oxygen consumed was shown to be proportional to
during degradation of hexadecane the substrate consumed and to the biomass produced
(Bouchez et al., 1997a). The experimentation covered a
Strain Solvent phase Floc Cell range of conditions, as presented in Table 2. C , used as
formation* hydrophobicity† sole source of carbon and energy, was added"'alone or
dissolved in HMN, and the effects of variations in initial
NapRu1 HMN j 99p1 concentations of C , in volumes of HMN and in stirring
C j 96p1 efficiencies were tested. These conditions resulted in
changes in the interfacial area between the hydrophobic
HdGe1 Silicone oil j 81p1 and aqueous phases, a key parameter in interfacial
Fo2 Silicone oil j 97p2 uptake (Westgate et al., 1995). Examples of the time
C j 95p2 courses of oxygen consumption curves obtained are
"' presented in Fig. 2. Kinetic analysis of the curves by
PyrGe1 C j 93p2 plotting the oxygen consumed versus time in a semi-
Silicone oil – 77p2 logarithmic scale led to characterization of a first short
* j, Flocs observable by visual and microscopic examination ; –, phase of exponential growth with a good correlation
no flocs observed. coefficient (0n9937 r# 0n9999). This phase rep-
† Values are meansp.
resented around 10 % of total growth (expressed as the
percentage of total oxygen consumed) when the initial
C concentration was 1000 mg l−", irrespective of the
volume of the solvent phase. The maximal specific
growth rate (µ) of strain NapRu1 growing on C (Table
different situation was observed with strain PyrGe1 2) was not dependent on the conditions tested as "' shown
when grown on C dissolved in silicone oil. In these by an analysis of variance (P l 0n96). A mean µ value of
"' formed visually homogeneous
conditions, this strain 0n20p0n02 h−" was recorded for all the conditions. After
cultures with a large proportion of bacteria dispersed in the first exponential phase, a phase of limited growth
the aqueous medium. This strain exhibited a lower cell was observed in all cases : when plotting the oxygen
hydrophobicity when growing on C dissolved in consumed versus time on a direct scale, portions of
silicone oil (77 %) than on C alone (93"'%). straight lines were obtained with excellent correlation
coefficients (0n9985 r# 0n9999). The values of linear
O consumption rates are presented in Table 2. A
Kinetic study of growth of flocculating strains on #
Student’s t-test performed on these values after analysis
hexadecane of variance showed that they were significantly different
Several properties of cultures relating to the mode of (P 0n05) when the stirring efficiency varied, but not
substrate uptake are presented in Fig. 1 for strain when the volume of the solvent phase and the initial
concentration of C were varied. Rate values were
16p1 mg O l−" h−""' when small magnetic bars were
NapRu1 during growth in fermenters with C as sole
source of carbon and energy and nitrate as"'limiting
used and a # mean of 30p3 mg O l−" h−" in other
nitrogen source. Determination of residual nitrate was
conditions. #
selected in order to follow the growth of the culture as
both biomass accretion and C consumption measure-
ments were hindered by heterogeneous dispersion in the The kinetics of growth of strains HdGe1, PyrGe1 and
medium. All the nitrate was consumed, which cor- Fo2 in flocculating conditions (Table 1) were studied for
responded to a biomass production of about 5 g l−" comparison with strain NapRu1. Kinetic patterns simi-
(assuming a nitrogen\biomass ratio of 10 %). The lar to those presented for strain NapRu1 were obtained
results also show that during the growth period, cell and Table 3 summarizes the resulting data. An initial
hydrophobicity remained high, an important charac- exponential phase was clearly observed (0n9857 r#
teristic for efficient interfacial uptake. Interfacial and 0n9991) during 5 % of the growth period for strains
surface tensions of culture supernatant fluids also HdGe1 and PyrGe1 and during 10 % of the growth
period for strain Fo2, when C was added at the initial
concentration of 1000 mg l−". "'For all strains, the maxi-
remained high, proving the absence of surfactant pro-
duction, even in conditions of nitrogen limitation which
are known to promote biosurfactant production (Desai mum specific growth rates were not dependent on the
& Banat, 1997). This strongly suggests that strain conditions tested (P  0n37 in all cases in analyses of
NapRu1 utilized C by a strictly interfacial uptake variance performed on µ values of each of the different
mechanism. "' strains). It is notable that strains HdGe1 and Fo2 grew at
a rate (0n18p0n01 h−" in both cases) similar to that of
A kinetic study of C degradation by strain NapRu1 strain NapRu1, whereas strain PyrGe1 showed a lower
was carried out. An "' excess of ammonium sulfate was µ value (0n13p0n01 h−"). A second phase of limited
used as nitrogen source. Time courses of oxygen uptake growth was again observed. In most cases, it was linear
during degradation were recorded in respirometric (0n9987 r# 0n9999). For a given strain, the linear O
flasks. Continuous monitoring of oxygen consumption consumption rates, when observed, were found not to#
is well suited to kinetic studies in heterogeneous media be dependent on the conditions tested (P  0n52 in all

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90 2·5

interfacial and surface tensions (mN m–1)

Hydrophobicity (%), 10× pH, 80

Glycosides produced (g l–1),

70 2·0

residual nitrates (g l–1)


20 0·5 Fig. 1. Cultural characteristics of R. equi
10 NapRu1 during growth on hexadecane in a
fermenter. $, Surface tension and
interfacial tension of culture supernatants ;
24 48 72 96 120 144 168 192 #, glycosides produced ; >, cell hydropho-
Time (h) bicity ; =, residual nitrate ; , pH.

Table 2. Kinetic constants for the two phases of hexadecane degradation by R. equi

The maximal specific growth rate (µ) and the oxygen uptake rate during the linear growth phase
were determined under various experimental conditions.

HMN Concn of C16 Replicates* µ (h−1)† Rate of linear

volume [mg (l aqueous oxygen uptake
(ml) phase)−1] (mg O2 l−1 h−1)†

0 1000 4 0n23p0n03 28p2

0 6160 2 0n19p0n01 29p1
0 30800 2 0n19p0n01 28p0
0 67760 1 0n23 28
5 1000 2 0n22p0n01 28p1
11 1000 7 0n20p0n02 31p3
22 1000 7 0n19p0n02 31p3
44 1000 4 0n21p0n03 30p2
0‡ 6160‡ 2‡ 0n21p0n01‡ 16p1‡§

* In respirometric flasks as described in Methods.

† Values are meansprange when two experiments were carried out, and meansp in other cases.
‡ Stirring carried out with smaller magnetic bars than in other experiments to reduce agitation
§ Significantly different from other rates as determined by a Student’s t-test (P 0n05).

cases in analyses of variance performed for each strain) were quite different from those obtained with floc-
and depended only on the strain (Table 3). Specific culating cultures. A long exponential phase was ob-
dependence on the strain was also observed for the span served during at least 40 % of the degradation period
of this linear phase, corresponding to 70 % of the growth and the maximum specific growth rate appeared to
period for strains HdGe1 and Fo2 and 50 % for strain depend on the volume of the hydrophobic phase (0n11,
PyrGe1 (data not shown). 0n13 and 0n15 h−" for volumes of 2, 10 and 50 ml,
respectively). Linear growth was observed in a second
phase for silicone oil volumes of 2 and 10 ml. For a
Kinetics of growth of non-flocculating cultures on silicone oil volume of 50 ml, the specific growth rate
hexadecane decreased in a second phase but no linear growth was
observed. In these cultures, no clear relationship be-
The kinetics of degradation of C in silicone oil by tween the pattern of the second phase of growth and the
strain PyrGe1 monitored with the respirometer (Fig. 3) volume of the organic phase was observed.

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Kinetics of interfacial hexadecane uptake

Oxygen consumed [mg(l aqueous medium)–1]

2500 2500
(a) (b)

2000 2000

1500 .....................................................................................................
Fig. 2. Time courses of oxygen consumption
during hexadecane degradation by R. equi
1000 NapRu1. Experiments were conducted in
respirometric flasks as described in Methods.
(a) C16 was added alone at concentrations of
1000 mg l−1 (——), or 6160 mg l−1 with a
500 500 normal-sized magnetic stirrer (– – –) or with
a smaller stirrer to reduce agitation efficiency
(:–:). (b) C16 (1000 mg l−1, expressed with
respect to the volume of the aqueous phase)
50 100 150 200 250 50 100 150 200 250 was added dissolved in 5 ml (---), 11 ml
Time (h) (——) or 22 ml (:–:) HMN.

Table 3. Kinetic constants for the two phases of hexadecane degradation by various
flocculating R. equi strains

Strain Silicone oil Concentration of Replicates* µ (h−1)† Rate of linear

volume (ml) C16 [mg (l aqueous oxygen uptake
phase)−1] (mg O2 l−1 h−1)†

HdGe1 2 1000 2 0n19p0n01 28p3

10 1000 2 0n18p0n02 27p2
50 1000 1 0n19 28
Fo2 2 1000 2 0n17p0n01 19p2
10 1000 3 0n19p0n01 20p1
0 6160 2 0n18p0n01 18p1
0 30800 2 0n18p0n00 None‡
0 61600 2 0n19p0n02 None‡
PyrGe1 0 6160 4 0n12p0n01 21p2
0 30800 3 0n13p0n01 23p3

* In respirometric flasks as described in Methods.

† Values are meansprange when two experiments were carried out, and meansp in other cases.
‡ Non-linear limited growth.


[mg(l aqueous medium)–1]
Oxygen consumed


400 Fig. 3. Time courses of oxygen consumption
during hexadecane degradation by R. equi
PyrGe1. C16 (1000 mg l−1, expressed with
respect to the volume of the aqueous phase)
was dissolved in 2 ml (——), 10 ml (-----)
and 50 ml (– – –) silicone oil. Experiments
10 20 30 40 50 60 70 80 90 100 were conducted in respirometric flasks as
Time (h) described in Methods.

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DISCUSSION the parameters that modify the aqueous and solvent

interface (such as the volume of the solvent phase) but
This study illustrates the convenience of respirometry to
by those that modify floc formation, since floc size
obtain detailed kinetic data on substrate degradation.
determines cell access to the substrate. In such a case,
Moreover, the use of a diphasic system with the
stirring is a key parameter because it controls floc size,
hydrophobic substrate dissolved in an inert organic
higher stirring rates promoting smaller flocs and thus
solvent is, as pointed out by Westgate et al. (1995) and
higher transfer rates. This point is in agreement with the
by Bouchez (1995), a valuable technique to study
slower degradation kinetics observed in conditions of
interfacial uptake as it allows the characterization of the
lower stirring efficiency. Cell aggregation can explain
dependence of uptake on the volume of solvent, i.e. on
the onset of the linear phase. The tendency of the cells to
interfacial area. In an interfacial model of uptake such as
agglomerate in flocs increases with increasing biomass.
that proposed by Dunn (1968), there is an equilibrium
The transition occurs when agglomeration pre-
between the bacterial cells adsorbed at the interface of
dominates over the turbulence of the medium, which
hydrophobic and aqueous phases and the cells in the
breaks the floc particles. Growth in cell aggregates
aqueous medium. Only the bacteria with access to the
accounts for the long linear phase of degradation
interface can take up the alkane and grow. Growth is
observed with these cultures. Differences in degradation
exponential until the interfacial area is covered with
rates during this phase appear to be essentially related,
cells. For this reason, exponential growth lasts longer
for a given rate of stirring, to differences in cell
when greater interfacial area is available. Moreover, the
aggregation properties among strains. The floc for-
specific growth rate during exponential growth is higher
mation observed here in almost all cases with Rhodo-
for larger interfacial areas unless cell adsorption at the
coccus strains leads us to suggest that the analysis given
interface is so strong that practically all cells are
by Blanch & Einsele (1973) for C. tropicalis may be
adsorbed at the interface. When all the interfacial area is
commonly applicable to micro-organisms growing on
covered with cells, a second phase where growth is
long-chain alkanes by strictly interfacial uptake. Ac-
linear is observed. During linear growth, the rate will be
tually, the propensity to aggregate appears quite signifi-
higher for larger interfacial areas which can support
cant among alkane-degrading micro-organisms and is
greater amounts of growing biomass. The dependence
obviously related to the high cell hydrophobicity of the
on interfacial area was well illustrated in a previous
strains concerned. Non-flocculent PyrGe1 cultures corre-
study of the interfacial uptake kinetics of pyrene by a
sponded to the lowest values of cell hydrophobicity. In
Rhodococus strain (Bouchez et al., 1997b). In the present
fact, because of their hydrophobicity, all our strains
study, the degradation kinetics of C corresponded to
the model for interfacial uptake only "' in the case of except HdGe1 exhibited some cell agglomeration in
cultures on soluble substrates such as succinate, glucose
PyrGe1 growing on C in silicone oil, where a long
exponential phase was"'observed. The specific growth
or glycerol, and the growth kinetics of strain NapRu1 on
glucose did not show a regular exponential growth
rates measured during this phase were found to be
pattern. However, growth of these strains on hexa-
higher in cultures with larger solvent volumes. The
decane clearly promoted cell aggregation.
characteristics of the cultures of strain PyrGe1 on C in
silicone oil were in good agreement with the growth "' Thus, the studies confirmed the diagnosis of an inter-
kinetics observed. The culture was not flocculent and facial uptake mechanism for the four strains studied, but
cell adsorption at the interface of solvent and aqueous showed that aggregation of these hydrophobic cells was
medium was partial. a major parameter that determined the degradation
kinetics. For one strain, PyrGe1, interfacial uptake was
A different situation, however, prevailed in other cases. either controlled or not by floc formation depending the
Flocculation of the cultures was always observed. Here, organic phase used. The results help to clarify the
the model for interfacial uptake can account for the hitherto insufficiently understood characteristics of
existence of a first phase of exponential growth. Higher interfacial uptake of alkanes. The phenomenon of
solvent volumes did not increase the values of the flocculation has significant ecological implications. It is
specific growth rates, but this can be explained by the also important for biotechnological applications. The
strong adsorption of the bacteria to their substrate, a case of micro-organisms producing biosurfactants is
well-recognized property of many alkane-degrading quite different, not only because biosurfactants promote
micro-organisms (Rosenberg, 1992). The interfacial the dispersion of the hydrophobic substrate as emulsions
uptake model, however, did not account for the fact that or as micelles, but also because they affect cell hydro-
the exponential phase was very short and that the phobicity and bacterial adherence to hydrocarbons, as
degradation rate during the subsequent phase of linear shown by Zhang & Miller (1994) for Pseudomonas
growth was not dependent on the solvent volume. The aeruginosa.
biological flocs formed were likely to be responsible for
these characteristics. Indeed, a similar situation was
discussed by Blanch & Einsele (1973) in the case of ACKNOWLEDGEMENTS
growth of the yeast Candida tropicalis on C . In flocs,
contact with hydrocarbon or dissolved oxygen "' is rate- We thank Bertrand Heyd for performing and interpreting
statistic analyses. We also thank Hanintsoa Rakotozafy for
limiting for cells inside the aggregates. Interfacial fermentations on hexadecane and Anne Regourd for cell
substrate uptake in this case is no longer controlled by hydrophobicity measurements.

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Kinetics of interfacial hexadecane uptake

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