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Applied and Environmental Microbiology, April 1999, p. 1658-1661, Vol. 65, No. 4
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Effects of Surfactant Mixtures, Including Corexit
9527, on Bacterial Oxidation of Acetate and Alkanes in Crude
Oil
Per
Bruheim,*
Harald
Bredholt, and
Kjell
Eimhjellen
Department of Biotechnology, Faculty of
Chemistry and Biology, The Norwegian University of Science and
Technology, N-7491 Trondheim, Norway
Received 22 July 1998/Accepted 27 January 1999
 |
ABSTRACT |
Mixtures of nonionic and anionic surfactants, including Corexit
9527, were tested to determine their effects on bacterial oxidation of
acetate and alkanes in crude oil by cells pregrown on these substrates.
Corexit 9527 inhibited oxidation of the alkanes in crude oil by
Acinetobacter calcoaceticus ATCC 31012, while Span 80, a
Corexit 9527 constituent, markedly increased the oil oxidation rate.
Another Corexit 9527 constituent, the negatively charged dioctyl
sulfosuccinate (AOT), strongly reduced the oxidation rate. The
combination of Span 80 and AOT increased the rate, but not as much as
Span 80 alone increased it, which tentatively explained the negative
effect of Corexit 9527. The results of acetate uptake and oxidation
experiments indicated that the nonionic surfactants interacted with the
acetate uptake system while the anionic surfactant interacted with the
oxidation system of the bacteria. The overall effect of Corexit 9527 on
alkane oxidation by A. calcoaceticus ATCC 31012 thus seems
to be the sum of the independent effects of the individual surfactants
in the surfactant mixture. When Rhodococcus sp. strain 094 was used, the alkane oxidation rate decreased to almost zero in the
presence of a mixture of Tergitol 15-S-7 and AOT even though the
Tergitol 15-S-7 surfactant increased the alkane oxidation rate and AOT
did not affect it. This indicated that there was synergism between the
two surfactants rather than an additive effect like that observed for
A. calcoaceticus ATCC 31012.
| |
INTRODUCTION |
When surfactants are applied,
mixtures are often used because they perform better than the individual
components (7). The exact formulations of commercial
dispersants are proprietary, but the general guidelines indicate that
two or more nonionic surfactants with different water solubilities and
one or more charged surfactants, preferably anionic, are used and that
all of the compounds are dissolved in a solvent consisting of water, water-miscible hydroxy compounds, or hydrocarbons (5).
Corexit 9527, a frequently mentioned oil spill dispersant, was
developed for use on open sea oil slicks. This dispersant is composed
of about 48% nonionic surfactants, including ethoxylated sorbitan mono- and trioleates (Tween 80 and Tween 85) and sorbitan monooleate (Span 80), about 35% anionic surfactants, including sodium dioctyl sulfosuccinate (AOT), and about 17% ethylene glycol monobutyl ether as
a solvent (13). There have been reports of both negative and
positive effects of Corexit 9527 on bacterial degradation of crude oil
(6, 11, 14). The explanations given for the effect of this
surfactant mixture vary from a negative effect on the hydrocarbon
uptake rate to a positive effect due to increased surface area of the
substrate (12).
In recent reports there has been a strong emphasis on studying
surfactant-bacterial cell interactions to determine the influence of
surfactants on alkane oxidation (2-4). In the present
study, we compared surfactant mixtures like oil spill dispersant
mixtures with the individual components of the mixtures. The effects of the surfactants on acetate oxidation rates and uptake rates were also
investigated since the results could provide information about how the
individual surfactants and mixtures of surfactants affect cell
processes. This was important since in previous work (2, 4)
researchers focused on the physicochemical functions of the
surfactants; in this study we examined the interactions of the
surfactants with bacterial cells.
| |
MATERIALS AND METHODS |
Bacterial isolates.
Rhodococcus sp. strain 094 was
obtained from the FINA Culture Collection kept at SINTEF Applied
Chemistry, Group of Biotechnology, Trondheim, Norway. This isolate was
obtained from enrichment cultures by using inocula from Norwegian
coastal waters and was an alkane oxidation-positive organism (1,
9). Acinetobacter calcoaceticus ATCC 31012 was
purchased from the American Type Culture Collection (Rockville, Md.).
Suspensions of oil-grown and acetate-grown bacteria in 15% glycerol
were stored in 1-ml cryotubes at 
80°C.
Media.
The seawater medium used has been described
previously (2). The concentration of crude oil or acetate
was 0.5%.
Compounds.
Tergitol 15-S-3
(C11-15E3, HLB 8.0), Tergitol 15-S-7
(C11-15E7, HLB 12.1), Tergitol 15-S-15
(C11-15E15, HLB 115.4), and Tergitol 15-S-30
(C11-15E30, HLB 20.6) are
polyglycolether surfactants. Span 20 (HLB 8.6) and Span 80 (HLB 4.3)
are laureate and stearate sorbitan fatty acid esters, respectively.
Tween 85 (HLB 11.0) is an (ethoxy)20 sorbitan trioleate
ester, while Tween 80 (HLB 15.6) is the monooleate ester. The Tergitol,
Span, Tween, AOT, and sodium dodecyl sulfate surfactants were purchased
from Sigma Chemical Co., St. Louis, Mo. Corexit 9527 was kindly
provided by P. J. Brandvik, SINTEF, Trondheim, Norway.
[1-14C]hexadecane and [2-14C]acetate were
purchased from Amersham, Little Chalfont, United Kingdom. The medium
constituents were obtained from Merck, Darmstadt, Germany.
Oxidation rate measurement.
The protocol which we used to
measure oxidation rates has been described previously (2).
Oxidation rates (in microliters of O2 per hour per
milligram [dry weight]) were determined by Warburg respirometry. The
cells were pregrown for 48 h (to the early stationary phase) in
500-ml shake flasks containing 100 ml of medium at 25°C, centrifuged
at 15,000 × g, and washed twice in N-free mineral
medium. A 150-µl portion of each cell suspension (5 to 10 mg [dry
weight]/ml) was transferred to the side arm of a Warburg flask (20 ml). The standard concentrations used were 0.5% (wt/vol) oil and
0.01% (wt/vol) surfactant. Surfactant-treated oil and N-free mineral
medium (1 ml) were premixed in the central compartment during 30 min of
temperature equilibration (25°C) before the cells were added. In some
experiments the crude oil was replaced with 10 µM acetate as the
substrate. Mineralization of acetate and alkanes was assessed by
determining the amount of 14CO2 produced from
[2-14C]acetate (150,000 dpm/flask) or from
[1-14C]hexadecane (50,000 dpm/flask) present in the oil.
The contents of the CO2 trap (0.1 ml of 2 M NaOH) in the
center well were transferred to Opti-Fluor scintillation cocktail
(Packard) and counted with a Wallac model s1410 scintillation counter.
Every experiment was performed at least twice with three flasks for
every condition. The results of one representative experiment are
presented below, and the statistical variations are indicated by the
standard deviations.
[14C]acetate uptake.
Cells were pregrown and
washed cell suspensions were prepared as described above for the
oxidation studies. Twenty milliliters of a cell suspension (5 to 10 mg
[dry weight]/ml) containing surfactants was mixed with 10 µM
[14C]acetate (150,000 dpm/ml). After 5 and 15 min three
2-ml aliquots were removed and filtered with a type GF/F 47-mm-diameter
Whatman microfiber filter. The filters were washed with 10 ml of
mineral medium and transferred to scintillation vials containing 10 ml of Hisafe III scintillation fluid (Pharmacia). After 2 h of
equilibration, the radioactivity was measured with the Wallac model
s1410 scintillation counter. Heat-inactivated Rhodococcus
sp. strain 094 cells were warmed to 100°C and cooled rapidly to room
temperature in a water bath. Viable counting indicated that less than
0.5% of the cells survived.
| |
RESULTS AND DISCUSSION |
A. calcoaceticus ATCC 31012.
The oxidation rates
of A. calcoaceticus ATCC 31012 were determined by Warburg
respirometry as described previously (2). These rates were
corrected for O2 uptake by using cell suspensions containing surfactants but no crude oil. In each case the presence of
surfactants resulted in a small increase in the respiration rate, but
this increase did not exceed two times the endogenous respiration rate.
Corexit 9527 decreased the rate of oxidation of alkanes in crude oil by
A. calcoaceticus ATCC 31012 rather strongly (Table 1). On the other hand, sorbitan
monooleate (Span 80, a Corexit 9527 constituent) increased the
oxidation rate very markedly. Tween 85 and Tween 80, the two other
surfactant components of Corexit 9527, did not affect and slightly
increased the oil oxidation rate, respectively. AOT, the prominent
anionic surfactant constituent of Corexit 9527, had a very strong
negative effect on the oil oxidation rate. The combination of Span 80 and AOT increased the oxidation rate, but not as much as Span 80 alone
increased it. The correlation between Corexit 9527 and the mixture
containing Span 80 and AOT was not quantitatively substantiated, but
this may have been due to differences in surfactant concentrations and
the presence of Tween 80, Tween 85, and other anionic surfactants in
Corexit 9527. The mineralization data, expressed as endpoint values for
the amount of 14CO2 that evolved from
[1-14C]hexadecane-spiked oil, validated the oxidation
results. The solvent of Corexit 9527, ethylene glycol monobutyl ether,
had no effect on the oxidation rate (data not shown).
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TABLE 1.
Effects of Corexit 9527, four of its component
surfactants, and one mixture on crude oil oxidation by oil-grown
A. calcoaceticus ATCC 31012
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In experiments performed with acetate as the substrate Span 80 was
replaced by Span 20 due to the very poor water solubility
of the former
compound. Span 20 had the same positive effect on
the oil oxidation
rate that Span 80 had (
4). Oil-grown
A. calcoaceticus ATCC 31012 cells had a very low specific oxidation
rate for acetate
(Table
2). In the
presence of Span 20 the oxidation rate increased
almost six times. This
was not due to oxidation of Span 20 but
was due to increased oxidation
of acetate, as confirmed by
14CO
2 recovery data
obtained with [2-
14C]acetate. The other sorbitan
surfactants and Corexit 9527 also
increased the rate of oxidation of
acetate to the same degree,
in contrast to the situation for oil
oxidation, where only Span
20 increased the oxidation rate.
Furthermore, the negatively charged
surfactant AOT drastically
decreased the acetate oxidation rate,
and the positive effect of the
Span 20-AOT mixture was much less
than the positive effect of Span 20 alone. Span 20-AOT mixtures
thus had very similar effects on the
oxidation of alkanes and
the oxidation of acetate in
A. calcoaceticus ATCC 31012.
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TABLE 2.
Effects of Corexit 9527 and four surfactants on acetate
oxidation by oil-grown A. calcoaceticus ATCC 31012 cells in
the stationary phase of growth
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The acetate oxidation data were correlated with acetate uptake rates.
The uptake of [2-
14C]acetate increased significantly in
the presence of the nonionic
surfactants and Corexit 9527 (Table
3). AOT had very little effect
on the
rate of uptake of acetate. Therefore, AOT had a strong
negative effect
on oxidation of acetate but not on transport of
acetate, while the
nonionic surfactants and Corexit 9527 increased
the rate of acetate
oxidation, probably by increasing the transport
rates. AOT did not
influence the effect of Span 20 on the acetate
uptake rate, which
contrasts with the effect of the mixture on
both the alkane and acetate
oxidation rates. It seems, therefore,
that the effect of the surfactant
mixture on acetate oxidation
was the sum of two independent effects,
the effect of AOT on the
oxidation machinery (a negative effect) and
the effect of Span
20 or Span 80 on the transport machinery (a positive
effect).
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TABLE 3.
Effects of Corexit 9527 and four surfactants on
[2-14C]acetate uptake by oil-grown A. calcoaceticus ATCC 31012
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Only two of the nonionic surfactants examined, Span 20 and Span 80, increased the alkane oxidation rates. This indicates that
the effects
of the nonionic surfactants on the alkane oxidation
rate were not due
to the general amphiphilic properties of the
surfactants but rather to
a specific interaction determined by
both the chemical structures and
the physicochemical properties
of the surfactants. In addition, the
effects of the surfactants
are also probably determined in part by the
structure of the components
in the bacterial cell envelope. Based on
these findings and the
acetate uptake and oxidation results, it may be
hypothesized that
the overall effect of Corexit 9527 on alkane
oxidation, as well
as acetate oxidation, is the sum of independent
effects exerted
by the individual surfactants in the surfactant
mixture.
Rhodococcus sp. strain 094.
The mixture containing
Span 20 and AOT and the individual surfactants were also tested with
the gram-positive organism Rhodococcus sp. strain 094. Span
20 slightly increased the alkane oxidation rate, while AOT and the
mixture containing the two surfactants had little or no effect on the
oxidation rate (Table 4, experiment A).
Span 20 was replaced by Tergitol 15-S-7, which is known to increase the
alkane oxidation rate in Rhodococcus sp. strain 094 (2). Tergitol 15-S-7 caused a threefold increase in the oil oxidation rate in oil-grown cells (Table 4, experiment B). AOT alone
slightly increased the oxidation rate. Mixing the two surfactants, however, resulted in almost complete cessation of alkane oxidation. The
endogenous respiration of the cells in the presence of the surfactant
mixture was also severely reduced (data not shown). Tergitol 15-S-7
interacted strongly with Rhodococcus sp. strain 094 cells
since it strongly increased the oil oxidation rate. In a mixture with
AOT, Tergitol 15-S-7 may decrease the expected repulsion between the
negatively charged bacterial cells and the negatively charged compound
AOT. This may give AOT access to structures in the cell envelope that
are not available to AOT alone and thus may explain the observed
synergistic effect. Span 20 did not influence the positive effect of
Tergitol 15-S-7 (Table 4, experiment C), which may indicate that Span
20 interacts much more weakly than Tergitol 15-S-7 with cell
structures. Therefore, as shown in Table 4 (experiment A), Span 20 cannot facilitate AOT's access to cell structures that are critical
for the integrity of the cells. This may also explain the observed
effects of the homologous Tergitol compounds shown in Table 4
(experiment D). The two more hydrophobic surfactants, Tergitol 15-S-7
and Tergitol 15-S-3, increased the rate of alkane oxidation by
Rhodococcus sp. strain 094 cells grown from the stationary
phase (2). The strong interactions between the surfactants
and the cells resulted in almost complete cessation of alkane oxidation
when the two surfactants were mixed with AOT (Table 4, experiment D).
The two more hydrophilic surfactants, Tergitol 15-S-15 and Tergitol
15-S-30, did not significantly increase the rate of alkane oxidation by
Rhodococcus sp. strain 094 cells grown from the stationary
phase (2). When Tergitol 15-S-15 and Tergitol 15-S-30 were
mixed with AOT, the decreases in the oxidation rate were much less than
the decreases observed with Tergitol 15-S-3 and Tergitol 15-S-7, in
accordance with the weaker interactions of the former nonionic
surfactants with the cells.
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TABLE 4.
Effects of surfactant mixtures on crude oil and acetate
oxidation by oil-grown Rhodococcus sp.
strain 094a
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Separately, Tergitol 15-S-7 and AOT decreased the rate of oxidation of
acetate by 30 to 40%, whereas a mixture containing
both of these
compounds decreased the oxidation rate to almost
zero (Table
4,
experiment E). The rates of uptake of [2-
14C]acetate by
Rhodococcus sp. strain 094 cells (Table
5) in the
presence of Tergitol 15-S-7 or
AOT were positively correlated
with the acetate oxidation data shown in
Table
4 (experiment
E). Tergitol 15-S-7 and AOT separately affected
acetate oxidation
by reducing the specific transport of acetate. The
Tergitol 15-S-7-AOT
mixture resulted in uptake of acetate
corresponding to the uptake
by heat-inactivated cells.
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TABLE 5.
Effects of two surfactants and mixtures on the uptake of
[2-14C]acetate by oil-grown Rhodococcus sp.
strain 094
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Comparison of acetate- and oil-grown A. calcoaceticus
ATCC 31012 and Rhodococcus sp. strain 094.
The alkane
oxidation genes are not constitutively expressed in most gram-positive
and gram-negative bacteria. There is a necessary induction period prior
to growth on alkanes, and there is derepression of the alkane oxidation
system, as well as a system for uptake of and adhesion to the
hydrophobic substrate (8). The latter very often coincides
with synthesis of biosurfactants, which alter the cell surface topology
of the degrading cells (10). Rhodococcus sp.
strain 094 gains a hydrophobic surface and adheres to the hexadecane
phase when it is transferred from acetate-containing medium to
hexadecane-containing medium (1). A. calcoaceticus Rag-1 generally is very hydrophobic during growth on
hexadecane and produces a water-bound heteropolysaccharide
bioemulsifier named emulsan. A comparative study of the effects of
surfactants on acetate- and oil-grown cells might provide information
about the dissimilarities of these two types of cells.
Oil-grown
A. calcoaceticus ATCC 31012 cells had a very low
specific activity for acetate oxidation compared to acetate-grown
cells
(5 versus 42 µl of O
2/h mg [dry weight]
1)
(Table
2; data not shown). In the presence of Corexit 9527,
Span 20, Tween 80, and Tween 85 the specific rate of acetate oxidation
in
oil-grown cells increased to approximately the rate in acetate-grown
cells. This indicated that there was surface restriction of acetate
transport that was circumvented by the added surfactants. AOT
affected
acetate oxidation and alkane oxidation in the same negative
way in
oil-grown cells, and the presence of an interacting nonionic
surfactant
partially counteracted the action of AOT (Tables
1 and
2). This may
indicate that overall oxidation of acetate in
oil-grown cells of
A. calcoaceticus ATCC 31012 is restricted by
the specific
surface conditions of cells induced to grow on hydrophobic
substrates.
In acetate-grown cells of
A. calcoaceticus ATCC 31012, Span
20 caused a moderate (10%) decrease in the acetate oxidation rate,
and
AOT and the mixture of the two compounds decreased the acetate
oxidation rate by 20% (data not shown), in sharp contrast to the
results obtained for the oil-grown cells. These findings illustrate
the
marked difference between oil-grown and acetate-grown cells
of this
gram-negative bacterium, which most likely is linked to
differences in
surface structure or
topography.
A mixture of Tergitol 15-S-7 and AOT affected acetate oxidation in
acetate-grown cells of
Rhodococcus sp. strain 094 in the
same way (data not shown) that it affected acetate oxidation in
oil-grown cells (Table
4, experiment E); however, when tested
separately, the surfactants had no effect on acetate-grown cells,
in
contrast to the marked negative effect that they had on oil-grown
cells. While Tergitol 15-S-7 markedly affected acetate oxidation
in
oil-grown cells, the results suggest that there was only a
weak
interaction in acetate-grown cells, which clearly indicated
that there
are structural differences between the two types of
cells. The weak
interaction of Tergitol 15-S-7 with acetate-grown
cells was, however,
sufficient for the dramatic negative synergistic
effect with AOT to
take
place.
In summary, we found that the effects of surfactant mixtures on
bacterial metabolism may not always be easily predicted on
the basis of
the effects of the individual surfactants in the
mixtures. Admittedly,
our information is limited, but two main
conclusions appear to be
relevant. The surfactants in a mixture
may independently affect various
sites in the cell and have an
overall effect which is additive. This
seems to be case for
A. calcoaceticus ATCC 31012. Alternatively, surfactants may influence
each other's interactions
with cells, resulting in synergistic
effects. This seems to be the case
for the gram-positive organism
Rhodococcus sp. strain
094.
| |
ACKNOWLEDGMENTS |
This work was supported by The Research Council of Norway and by
Fina Exploration Norway.
We thank P. J. Brandvik for providing Corexit 9527 and the Group
of Biotechnology, SINTEF Applied Chemistry, for providing Statfjord
crude oil.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Biotechnology, Faculty of Chemistry and Biology, The Norwegian
University of Science and Technology, Sem Selands vei 6/8, N-7491
Trondheim, Norway. Phone: 47-73593104. Fax: 47-73591283. E-mail:
bruheim{at}chembio.ntnu.no.
| |
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Applied and Environmental Microbiology, April 1999, p. 1658-1661, Vol. 65, No. 4
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
