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.
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).
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
| Prepn | Oxidation rate (μl of O2/h/mg [dry wt])a | 14CO2 radioactivity (dpm)b |
|---|---|---|
| Oil | 16.7 ± 1.4 | 400 ± 50 |
| Oil + Corexit 9527 (0.01%) | 9.7 ± 0.5 | 250 ± 150 |
| Oil + Span 80 (0.01%) | 41.1 ± 1.0 | 1,800 ± 200 |
| Oil + Tween 85 (0.01%) | 19.0 ± 2.4 | 700 ± 250 |
| Oil + Tween 80 (0.01%) | 16.0 ± 0.7 | 300 ± 200 |
| Oil + AOT (0.005%) | 6.1 ± 2.5 | |
| Oil + Span 80 (0.01%) + AOT (0.005%) | 30.3 ± 0.9 |
Oxidation in the presence of 0.5% (wt/vol) crude oil and 0.01 or 0.005% (wt/vol) surfactant in artificial seawater without nitrogen. The endogenous respiration rate with oil was 3.0 ± 0.3 μl of O2/h/mg (dry weight).
Amount of 14CO2 recovered from the NaOH trap at the end of the experiment.
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 14CO2 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.
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
| Prepn | Oxidation rate (μl of O2/h/mg [dry wt])a | 14CO2 radioactivity (dpm) |
|---|---|---|
| Acetate | 5.0 ± 0.3 | 2,300 ± 500 |
| Acetate + Span 20 (0.01%) | 32.3 ± 1.7 | 14,950 ± 2,500 |
| Acetate + Tween 85 (0.01%) | 31.0 ± 3.0 | 14,700 ± 550 |
| Acetate + Tween 80 (0.01%) | 26.7 ± 2.7 | 14,900 ± 350 |
| Acetate + Corexit 9527 (0.01%) | 25.9 ± 3.2 | 15,350 ± 550 |
| Acetate + AOT (0.005%) | 0.5 ± 1.7 | |
| Acetate + Span 20 (0.01%) + AOT (0.005%) | 15.3 ± 1.7 |
The endogenous respiration rate with acetate was 2.0 ± 0.7 μl of O2/h/mg (dry weight).
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).
TABLE 3.
Effects of Corexit 9527 and four surfactants on [2-14C]acetate uptake by oil-grown A. calcoaceticus ATCC 31012
| Prepn | [2-14C]acetate uptake (dpm)
|
|
|---|---|---|
| 5 min | 15 min | |
| Acetate | 2,100 ± 100 | 4,500 ± 400 |
| Acetate + Span 20 (0.01%) | 5,700 ± 500 | 33,000 ± 1,000 |
| Acetate + Tween 85 (0.01%) | 5,000 ± 750 | 41,000 ± 2,500 |
| Acetate + Tween 80 (0.01%) | 3,500 ± 1,300 | 23,900 ± 500 |
| Acetate + Corexit 9527 (0.01%) | 5,000 ± 1,200 | 33,000 ± 1,500 |
| Acetate + AOT (0.005%) | 1,850 ± 50 | 4,500 ± 50 |
| Acetate + Span 20 (0.01%) + AOT (0.005%) | 5,300 ± 300 | 35,100 ± 700 |
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.
TABLE 4.
Effects of surfactant mixtures on crude oil and acetate oxidation by oil-grown Rhodococcus sp. strain 094a
| Expt | Prepn | Oxidation rate (μl of O2/h/mg [dry wt]) |
|---|---|---|
| Ab | Oil | 5.1 ± 0.3 |
| Oil + Span 20 (0.01%) | 6.1 ± 0.1 | |
| Oil + AOT (0.005%) | 5.5 ± 0.4 | |
| Oil + Span 20 + AOT | 5.8 ± 0.1 | |
| Bc | Oil | 3.5 ± 0.4 |
| Oil + Tergitol 15-S-7 (0.01%) | 10.9 ± 0.5 | |
| Oil + AOT (0.005%) | 5.0 ± 0.1 | |
| Oil + Tergitol 15-S-7 + AOT | 0.3 ± 0.2 | |
| Cd | Oil | 3.4 ± 0.1 |
| Oil + Tergitol 15-S-7 (0.01%) | 8.4 ± 0.5 | |
| Oil + Span 20 (0.005%) | 5.0 ± 1.3 | |
| Oil + Tergitol 15-S-7 + Span 20 | 8.4 ± 0.9 | |
| De | Oil | 6.2 ± 0.5 |
| Oil + Tergitol 15-S-7 (0.01%) | 9.5 ± 0.2 | |
| Oil + AOT (0.005%) | 7.0 ± 0.3 | |
| Oil + Tergitol 15-S-7+ AOT | 1.1 ± 0.2 | |
| Oil + Tergitol 15-S-3+ AOT | 1.2 ± 0.2 | |
| Oil + Tergitol 15-S-15+ AOT | 4.2 ± 2.0 | |
| Oil + Tergitol 15-S-30+ Span 20 | 5.2 ± 0.9 | |
| Ef | Acetate | 21.5 ± 1.4 |
| Acetate + Tergitol 15-S-7 (0.01%) | 15.5 ± 1.0 | |
| Acetate + AOT (0.005%) | 13.6 ± 2.0 | |
| Acetate + Tergitol 15-S-7 + AOT | 0.5 ± 0.6 |
For experimental conditions see Table 1.
The endogenous respiration rate with oil was 2.4 ± 0.1 μl of O2/h/mg (dry weight).
The endogenous respiration rate with oil was 1.5 ± 0.2 μl of O2/h/mg (dry weight).
The endogenous respiration rate with oil was 1.3 ± 0.1 μl of O2/h/mg (dry weight).
The endogenous respiration rate with oil was 2.1 ± 0.3 μl of 02/h/mg (dry weight).
The endogenous respiration rate with acetate was 1.9 ± 0.4 μl of O2/h/mg (dry weight).
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.
TABLE 5.
Effects of two surfactants and mixtures on the uptake of [2-14C]acetate by oil-grown Rhodococcus sp. strain 094
| Prepn | [2-14C]acetate uptake (dpm)
|
|
|---|---|---|
| 5 min | 15 min | |
| Acetate | 16,800 ± 900 | 47,200 ± 600 |
| Acetate + Tergitol 15-S-7 (0.01%) | 9,250 ± 1,200 | 18,500 ± 400 |
| Acetate + AOT (0.01%) | 9,900 ± 50 | 18,600 ± 700 |
| Acetate + Tergitol + AOT | 900 ± 150 | 1,450 ± 100 |
| Acetate + heat-inactivated cells | 800 ± 250 | 1,100 ± 100 |
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 O2/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.
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