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Alkane biodegradation by a microbial community from contaminated sediments in Patagonia, Argentina
International Biodeterioration & Biodegradation, Vol. 40, No. 1 (1997) 75-19 0 1997 Elwier Science Limited AU rights reserved. Printed in Great Britain
PII:SO964-8305(97)00065-6
ELSEVIER
09&l-8305/97 $17.00+0.00
Alkane Biodegradation by a Microbial Community from Contaminated Sediments in Patagonia, Argentina Nelda L. Olivera*, Jo.4 L. Esteves & Marta G. Commendatore Centro National Patagdnico (CONICET),
Bv. Brown s/n, 9120 Puerto Madryn, Chubut, Argentina
Biodegradation of a mix of normal alkanes (decane, dodecane, tetradecane, hexadecane, octadecane and eicosane) was studied in batch cultures after inoculating with microbial communities from pristine and hydrocarbon contaminated sediments. Analysis showed that the community from polluted sediments reduced the concentrations of all alkanes to < 5 mg 1-l after a 240-h incubation period (< 5% initial concentration), while the control community only degraded 510% of them. The hydrocarbon adapted community showed a lag phase of 48 h, in which no alkane biodegradation was found, followed by a 96-h growth period and a stationary phase from that moment, whereas the control community grew poorly. Isolated strains were mainly Gram-negative, motile and non-glucose fermenter rods. Based on these results, it could be concluded that the hydrocarbon adaptation of microorganisms led to an increase in alkane biodegradation. This capacity could be useful to improve biodegradation of hydrocarbon regional wastes. 0 1997 Elsevier Science Ltd. All rights reserved
INTRODUCTION
microorganisms to biodegrade them (Alvarez & Pucci, 1993). The aim of this paper is to evaluate the degradation of normal alkanes by microbial communities from pristine and hydrocarbon polluted sediments.
It has been well documented that microorganisms are responsible for part of the removal of hydrocarbons from the environment (Atlas, 1981; Rosemberg, 1991; Cerniglia, 1992; Wilson & Jones, 1993). This degradative potential has been used in the bioremediation of oil-contaminated areas; the effectiveness of such processes under field conditions is affected by many factors (Swannell et al., 1996). A number of oil development activities that involve hydrocarbon spill risks, including exploration, exploitation, and marine transport, have been taking place along the Patagonian coast for several decades. This coastal ecosystem is liable to chronic petroleum pollution, which causes not only damage to fauna and flora but might also negatively affect ecotourism activities. A few studies have analysed the presence of hydrocarbons in the Patagonian coasts (Esteves & Commendatore, 1993; Esteves et al., 1993), and the potential capacity of autochthonous
MATERIALS AND METHODS Sampling Sediment samples were taken using sterile flasks from two stations: (1) a chronically polluted beach, situated near a waste water stream with hydrocarbons (45” 49’ S, 67” 27’ W), north of the city of Comodoro Rivadavia, Golfo San Jorge, Patagonia (community A). Salinity 33.5 gl-‘, winter temperature 9°C and summer temperature 12°C (Piola & Garcia, 1993). (2) a non-polluted beach, situated 6 km north of the city of Puerto Madryn (42” 40’ S, 64” 59’ W), Golfo Nuevo, Patagonia (community B). Salinity 33.9 g l-‘, winter temperature 12°C and summer temperature 15°C.
*To whom correspondence should be addressed at: Centro National Patagonico (CONICET), Bv. Brown s/n, 9120 Puerto Madryn, Chubut, Argentina. 75
76
N. L. Olivera et al.
Inocolmn preparation
Other methods
A sediment sample (100 g) was placed into 500 ml Erlenmeyer flasks with 150 ml of sterile synthetic sea water medium (SWM) with the following composition (gl-‘): Tris 12.1; NaCl, 23; KCl, 0.75; CaC12.2H20, 1.47; MgClZ.6H20, 6.16; NH&l, 3.74; NaNOs, 2.97 at pH= 7.3. After autoclaving for 20 min at 12O”C, 2 ml 1-l and 4 ml 1-l of sterile solutions of FeS04 (6.6 mM) and sodium phosphate (Na2HP04. 12HzO + NaH2P04; 0.07 M and 0.03 M), respectively, were added. These flasks were shaken for 2min to detach the bacteria fixed at the sediment.
Microbial growth was determined at different times by spectrophotometry (Hitachi llOA), at 450 nm. Samples of the cultures were transferred onto nutrient agar plates prepared with seawater. Isolates were tested for morphology and mobility (microscopically), Gram reaction, catalase and oxidase (Bactident Oxidase, Merck) production, and glucose utilization (OF Basal Medium Hugh and Leifson, Merck, with a final concentration of 1.O% of glucose).
Cultures
RESULTS AND DISCUSSION
The studies were performed in 250ml Erlenmeyer flasks with 90ml SWM and lOm1 of inoculum. A solution of normal alkanes in pentane was added as a carbon source to reach the following concentrations in the cultures (mgl-‘): decane 87.6, dodecane 87.8, tetradecane 91.5, hexadecane 92.8, octadecane 80.0, and eicosane 80.0 (SIGMA Standards for GLC). Eight replicates from each inoculum were prepared (A and B), two of which were used to measure growth and the rest to recover residual hydrocarbons. Flasks were incubated for 10 days in a reciprocal shaker at 25°C. Sterile controls evaluate hydrocarbon were prepared to evaporation.
Residual alkanes were determined at 48, 120, and 240 h in cultures inoculated with the community from a polluted beach (A) and from a pristine one (B). After the first 48 h of incubation similar hydrocarbon concentrations were found in cultures and sterile controls, possibly due to evaporation. This period matched the lag phase in community ‘A’ cultures. After which a growth period of 96 h, followed by stabilization, was This reduced community the found. concentration of all the alkanes to < 1Omg 1-l after 120 h of incubation, while the community sediments showed significant from pristine concentrations of undegraded alkanes even at the end of the experiment, except for dodecane, which was the most volatile (Fig. 1). Community ‘A’ cultures showed important growth in comparison with that of the community not exposed to contamination (‘B’). Within 72 h, community ‘A’ culture turbidities were substantially greater than those of community ‘B’. The h5snm of community ‘A’ continued to increase throughout the 240-h time course. In contrast, the &sonm of community ‘B’ remained essentially constant at approximately 0.1 (Fig. 2). summarizes evaporated, Figure 3(a) and recovered hydrocarbon biodegraded concentrations of each alkane after a 240-h incubation period for community ‘A’. At this time, all alkanes had been reduced to < 5 mg l-‘, indicating that only about 4% of them remained in the system. Eicosane biodegradation was 58%, while octadecane, hexadecane, and tetradecane were removed by 42%, 35% and 28%, respectively. Dodecane biodegradation was 7.5% because of its rapid evaporation. Decane loss due
Hydrocarbon degradation
In order to recover residual hydrocarbons after cultures and sterile microbial degradation, controls were collected at different times, centrifuged at 3000rpm, and extracted twice with methylene chloride (50ml). Both extracts were combined and evaporated at room temperature. The alkane concentrations were analysed using a gas chromatograph (KONIK-3000), equipped with a flame ionization detector and a splitless injector. A column of 30mx0.25 mm i.d. coated with DB-1 (film thickness 0.25pm) was used. Nitrogen was the carrier gas (1 mlmin-‘). The temperature was programmed from 60°C to 290°C at 8°C min-’ . Injector and detector temperatures were 200°C and 32O”C, respectively. Identification and quantification of hydrocarbons were made by comparison with external standard mixtures of alkanes.
77
Alkane biodegradation
80
20
0
(b)
48
96
144
192
240
Hours 100
,
80
60
O-
’
a8
’
66
’
Ii4
’
1 192
I 240
Hours
Concentrations of (B) dodecane, (0) tetradecane, (v) hexadecane, (A) octadecane, and (0) eicosane after 48, 120, and 240 h of incubation of the cultures with: (a) microbial community from contaminated sediments and (b) community from pristine sediments. Each point represents the arithmetic mean for duplicates, error bars indicate &l standard deviation, in figure: (a) at 48 h SD varies between 7.5 and 0.75mg/l; at 120 and 240 h SD<2.2mg/l; (b) at 48 h SD varies between 9.3 and Omg/l; at 120 and 240 h SDs3.2 mg/l.
Fig. 1.
to evaporation was almost complete at 48 h, so no conclusion about its degradation can be drawn. Community ‘B’, from pristine sediments, showed limited alkane biodegradation capacity, about 5510% after 240 h (Fig. 3b). However, it is important to point out that even though this an area free of community comes from anthropogenic hydrocarbon contributions (Esteves & Commendatore, 1993) it shows some potential to degrade alkanes. Studies carried out with Antarctic microbial populations, after the Bahia Paraiso diesel fuel arctic (DFA) spill, showed that both impacted populations and controls had some hydrocarbon oxidation potential (Karl, 1992). The strains isolated from the cultures of both communities were mainly Gram-negative rods,
motile, non-glucose fermenters, and catalase and oxidase positives. Yeast strains were also found in ‘A’ cultures. Other researchers have found that predominate in bacteria Gram-negative recoverable hydrocarbon communities of oxidizing organisms (Westlake et al., 1974; Alvarez & Pucci, 1993). Based on these degradation studies, it could be concluded that, after controlling for physical and chemical factors under laboratory conditions, the hydrocarbon adaptation of microorganisms led to an increase in alkane biodegradation. This agrees with observations made by other authors, who found that hydrocarbon presence in the selective environment often produces a enrichment of microorganisms with the potential
78
0
N. L. Olivera et
al.
96
144
48
192
240
Hours Fig.
2.
Growth curves of the cultures with microorganisms from: (W) polluted and (0) pristine sediments. Each point represents the average absorbance (wavelength 450nm) for duplicates, error bars indicate fl SD.
(4 20
@I 20
12 0
50
recovered
n
mgn
biodegraded
fq ..~~~~ evaporated
Fig. 3. Evaporated, biodegraded, and recovered concentrations of each alkane (dodecane, tetradecane, hexadecane, octadecane and eicosane) after 240 h of incubation of the cultures with: (a) community adapted to hydrocarbon pollution, and (b) pristine sediment community.
to attack them (Leahy & Colwell, 1990; Rosemberg, 1991). These preliminary studies also suggest the possibility of using community ‘A’ strains to degrade hydrocarbon regional wastes. Current research is being conducted to improve
biodegradation of ship bilge residues by means of bioaugmentation with combinations of these strains. In our case, these kind of residues are mainly composed by aliphatic hydrocarbons including continuous homologous series of y1alkanes.
Alkane biodegradation
ACKNOWLEDGEMENTS
The authors gratefully acknowledge Dr F. Siiieriz for critical revision of this paper and Dr P. Yorio for the correction of the English manuscript. This work was supported by the Patagonian Coastal Plan Zone Management (GEF/PNUD), implemented by Fundacion Patagonia Natural and Wildlife Conservation Society, and grants from Universidad National de la Patagonia San Juan Bosco and Centro National Patagonico (CONICET).
REFERENCES Alvarez, H. M. & Pucci, 0. H. (1993) Biodegradation de hidrocarburos en condiciones de baja temperatura. Petrotecnia, 34(2), 3 I-34.
Atlas, R. M. (1981) Microbial degradation of petroleum hydrocarbons: an environmental perspective. Microbiological Reviews 45, 180-209. Cerniglia, C. E. (1992) Biodegradation of polycyclic aromatic hydrocarbons. Biodegradation 3, 351-368.
79
Esteves, J. L. & Commendatore, M. G. (1993) Total aromatic hydrocarbons in water and sediments in a coastal zone of Patagonia, Argentina. Marine Pollution Bulletin 26, 341342.
Esteves, J. L., Commendatore, M. G., Solis, M., Gil, M. 8z Olivera, N. (1993) Investigation en Costas de1 Atlantic0 Sur. Petrotecnia, 3, pp. 26-32. Karl, D. M. (1992) The grounding of the Bahia Paraiso: Microbial ecology of the 1989 Antarctic oil spill. Microbial Ecology 24, 77-89.
Leahy, J. G. & Colwell, R. R. (1990) Microbial degradation of hydrocarbons in the environment. Microbiological Reviews 54,305-31.5.
Piola, A. R. & Garcia, 0. A. (1993) Atlas Oceanogrhjko de la Cuenca Argentina Occidental y de la Plataforma Continental Lindera. Servicio de Hidrografia Naval, Publication H-670.
Rosemberg, E. (199 1) Hydrocarbon-oxidizing bacteria. In The Prokaryotes, ed. A. Ballows, pp. 441459. Springer--Verlag, Berlin. Swannell, R. P. J., Lee, K. & McDonagh, M. (1996) Field evaluations of marine oil spill bioremediation. Microbiological Reviews 60, 342-365.
Westlake, D. W. S., Jobson, A., Phillippe, R. & Cooke, F. D. (1974) Biodegradability and crude oil composition. Canadian Journal of Microbiology 20, 915-928.
Wilson, C. S. & Jones, K. C. (1993) Bioremediation of soil contaminated with polynuclear aromatic hydrocarbons (PAH,): a review. Environmental Pollution 81, 229-249.
PII:SO964-8305(97)00065-6
ELSEVIER
09&l-8305/97 $17.00+0.00
Alkane Biodegradation by a Microbial Community from Contaminated Sediments in Patagonia, Argentina Nelda L. Olivera*, Jo.4 L. Esteves & Marta G. Commendatore Centro National Patagdnico (CONICET),
Bv. Brown s/n, 9120 Puerto Madryn, Chubut, Argentina
Biodegradation of a mix of normal alkanes (decane, dodecane, tetradecane, hexadecane, octadecane and eicosane) was studied in batch cultures after inoculating with microbial communities from pristine and hydrocarbon contaminated sediments. Analysis showed that the community from polluted sediments reduced the concentrations of all alkanes to < 5 mg 1-l after a 240-h incubation period (< 5% initial concentration), while the control community only degraded 510% of them. The hydrocarbon adapted community showed a lag phase of 48 h, in which no alkane biodegradation was found, followed by a 96-h growth period and a stationary phase from that moment, whereas the control community grew poorly. Isolated strains were mainly Gram-negative, motile and non-glucose fermenter rods. Based on these results, it could be concluded that the hydrocarbon adaptation of microorganisms led to an increase in alkane biodegradation. This capacity could be useful to improve biodegradation of hydrocarbon regional wastes. 0 1997 Elsevier Science Ltd. All rights reserved
INTRODUCTION
microorganisms to biodegrade them (Alvarez & Pucci, 1993). The aim of this paper is to evaluate the degradation of normal alkanes by microbial communities from pristine and hydrocarbon polluted sediments.
It has been well documented that microorganisms are responsible for part of the removal of hydrocarbons from the environment (Atlas, 1981; Rosemberg, 1991; Cerniglia, 1992; Wilson & Jones, 1993). This degradative potential has been used in the bioremediation of oil-contaminated areas; the effectiveness of such processes under field conditions is affected by many factors (Swannell et al., 1996). A number of oil development activities that involve hydrocarbon spill risks, including exploration, exploitation, and marine transport, have been taking place along the Patagonian coast for several decades. This coastal ecosystem is liable to chronic petroleum pollution, which causes not only damage to fauna and flora but might also negatively affect ecotourism activities. A few studies have analysed the presence of hydrocarbons in the Patagonian coasts (Esteves & Commendatore, 1993; Esteves et al., 1993), and the potential capacity of autochthonous
MATERIALS AND METHODS Sampling Sediment samples were taken using sterile flasks from two stations: (1) a chronically polluted beach, situated near a waste water stream with hydrocarbons (45” 49’ S, 67” 27’ W), north of the city of Comodoro Rivadavia, Golfo San Jorge, Patagonia (community A). Salinity 33.5 gl-‘, winter temperature 9°C and summer temperature 12°C (Piola & Garcia, 1993). (2) a non-polluted beach, situated 6 km north of the city of Puerto Madryn (42” 40’ S, 64” 59’ W), Golfo Nuevo, Patagonia (community B). Salinity 33.9 g l-‘, winter temperature 12°C and summer temperature 15°C.
*To whom correspondence should be addressed at: Centro National Patagonico (CONICET), Bv. Brown s/n, 9120 Puerto Madryn, Chubut, Argentina. 75
76
N. L. Olivera et al.
Inocolmn preparation
Other methods
A sediment sample (100 g) was placed into 500 ml Erlenmeyer flasks with 150 ml of sterile synthetic sea water medium (SWM) with the following composition (gl-‘): Tris 12.1; NaCl, 23; KCl, 0.75; CaC12.2H20, 1.47; MgClZ.6H20, 6.16; NH&l, 3.74; NaNOs, 2.97 at pH= 7.3. After autoclaving for 20 min at 12O”C, 2 ml 1-l and 4 ml 1-l of sterile solutions of FeS04 (6.6 mM) and sodium phosphate (Na2HP04. 12HzO + NaH2P04; 0.07 M and 0.03 M), respectively, were added. These flasks were shaken for 2min to detach the bacteria fixed at the sediment.
Microbial growth was determined at different times by spectrophotometry (Hitachi llOA), at 450 nm. Samples of the cultures were transferred onto nutrient agar plates prepared with seawater. Isolates were tested for morphology and mobility (microscopically), Gram reaction, catalase and oxidase (Bactident Oxidase, Merck) production, and glucose utilization (OF Basal Medium Hugh and Leifson, Merck, with a final concentration of 1.O% of glucose).
Cultures
RESULTS AND DISCUSSION
The studies were performed in 250ml Erlenmeyer flasks with 90ml SWM and lOm1 of inoculum. A solution of normal alkanes in pentane was added as a carbon source to reach the following concentrations in the cultures (mgl-‘): decane 87.6, dodecane 87.8, tetradecane 91.5, hexadecane 92.8, octadecane 80.0, and eicosane 80.0 (SIGMA Standards for GLC). Eight replicates from each inoculum were prepared (A and B), two of which were used to measure growth and the rest to recover residual hydrocarbons. Flasks were incubated for 10 days in a reciprocal shaker at 25°C. Sterile controls evaluate hydrocarbon were prepared to evaporation.
Residual alkanes were determined at 48, 120, and 240 h in cultures inoculated with the community from a polluted beach (A) and from a pristine one (B). After the first 48 h of incubation similar hydrocarbon concentrations were found in cultures and sterile controls, possibly due to evaporation. This period matched the lag phase in community ‘A’ cultures. After which a growth period of 96 h, followed by stabilization, was This reduced community the found. concentration of all the alkanes to < 1Omg 1-l after 120 h of incubation, while the community sediments showed significant from pristine concentrations of undegraded alkanes even at the end of the experiment, except for dodecane, which was the most volatile (Fig. 1). Community ‘A’ cultures showed important growth in comparison with that of the community not exposed to contamination (‘B’). Within 72 h, community ‘A’ culture turbidities were substantially greater than those of community ‘B’. The h5snm of community ‘A’ continued to increase throughout the 240-h time course. In contrast, the &sonm of community ‘B’ remained essentially constant at approximately 0.1 (Fig. 2). summarizes evaporated, Figure 3(a) and recovered hydrocarbon biodegraded concentrations of each alkane after a 240-h incubation period for community ‘A’. At this time, all alkanes had been reduced to < 5 mg l-‘, indicating that only about 4% of them remained in the system. Eicosane biodegradation was 58%, while octadecane, hexadecane, and tetradecane were removed by 42%, 35% and 28%, respectively. Dodecane biodegradation was 7.5% because of its rapid evaporation. Decane loss due
Hydrocarbon degradation
In order to recover residual hydrocarbons after cultures and sterile microbial degradation, controls were collected at different times, centrifuged at 3000rpm, and extracted twice with methylene chloride (50ml). Both extracts were combined and evaporated at room temperature. The alkane concentrations were analysed using a gas chromatograph (KONIK-3000), equipped with a flame ionization detector and a splitless injector. A column of 30mx0.25 mm i.d. coated with DB-1 (film thickness 0.25pm) was used. Nitrogen was the carrier gas (1 mlmin-‘). The temperature was programmed from 60°C to 290°C at 8°C min-’ . Injector and detector temperatures were 200°C and 32O”C, respectively. Identification and quantification of hydrocarbons were made by comparison with external standard mixtures of alkanes.
77
Alkane biodegradation
80
20
0
(b)
48
96
144
192
240
Hours 100
,
80
60
O-
’
a8
’
66
’
Ii4
’
1 192
I 240
Hours
Concentrations of (B) dodecane, (0) tetradecane, (v) hexadecane, (A) octadecane, and (0) eicosane after 48, 120, and 240 h of incubation of the cultures with: (a) microbial community from contaminated sediments and (b) community from pristine sediments. Each point represents the arithmetic mean for duplicates, error bars indicate &l standard deviation, in figure: (a) at 48 h SD varies between 7.5 and 0.75mg/l; at 120 and 240 h SD<2.2mg/l; (b) at 48 h SD varies between 9.3 and Omg/l; at 120 and 240 h SDs3.2 mg/l.
Fig. 1.
to evaporation was almost complete at 48 h, so no conclusion about its degradation can be drawn. Community ‘B’, from pristine sediments, showed limited alkane biodegradation capacity, about 5510% after 240 h (Fig. 3b). However, it is important to point out that even though this an area free of community comes from anthropogenic hydrocarbon contributions (Esteves & Commendatore, 1993) it shows some potential to degrade alkanes. Studies carried out with Antarctic microbial populations, after the Bahia Paraiso diesel fuel arctic (DFA) spill, showed that both impacted populations and controls had some hydrocarbon oxidation potential (Karl, 1992). The strains isolated from the cultures of both communities were mainly Gram-negative rods,
motile, non-glucose fermenters, and catalase and oxidase positives. Yeast strains were also found in ‘A’ cultures. Other researchers have found that predominate in bacteria Gram-negative recoverable hydrocarbon communities of oxidizing organisms (Westlake et al., 1974; Alvarez & Pucci, 1993). Based on these degradation studies, it could be concluded that, after controlling for physical and chemical factors under laboratory conditions, the hydrocarbon adaptation of microorganisms led to an increase in alkane biodegradation. This agrees with observations made by other authors, who found that hydrocarbon presence in the selective environment often produces a enrichment of microorganisms with the potential
78
0
N. L. Olivera et
al.
96
144
48
192
240
Hours Fig.
2.
Growth curves of the cultures with microorganisms from: (W) polluted and (0) pristine sediments. Each point represents the average absorbance (wavelength 450nm) for duplicates, error bars indicate fl SD.
(4 20
@I 20
12 0
50
recovered
n
mgn
biodegraded
fq ..~~~~ evaporated
Fig. 3. Evaporated, biodegraded, and recovered concentrations of each alkane (dodecane, tetradecane, hexadecane, octadecane and eicosane) after 240 h of incubation of the cultures with: (a) community adapted to hydrocarbon pollution, and (b) pristine sediment community.
to attack them (Leahy & Colwell, 1990; Rosemberg, 1991). These preliminary studies also suggest the possibility of using community ‘A’ strains to degrade hydrocarbon regional wastes. Current research is being conducted to improve
biodegradation of ship bilge residues by means of bioaugmentation with combinations of these strains. In our case, these kind of residues are mainly composed by aliphatic hydrocarbons including continuous homologous series of y1alkanes.
Alkane biodegradation
ACKNOWLEDGEMENTS
The authors gratefully acknowledge Dr F. Siiieriz for critical revision of this paper and Dr P. Yorio for the correction of the English manuscript. This work was supported by the Patagonian Coastal Plan Zone Management (GEF/PNUD), implemented by Fundacion Patagonia Natural and Wildlife Conservation Society, and grants from Universidad National de la Patagonia San Juan Bosco and Centro National Patagonico (CONICET).
REFERENCES Alvarez, H. M. & Pucci, 0. H. (1993) Biodegradation de hidrocarburos en condiciones de baja temperatura. Petrotecnia, 34(2), 3 I-34.
Atlas, R. M. (1981) Microbial degradation of petroleum hydrocarbons: an environmental perspective. Microbiological Reviews 45, 180-209. Cerniglia, C. E. (1992) Biodegradation of polycyclic aromatic hydrocarbons. Biodegradation 3, 351-368.
79
Esteves, J. L. & Commendatore, M. G. (1993) Total aromatic hydrocarbons in water and sediments in a coastal zone of Patagonia, Argentina. Marine Pollution Bulletin 26, 341342.
Esteves, J. L., Commendatore, M. G., Solis, M., Gil, M. 8z Olivera, N. (1993) Investigation en Costas de1 Atlantic0 Sur. Petrotecnia, 3, pp. 26-32. Karl, D. M. (1992) The grounding of the Bahia Paraiso: Microbial ecology of the 1989 Antarctic oil spill. Microbial Ecology 24, 77-89.
Leahy, J. G. & Colwell, R. R. (1990) Microbial degradation of hydrocarbons in the environment. Microbiological Reviews 54,305-31.5.
Piola, A. R. & Garcia, 0. A. (1993) Atlas Oceanogrhjko de la Cuenca Argentina Occidental y de la Plataforma Continental Lindera. Servicio de Hidrografia Naval, Publication H-670.
Rosemberg, E. (199 1) Hydrocarbon-oxidizing bacteria. In The Prokaryotes, ed. A. Ballows, pp. 441459. Springer--Verlag, Berlin. Swannell, R. P. J., Lee, K. & McDonagh, M. (1996) Field evaluations of marine oil spill bioremediation. Microbiological Reviews 60, 342-365.
Westlake, D. W. S., Jobson, A., Phillippe, R. & Cooke, F. D. (1974) Biodegradability and crude oil composition. Canadian Journal of Microbiology 20, 915-928.
Wilson, C. S. & Jones, K. C. (1993) Bioremediation of soil contaminated with polynuclear aromatic hydrocarbons (PAH,): a review. Environmental Pollution 81, 229-249.