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Biodegradation of crude oil in experimentally-polluted clayey and sandy mangrove soils
Oil & Chemical Pollution 6 (1990) 163-176
Biodegradation of Crude Oil in Experimentally-Polluted Clayey and Sandy Mangrove Soils
P. S c h e r r e r Institut de la Carte Internationale de la V6g6tation, Universit6 Paul Sabatier, 39 all6es J. Guesde, 31062 Toulouse C6dex, France
&
G. Mille* Centre de Spectroscopie Mol6culaire, Facult6 des Sciences et Techniques de St J6r6me, Avenue Escadrille Normandie-Niemen, 13397 Marseille C6dex 13, France (Received 4 January 1989; accepted 24 July 1989)
ABSTRACT Comparative study of oil biodegradation in clayey and sandy mangrove soils shows that, during their emergence period, microorganism activity is related to the soil water content. Drying beyond a certain point prevents good nutrient circulation which may lead to a lack of nutrients at the soil-oil interface. An oleophilic fertiliser supplies nutrients which stimulate the biodegradation process. When the soil is swamped, oxygen is the main limiting factor for biodegradation. Thus, water level variation and soil porosity determine conditions which control the biodegradation activity of aerobic microorganisms.
INTRODUCTION M a n g r o v e is a coastal ecosystem regularly polluted b y accidental oil spills (Getter et al., 1981). T h e forest cover o f these low energy tropical *To whom correspondence should be addressed. 163 Oil & Chemical Pollution 0269-8579/90/$03.50 © 1990 Elsevier Science Publishers Ltd, England. Printed in Ireland.
P. Scherrer, G. Mille
164
areas is particularly vulnerable to oil pollution, since oil is very likely to accumulate and stagnate in their substrata (Scherrer, 1988). Therefore, the long-range future of a polluted mangrove directly depends on the transformation rate of the oil trapped in the soil. This oil evolution process has often been studied in temperate coastal ecosystems (Oudot, 1984; Mulyono, 1985) but there are very few data on biodegradation in mangrove, where experimental conditions are particularly difficult. In this paper we report a study on the role of environmental factors on oil biodegradation in two typical soils from the West Indian mangroves (Imbert, 1985).
MATERIALS AND METHODS Two distinct stations, one clayey the other one sandy, were selected for in situ experiments in the mangrove of Petit Bourg, Guadeloupe, France. The two experimental stations differ from each other by their distance from the river and by their topographic level. Each station may be characterised by specific emergence-submergence cycles (Fig. 1) and by its soil granulometry and organic matter content. These soils are sulphaquants according to the USDA classification. In each station two plots of 2 m 2 were contaminated with oil. l
40
l ...... sandy station clayey station
20 ,3u
0
.-
~, - 2 0
,
',l//
,
-40 --60
/
¢
-80 0
t 60
T 120
I 180
I 240
I 300
I 360
I 420
I 480
Time (doya) 12.3
1986
1987
Fig. 1. Water level variations with time at the two stations.
29.7
Biodegradation of crude oil in experimentally-polluted soils
165
Clayey station The soil was essentially composed of about 70% mineral matter, predominantly clay, associated with about 30% organic matter (peat). Rhizophora mangle was the prevailing vegetation. During the rainy season (May to November), this station is almost permanently covered with a few centimetres of water and the soil is in a reduced state. During the dry season, the water level falls by as much as 60 cm and the emergence period lasts 2-3 months (Fig. 1). Above the water level, redox potentials were generally positive: Two plots were polluted with 5 litres of Light Arabian Crude Oil (LAC) per m 2. In one case, the oil was premixed with 10% bioactivator (oleophilic fertiliser) in order to assess the possible effect of this product on the biodegradation process. For each plot, samples were collected with a graduated mud shovel after periods of 3 days, 3 months and 11 months. Samples were stored at -20°C. The 0-5 cm, 5-10 cm and 10-20 cm levels were analysed separately.
Sandy station This station stood at a slightly higher level than the previous one and was consequently less often covered with water. During the rainy season the water level is often at ground level or even a few centimetres below (Fig. 1). During the dry season, the water level can fall to 70 cm depth. Redox potentials were thus often positive in the top decimetres. The soil was composed of about 80% fine sand (< 150/Jm) and less than 10% decomposed organic matter. Two plots were contaminated as for the clayey station plots. The uppermost 30 cm of the soil was sampled after 3 and 11 months. The prevailing species of vegetation was Avicennia germinans.
Sample analysis The organic matter extraction was performed by alkaline digestion with methanolic KOH and toluene. Solvents were separated from the soil by centrifugation. Unsaponifiable lipids and hydrocarbons were extracted from the organic phase with hexane. The hexane fraction was evaporated and then dry weighed. The extracted organic matter concentrations were expressed in g/kg of dry soil. Separation of the alkanes was achieved by elution of the organic matter with hexane in a glass column filled with activated silica gel. The glassware was thoroughly washed and rinsed with alcohol and the solvents used. The
166
P Seherrer, G. Mille
solvents were of chromatographic quality and their purity was checked regularly. The saturated compounds were analysed by capillary gas chromatography using a Carlo Erba 4160 gas chromatograph equipped with a flame ionisation detector (FID) and connected to an HP 1000 computer. The glass column (50 m X 0.25 mm i.d.) was coated with~OV1 stationary phase. Oven temperature was monitored from 80 to 300°C at the rate of 1.8°C/min. The injector and detector temperatures were 235°C and 320°C, respectively. Hydrogen was the carrier gas. The quality of the computer peak integration was checked on the screen, when necessary, and the peak areas were again determined using modified integration parameters. Further details have been published elsewhere (Scherrer, 1988).
RESULTS AND DISCUSSION In March 1986 we contaminated a clayey station and a sandy station in the West Indies as described in the Materials and Methods section.
Clayey station Figure 2 shows oil distribution in the soil of the two related plots. Biogenic organic matter corresponded to a concentration of 10 g/kg dry weight. The top 30 cm were contaminated with decreasing concentrations from top to bottom. TheLAC was similarly distributed in the 2 plots and no significant decontamination Of the oil was observed during the first year.
LAC plot During the dry season, the n-alkanes were partially biodegraded, as shown by the decrease of the n C~7/pristane and n C~Jphytane ratios with respect to the referelace values (Table 1). Microorganism action was most visible at a depth of :10-20 cm where the oil concentrations were the lowest. Nevertheless, biodegradation was likely to be quantitatively more important in the uppermost levels which were better oxygenated. Indeed, in these soil samples, the decrease of the characteristic biodegradation ratios was obvious in spite of oil accumulation. Between June 1986 and February 1987 biodegradation proceeded fu~rther in the uppermost,20 cm (Fig. 3 and Table 1), although the station was almost always flooded (Fig. 1). This result was unexpected, as most
Biodegradation of crude oil in experimentally-polluted soils
t
167
Concentration
120(g/G~Vkg)
Tirol (months) Depth
(cml
Concentrltlofl 201a/dr y kg)
tl 40
26 11
Fig. 2. Evolution of the extracted organic matter concentration with depth and time at the clayey station. A~ plot polluted with 5 litres ofoil per m2; B, plot polluted with 5 litres of oil premixed with 10% bioactivator per m 2.
168
P. Scherrer, G. Mille
~7
18 I i
L AC
LIL
O-Sere
1718
5-10cm
~~~11° /-~l:Ph
,
i
0"00
,
I
,
,
{
,
40"00 20"00
I
,
,
50~30 60"00
I
,
I
120.00 100.00
,o2ocm
160~30 140.00
Minutes
Fig. 3. Chromatograms of the oil saturated fraction: Light Arabian Crude Oil (LAC) from the clayey station after 11 months, at different depths.
169
Biodegradation of crude oiI in experimentally-polluted soils
17
LAC * Bioactivator
18
0-Scm
I
I
I
I
I
5-10cm
I
I
10-20cm
I
I
1718
I
~
I
I
I
J
i
t ,
0.00
40.00 20.00
60"00
Minutes
Fig. 3--contd.
[
120"00
8000 100"00
L
L
160<)0 140"00
P. Scherrer, G. Mille
170
TABLE 1 nCl7/Pristane and nCls/Phytane Ratios Calculated from the Chromatograms of the Saturated Fractions: Light Arabian Crude Oil (LAC) from the Clayey and Sandy Soils after 3 and 11 Months, at Different Depths
Date
Clayey station
Sandy station
Depth (cm)
LAC
LAC + bioaetivator
nClz/Pr
nCjs/Ph
nClz/Pr
nCls/Ph
18 June 1986 (3 months)
0-5 5-10 10-20
3.1 3.5 2.5
1.7 2.0 1.5
2.8 2.0 1-7
1.6 1.1 1.0
17 Feb. 1987 (11 months)
0-5 5-10 10-20
1.7 1.2 1.0
1.1 0.7 0.6
2.6 1.0 1.2
1.4 0.5 0.7
18 June 1986 (3 months)
0-5 5-10 10-20
4.1 4-4 4.2
2.3 2.7 2-6
2.1 2.8 3.4
1.3 1.8 2.1
17 Feb. 1987 (11 months)
0-5 5-10 10-20
0.9 1.1 1.2
0.5 0.7 0.7
1.4 1-0 0.5
0.7 0.6 0.3
Reference LAC: nClT/Pr = 5-1, nCi8/Ph = 2.9.
authors consider that rapid biodegradation processes are aerobic (Higgins & Gilbert, 1978; Ratledge, 1978; Cerniglia, 1984). Therefore, the station might have been more often in an emergent condition than can be deduced from Fig. 1. Water level fluctuation might have allowed enough oxygen diffusion in the first 20 cm of the soil to sustain an aerobic microbial activity. LAC + bioactivator plot During the first three months hydrocarbon biodegradation seemed more advanced, between 5 and 20 cm depth (Table 1), than in the previous plot. Bioactivator nutrients had probably stimulated an aerobic microbial activity. After 11 months, LAC transformation was more or less the same on the two plots of this station (Fig. 3 and Table 1). This 'catching up' could be explained by a decrease in microbial activity, linked to the progressive removal of the most biodegradable hydrocarbons. Indeed, biodegradation of other oil compounds, such as branched alkanes and aromatics, is generally lower than that of linear chains. It is also possible that
Biodegradation of crude oil in experimentally-polluted soils
171
microorganisms found it somewhat more difficult to carry on their activity in highly polluted areas, where oxygen diffusion was limited.
Sandy station Figure 4 shows crude oil distribution in the soil after 3 and 11 months. The concentrations were similar for both plots. The uppermost 30 cm
A -2.5 v
-7.5
e-., -15
............... ...........0""'"'"""'"
~/t
-25
5
10
15
20
25
30
Concentratlon(g/drykgJ
B 0 -2.5 u
-7.5 Q
-15
-25
5
I 10
( 15
..........LAC.bio. ,LAC 20
25
30
Concentration (g/drykg)
Fig. 4. Distribution of the extracted organic matterin the soil of the sandy station. A, at 3 months (18 June 1986); B, at 11 months (17 February 1987). LAC, plot polluted with 5 litres of oil per m2; LAC +bio., plot polluted with 5 litres of oil and 10% bioactivator per m2; biog.o.m., biogenic organic matter.
172
P. Scherrer, G. Mille
were polluted and the highest concentrations were at the soil surface. After 11 months, a significant soil decontamination was observed. This could be partly attributed to the disappearance of the most biodegradable compounds. LAC plot During the first 3 months when the water level was low, LAC biodegradation was very limited in the uppermost 20 cm (Table 1). As the soil was aerated, redox potential would not have restricted aerobic microbial activity. The lack of biode~gradation probably results from nutrient deficiency at the oil-soil interface. It may be explained by soil dryness, which prevents both a high level of organic matter mineralisation, and movement of nutrients. This is supported by the spectacular decrease in characteristic biodegradation ratios during the 8 month rainy season (Fig. 5 and Table 1). These results support the abovementioned hypothesis of a decrease in microbiological activity, due to dryness. In a swamped soil, microorganisms multiply easily in contact with the oil substratum, and oxygen is, possibly, their main limiting factor. Nutrients are probably in adequate supply since the soil is well mineralised. LAC + bioactivator plot The addition of bioactivator to the oil, undoubtedly promoted normal alkane biodegradation during the dry season (Table 1). Microorganism activity decreased from top to bottom. Therefore, in the presence of non-limiting amounts of nutrients, oxygen is the main factor controlling biodegradation. The stimulating effect of the bioactivator could be explained by nutrient release, directly within the oil. The oleophilic properties of this product make such a close association possible. These results support the view that nutrients become far less available during the dry season than during the rainy one. The oil biodegradation continued from June 1986 to February 1987. However, after one year, the oil content was rather similar to the one of the plot not supplied with bioactivator. This 'catching up', already observed at the clayey station, can be explained by the same arguments as before. The results obtained at the clayey and sandy stations reveal the influence of environmental factors on the oil biodegradation process. During the emergence period, the activity of aerobic microorganisms is much reduced, as the soil water content is low. Nutrients scarcely move about and become rapidly deficient at the water-soil interface. Bacteria multiply slowly on the substratum. During this period, oxygen is not the
Biodegradation of crude oil in experimentally-polluted soils
173
main limiting factor for biodegradation. The addition of an oleophilic fertiliser seems likely to make up for the non-availability of nutrients. These results can be compared to those obtained in a predominantly peaty mangrove soil (Scherrer & Mille, 1989). In such a substratum we have attributed the lack of biodegradation of the normal alkanes to a shortage of nutrients, related to the slowness of mineralisation of the organic matter. We also reveal the stimulating action of the bioactivator. In a swamped soil (rainy season), biodegradation does not seem to depend on nutrient availability, irrespective of the station, Oil biodegradation is more efficient because water level variations provide better sediment oxygenation. Thus, a sandy sediment, highly porous and frequently emerged, offers suitable conditions for rapid biodegradation of the normal alkanes. In such favourable conditions, the use of an oleophilic fertiliser is not necessary.
CONCLUSIONS The comparative studies that we carried out in the 'Petit-Bourg' mangrove, have shown the role of three environmental factors in hydrocarbon biodegradation: soil oxygenation, amount of nutrients related to soil mineralisation and availability (for microbes) of the nutrients at the soil-oil interface. For a given mangrove, very different situations may be encountered. The period of time for which a mangrove sediment remains polluted is directly linked to its soil and hydrodynamic characteristics. In some cases, an external nutrient supply (oleophilic fertiliser) might favour biodegradation of the hydrocarbons trapped in the soil, but in others this will serve no useful purpose. The results presented here are, to our knowledge, the first dealing with oil biodegradation in mangroves. However, this study and subsequent ones, will never give results applicable to all mangroves, in general, since their ecosystems differ greatly from one place to the other. Therefore, other studies are necessary to confirm these results and to understand oil evolution, especially in more dynamic systems.
ACKNOWLEDGMENTS The authors are very grateful to ELF Aquitaine Company for their logistic and analytical help. They also wish to thank Professor J. Portecop and Dr D. Imbert for their field cooperation.
P. Seherrer, G. Mille
174
LAC
,'
[
1~. Ph ,
101 1
I
f
I
.
~
i
r
'
,
'
_J
,
I
,
I
,
I
'r~118 '
'
l
5-10cm
~
1
I
I
I , [
I
i
l
r
,J
1718
10-20cm
i, __~
l
0.00
J'l I
I
40-O0 20.00
f
I
6000
]
120-00
80.00 100-00
J
160"00
140.00
Minutes Fig. 5. Chromatograms of the oil saturated fraction: Light Arabian Crude Oil (LAC) from the sandy station after 11 months, at different depths. (Numbers over the peaks represent the number of carbon atoms in the n-alkane chain. Pr, pristane; Ph, phytane).
Biodegradation of crude oil in experimentally-polluted soils
175
LAC* BioactivatoP r 18
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5-10cm
14 I
•
I
t
t
Ph
10-20cm
2, I
I
I,
20"00
I
I
80.00
40-00
0"00
6000 Minutes
Fig. 5--contd
I
I
12000 100<)0
I
160.00 140-00
176
P. Scherrer, G. Mille REFERENCES
Cerniglia, C. E. (1984). Microbial transformation of aromatic hydrocarbons. In Petroleum Microbiology, ed. R. M. Atlas. Macmillan Publishing Company, New York, pp. 99-128. Getter, C. D., Scott, G. I. & Michel, J. (1981 ). The effects of oil spills on mangrove forests: a comparison of fire oil spill sites in the Gulf of Mexico and the Caribbean Sea. In Proceedings of the 1981 Oil Spill Conference. American Petroleum Institute, Washington, DC, pp. 535-40. Higgins, I. J. & Gilbert, P. D. (1978). The biodegradation of hydrocarbons. In The Oillndustry and Microbial Ecosystems, ed. K. W. A. Chater & H. J. Somerville. Heyden and Son Ltd, London, pp. 80-117. Imbert, D. (1985). Organisation spatio-temporelle des communautrs vrgrtales dans la mangrove du Grand Cul-de-Sac Marin (Guadeloupe). Th~se, Universit6 des Sciences et Techniques du Languedoc, France, 132 pp. + annexes. Mulyono, M. (1985). Evolution in situ et in vitro des hydrocarbures dans des srdiments marins massivement contaminrs. Thrse, Universit6 de Droit, d'Economie et des Sciences d'Aix-Marseille, France, 212 pp. Oudot, J. (1984). La biodrgradation microbienne des hydrocarbures. Etudes du potentiel de biodrgradation et de son expression dans le milieu. Thrse de Docteur en Sciences, Universit6 Paris VII, France, 121 pp. Ratledge, C. (1978). Degradation of aliphatic hydrocarbons. In Developments in Biodegradation of Hydrocarbons--I, ed. J. R. Watkinson. Applied Science Publishers, London, pp. 1-46. Scherrer, P. (1988). La rrgrnrration de la mangrove aprrs un drversement accidentel d'hydrocarbures: phytotoxicit6 et 6volution physico-chimique du prtrole brut pirg6 dans le substrat. Thrse de Docteur en Sciences, Universit6 Paul Sabatier, Toulouse, France, 348 pp. (2 volumes). Scherrer, P. & Mille, G. (1989). Mar. Pollut. Bull., 20, 430-2.
Biodegradation of Crude Oil in Experimentally-Polluted Clayey and Sandy Mangrove Soils
P. S c h e r r e r Institut de la Carte Internationale de la V6g6tation, Universit6 Paul Sabatier, 39 all6es J. Guesde, 31062 Toulouse C6dex, France
&
G. Mille* Centre de Spectroscopie Mol6culaire, Facult6 des Sciences et Techniques de St J6r6me, Avenue Escadrille Normandie-Niemen, 13397 Marseille C6dex 13, France (Received 4 January 1989; accepted 24 July 1989)
ABSTRACT Comparative study of oil biodegradation in clayey and sandy mangrove soils shows that, during their emergence period, microorganism activity is related to the soil water content. Drying beyond a certain point prevents good nutrient circulation which may lead to a lack of nutrients at the soil-oil interface. An oleophilic fertiliser supplies nutrients which stimulate the biodegradation process. When the soil is swamped, oxygen is the main limiting factor for biodegradation. Thus, water level variation and soil porosity determine conditions which control the biodegradation activity of aerobic microorganisms.
INTRODUCTION M a n g r o v e is a coastal ecosystem regularly polluted b y accidental oil spills (Getter et al., 1981). T h e forest cover o f these low energy tropical *To whom correspondence should be addressed. 163 Oil & Chemical Pollution 0269-8579/90/$03.50 © 1990 Elsevier Science Publishers Ltd, England. Printed in Ireland.
P. Scherrer, G. Mille
164
areas is particularly vulnerable to oil pollution, since oil is very likely to accumulate and stagnate in their substrata (Scherrer, 1988). Therefore, the long-range future of a polluted mangrove directly depends on the transformation rate of the oil trapped in the soil. This oil evolution process has often been studied in temperate coastal ecosystems (Oudot, 1984; Mulyono, 1985) but there are very few data on biodegradation in mangrove, where experimental conditions are particularly difficult. In this paper we report a study on the role of environmental factors on oil biodegradation in two typical soils from the West Indian mangroves (Imbert, 1985).
MATERIALS AND METHODS Two distinct stations, one clayey the other one sandy, were selected for in situ experiments in the mangrove of Petit Bourg, Guadeloupe, France. The two experimental stations differ from each other by their distance from the river and by their topographic level. Each station may be characterised by specific emergence-submergence cycles (Fig. 1) and by its soil granulometry and organic matter content. These soils are sulphaquants according to the USDA classification. In each station two plots of 2 m 2 were contaminated with oil. l
40
l ...... sandy station clayey station
20 ,3u
0
.-
~, - 2 0
,
',l//
,
-40 --60
/
¢
-80 0
t 60
T 120
I 180
I 240
I 300
I 360
I 420
I 480
Time (doya) 12.3
1986
1987
Fig. 1. Water level variations with time at the two stations.
29.7
Biodegradation of crude oil in experimentally-polluted soils
165
Clayey station The soil was essentially composed of about 70% mineral matter, predominantly clay, associated with about 30% organic matter (peat). Rhizophora mangle was the prevailing vegetation. During the rainy season (May to November), this station is almost permanently covered with a few centimetres of water and the soil is in a reduced state. During the dry season, the water level falls by as much as 60 cm and the emergence period lasts 2-3 months (Fig. 1). Above the water level, redox potentials were generally positive: Two plots were polluted with 5 litres of Light Arabian Crude Oil (LAC) per m 2. In one case, the oil was premixed with 10% bioactivator (oleophilic fertiliser) in order to assess the possible effect of this product on the biodegradation process. For each plot, samples were collected with a graduated mud shovel after periods of 3 days, 3 months and 11 months. Samples were stored at -20°C. The 0-5 cm, 5-10 cm and 10-20 cm levels were analysed separately.
Sandy station This station stood at a slightly higher level than the previous one and was consequently less often covered with water. During the rainy season the water level is often at ground level or even a few centimetres below (Fig. 1). During the dry season, the water level can fall to 70 cm depth. Redox potentials were thus often positive in the top decimetres. The soil was composed of about 80% fine sand (< 150/Jm) and less than 10% decomposed organic matter. Two plots were contaminated as for the clayey station plots. The uppermost 30 cm of the soil was sampled after 3 and 11 months. The prevailing species of vegetation was Avicennia germinans.
Sample analysis The organic matter extraction was performed by alkaline digestion with methanolic KOH and toluene. Solvents were separated from the soil by centrifugation. Unsaponifiable lipids and hydrocarbons were extracted from the organic phase with hexane. The hexane fraction was evaporated and then dry weighed. The extracted organic matter concentrations were expressed in g/kg of dry soil. Separation of the alkanes was achieved by elution of the organic matter with hexane in a glass column filled with activated silica gel. The glassware was thoroughly washed and rinsed with alcohol and the solvents used. The
166
P Seherrer, G. Mille
solvents were of chromatographic quality and their purity was checked regularly. The saturated compounds were analysed by capillary gas chromatography using a Carlo Erba 4160 gas chromatograph equipped with a flame ionisation detector (FID) and connected to an HP 1000 computer. The glass column (50 m X 0.25 mm i.d.) was coated with~OV1 stationary phase. Oven temperature was monitored from 80 to 300°C at the rate of 1.8°C/min. The injector and detector temperatures were 235°C and 320°C, respectively. Hydrogen was the carrier gas. The quality of the computer peak integration was checked on the screen, when necessary, and the peak areas were again determined using modified integration parameters. Further details have been published elsewhere (Scherrer, 1988).
RESULTS AND DISCUSSION In March 1986 we contaminated a clayey station and a sandy station in the West Indies as described in the Materials and Methods section.
Clayey station Figure 2 shows oil distribution in the soil of the two related plots. Biogenic organic matter corresponded to a concentration of 10 g/kg dry weight. The top 30 cm were contaminated with decreasing concentrations from top to bottom. TheLAC was similarly distributed in the 2 plots and no significant decontamination Of the oil was observed during the first year.
LAC plot During the dry season, the n-alkanes were partially biodegraded, as shown by the decrease of the n C~7/pristane and n C~Jphytane ratios with respect to the referelace values (Table 1). Microorganism action was most visible at a depth of :10-20 cm where the oil concentrations were the lowest. Nevertheless, biodegradation was likely to be quantitatively more important in the uppermost levels which were better oxygenated. Indeed, in these soil samples, the decrease of the characteristic biodegradation ratios was obvious in spite of oil accumulation. Between June 1986 and February 1987 biodegradation proceeded fu~rther in the uppermost,20 cm (Fig. 3 and Table 1), although the station was almost always flooded (Fig. 1). This result was unexpected, as most
Biodegradation of crude oil in experimentally-polluted soils
t
167
Concentration
120(g/G~Vkg)
Tirol (months) Depth
(cml
Concentrltlofl 201a/dr y kg)
tl 40
26 11
Fig. 2. Evolution of the extracted organic matter concentration with depth and time at the clayey station. A~ plot polluted with 5 litres ofoil per m2; B, plot polluted with 5 litres of oil premixed with 10% bioactivator per m 2.
168
P. Scherrer, G. Mille
~7
18 I i
L AC
LIL
O-Sere
1718
5-10cm
~~~11° /-~l:Ph
,
i
0"00
,
I
,
,
{
,
40"00 20"00
I
,
,
50~30 60"00
I
,
I
120.00 100.00
,o2ocm
160~30 140.00
Minutes
Fig. 3. Chromatograms of the oil saturated fraction: Light Arabian Crude Oil (LAC) from the clayey station after 11 months, at different depths.
169
Biodegradation of crude oiI in experimentally-polluted soils
17
LAC * Bioactivator
18
0-Scm
I
I
I
I
I
5-10cm
I
I
10-20cm
I
I
1718
I
~
I
I
I
J
i
t ,
0.00
40.00 20.00
60"00
Minutes
Fig. 3--contd.
[
120"00
8000 100"00
L
L
160<)0 140"00
P. Scherrer, G. Mille
170
TABLE 1 nCl7/Pristane and nCls/Phytane Ratios Calculated from the Chromatograms of the Saturated Fractions: Light Arabian Crude Oil (LAC) from the Clayey and Sandy Soils after 3 and 11 Months, at Different Depths
Date
Clayey station
Sandy station
Depth (cm)
LAC
LAC + bioaetivator
nClz/Pr
nCjs/Ph
nClz/Pr
nCls/Ph
18 June 1986 (3 months)
0-5 5-10 10-20
3.1 3.5 2.5
1.7 2.0 1.5
2.8 2.0 1-7
1.6 1.1 1.0
17 Feb. 1987 (11 months)
0-5 5-10 10-20
1.7 1.2 1.0
1.1 0.7 0.6
2.6 1.0 1.2
1.4 0.5 0.7
18 June 1986 (3 months)
0-5 5-10 10-20
4.1 4-4 4.2
2.3 2.7 2-6
2.1 2.8 3.4
1.3 1.8 2.1
17 Feb. 1987 (11 months)
0-5 5-10 10-20
0.9 1.1 1.2
0.5 0.7 0.7
1.4 1-0 0.5
0.7 0.6 0.3
Reference LAC: nClT/Pr = 5-1, nCi8/Ph = 2.9.
authors consider that rapid biodegradation processes are aerobic (Higgins & Gilbert, 1978; Ratledge, 1978; Cerniglia, 1984). Therefore, the station might have been more often in an emergent condition than can be deduced from Fig. 1. Water level fluctuation might have allowed enough oxygen diffusion in the first 20 cm of the soil to sustain an aerobic microbial activity. LAC + bioactivator plot During the first three months hydrocarbon biodegradation seemed more advanced, between 5 and 20 cm depth (Table 1), than in the previous plot. Bioactivator nutrients had probably stimulated an aerobic microbial activity. After 11 months, LAC transformation was more or less the same on the two plots of this station (Fig. 3 and Table 1). This 'catching up' could be explained by a decrease in microbial activity, linked to the progressive removal of the most biodegradable hydrocarbons. Indeed, biodegradation of other oil compounds, such as branched alkanes and aromatics, is generally lower than that of linear chains. It is also possible that
Biodegradation of crude oil in experimentally-polluted soils
171
microorganisms found it somewhat more difficult to carry on their activity in highly polluted areas, where oxygen diffusion was limited.
Sandy station Figure 4 shows crude oil distribution in the soil after 3 and 11 months. The concentrations were similar for both plots. The uppermost 30 cm
A -2.5 v
-7.5
e-., -15
............... ...........0""'"'"""'"
~/t
-25
5
10
15
20
25
30
Concentratlon(g/drykgJ
B 0 -2.5 u
-7.5 Q
-15
-25
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172
P. Scherrer, G. Mille
were polluted and the highest concentrations were at the soil surface. After 11 months, a significant soil decontamination was observed. This could be partly attributed to the disappearance of the most biodegradable compounds. LAC plot During the first 3 months when the water level was low, LAC biodegradation was very limited in the uppermost 20 cm (Table 1). As the soil was aerated, redox potential would not have restricted aerobic microbial activity. The lack of biode~gradation probably results from nutrient deficiency at the oil-soil interface. It may be explained by soil dryness, which prevents both a high level of organic matter mineralisation, and movement of nutrients. This is supported by the spectacular decrease in characteristic biodegradation ratios during the 8 month rainy season (Fig. 5 and Table 1). These results support the abovementioned hypothesis of a decrease in microbiological activity, due to dryness. In a swamped soil, microorganisms multiply easily in contact with the oil substratum, and oxygen is, possibly, their main limiting factor. Nutrients are probably in adequate supply since the soil is well mineralised. LAC + bioactivator plot The addition of bioactivator to the oil, undoubtedly promoted normal alkane biodegradation during the dry season (Table 1). Microorganism activity decreased from top to bottom. Therefore, in the presence of non-limiting amounts of nutrients, oxygen is the main factor controlling biodegradation. The stimulating effect of the bioactivator could be explained by nutrient release, directly within the oil. The oleophilic properties of this product make such a close association possible. These results support the view that nutrients become far less available during the dry season than during the rainy one. The oil biodegradation continued from June 1986 to February 1987. However, after one year, the oil content was rather similar to the one of the plot not supplied with bioactivator. This 'catching up', already observed at the clayey station, can be explained by the same arguments as before. The results obtained at the clayey and sandy stations reveal the influence of environmental factors on the oil biodegradation process. During the emergence period, the activity of aerobic microorganisms is much reduced, as the soil water content is low. Nutrients scarcely move about and become rapidly deficient at the water-soil interface. Bacteria multiply slowly on the substratum. During this period, oxygen is not the
Biodegradation of crude oil in experimentally-polluted soils
173
main limiting factor for biodegradation. The addition of an oleophilic fertiliser seems likely to make up for the non-availability of nutrients. These results can be compared to those obtained in a predominantly peaty mangrove soil (Scherrer & Mille, 1989). In such a substratum we have attributed the lack of biodegradation of the normal alkanes to a shortage of nutrients, related to the slowness of mineralisation of the organic matter. We also reveal the stimulating action of the bioactivator. In a swamped soil (rainy season), biodegradation does not seem to depend on nutrient availability, irrespective of the station, Oil biodegradation is more efficient because water level variations provide better sediment oxygenation. Thus, a sandy sediment, highly porous and frequently emerged, offers suitable conditions for rapid biodegradation of the normal alkanes. In such favourable conditions, the use of an oleophilic fertiliser is not necessary.
CONCLUSIONS The comparative studies that we carried out in the 'Petit-Bourg' mangrove, have shown the role of three environmental factors in hydrocarbon biodegradation: soil oxygenation, amount of nutrients related to soil mineralisation and availability (for microbes) of the nutrients at the soil-oil interface. For a given mangrove, very different situations may be encountered. The period of time for which a mangrove sediment remains polluted is directly linked to its soil and hydrodynamic characteristics. In some cases, an external nutrient supply (oleophilic fertiliser) might favour biodegradation of the hydrocarbons trapped in the soil, but in others this will serve no useful purpose. The results presented here are, to our knowledge, the first dealing with oil biodegradation in mangroves. However, this study and subsequent ones, will never give results applicable to all mangroves, in general, since their ecosystems differ greatly from one place to the other. Therefore, other studies are necessary to confirm these results and to understand oil evolution, especially in more dynamic systems.
ACKNOWLEDGMENTS The authors are very grateful to ELF Aquitaine Company for their logistic and analytical help. They also wish to thank Professor J. Portecop and Dr D. Imbert for their field cooperation.
P. Seherrer, G. Mille
174
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Biodegradation of crude oil in experimentally-polluted soils
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P. Scherrer, G. Mille REFERENCES
Cerniglia, C. E. (1984). Microbial transformation of aromatic hydrocarbons. In Petroleum Microbiology, ed. R. M. Atlas. Macmillan Publishing Company, New York, pp. 99-128. Getter, C. D., Scott, G. I. & Michel, J. (1981 ). The effects of oil spills on mangrove forests: a comparison of fire oil spill sites in the Gulf of Mexico and the Caribbean Sea. In Proceedings of the 1981 Oil Spill Conference. American Petroleum Institute, Washington, DC, pp. 535-40. Higgins, I. J. & Gilbert, P. D. (1978). The biodegradation of hydrocarbons. In The Oillndustry and Microbial Ecosystems, ed. K. W. A. Chater & H. J. Somerville. Heyden and Son Ltd, London, pp. 80-117. Imbert, D. (1985). Organisation spatio-temporelle des communautrs vrgrtales dans la mangrove du Grand Cul-de-Sac Marin (Guadeloupe). Th~se, Universit6 des Sciences et Techniques du Languedoc, France, 132 pp. + annexes. Mulyono, M. (1985). Evolution in situ et in vitro des hydrocarbures dans des srdiments marins massivement contaminrs. Thrse, Universit6 de Droit, d'Economie et des Sciences d'Aix-Marseille, France, 212 pp. Oudot, J. (1984). La biodrgradation microbienne des hydrocarbures. Etudes du potentiel de biodrgradation et de son expression dans le milieu. Thrse de Docteur en Sciences, Universit6 Paris VII, France, 121 pp. Ratledge, C. (1978). Degradation of aliphatic hydrocarbons. In Developments in Biodegradation of Hydrocarbons--I, ed. J. R. Watkinson. Applied Science Publishers, London, pp. 1-46. Scherrer, P. (1988). La rrgrnrration de la mangrove aprrs un drversement accidentel d'hydrocarbures: phytotoxicit6 et 6volution physico-chimique du prtrole brut pirg6 dans le substrat. Thrse de Docteur en Sciences, Universit6 Paul Sabatier, Toulouse, France, 348 pp. (2 volumes). Scherrer, P. & Mille, G. (1989). Mar. Pollut. Bull., 20, 430-2.