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Cold Regions Science and Technology 40 (2004) 61–70
Effects of temperature warming during a
bioremediation study of natural and nutrient-amended
hydrocarbon-contaminated sub-Antarctic soils
Daniel Delillea,*, Frederic Coulona, Emilien Pelletierb
aObservatoire Oceanologique de Banyuls, Universite P. et M. Curie UMR-CNRS 7621, Laboratoire Arago 66650 Banyuls sur mer, Franceb Institut des Sciences de la Mer de Rimouski (ISMER), Universite du Quebec a Rimouski, 310 allee des Ursulines, Rimouski, Canada G5L 3A1
Received 30 January 2004; accepted 26 May 2004
Abstract
Although petroleum contamination is recognized as a significant threat to polar environments, documented research on
the environmental consequences of terrestrial spills in cold regions is still scarce. Full-scale in situ remediation of petroleum-
contaminated soils has not yet been used in Antarctica, partly because it has long been assumed that air and soil temperatures
are too low for an effective biodegradation. To test this assumption, the effects of temperature on the hydrocarbon
mineralization rate have been quantified during a field pilot study carried out on artificially contaminated sub-Antarctic soils.
The field study was initiated in December 2000 on two selected soils of the Grande Terre (Kerguelen Archipelago,
69j42VE–49j19VS). The first site supported an abundant vegetal cover, while the second one was a desert soil exempt of
plant material. Two series of five experimental plots (0.75� 0.75 m) were settled firmly into each of the studied soils. Each
plot received 500 ml of diesel fuel or Arabian light crude oil, and half of them were covered with a black plastic sheet. All
the plots were sampled on a regular basis over a 2-year period. Under natural sub-Antarctic conditions, the field tests
revealed that up to 95% of total hydrocarbons were degraded within 1 year, indicating that low temperatures (0–7 jC) canstill allow oil biodegradation by indigenous microorganisms. Soil coverage induced a small but permanent increase of the
temperature in the surface soil of 2 jC (annual mean) and favored the degradation of alkanes over aromatics. The present
observations increase the number of possible scenarios involving controlled temperature design and effects in future in situ
bioremediation strategies in sub-Antarctic soils.
D 2004 Elsevier B.V. All rights reserved.
Keywords: Temperature; Soil petroleum hydrocarbon; Bioremediation; Antarctica
1. Introduction
The Antarctic ecosystem has been considered as
the last remaining pristine zone, almost uncontami-
nated by anthropogenic hydrocarbons (Reinhardt and
0165-232X/$ - see front matter D 2004 Elsevier B.V. All rights reserved.
doi:10.1016/j.coldregions.2004.05.005
* Corresponding author. Fax: +33-04-68-50-49-51.
E-mail address: delille@obs-banyuls.fr (D. Delille).
VanVleet, 1986; Cripps, 1990, 1992; Berkman,
1992). However, the Antarctic can no longer be
considered as a pristine environment because con-
tamination has affected many coastal marine and
terrestrial areas (Karl, 1992; Bej et al., 2000; Delille
and Pelletier, 2002). Of all the different types of
contamination reported up to now in Antarctic sea
and on the continent, petroleum has been identified
Fig. 1. The Kerguelen archipelago with the location of the study
area. The bottom insert of the figure shows the relative position of
the contaminated plots.
D. Delille et al. / Cold Regions Science and Technology 40 (2004) 61–7062
as the most significant problem (Snape et al., 2001).
In recent years, the microbial decontamination (bio-
remediation) of oil-polluted soils was often claimed
to be an efficient, economic and versatile alternative
to physical and chemical treatments (Atlas, 1981;
Joergensen et al., 1995; Møller et al., 1996). Once a
spill has occurred in Antarctica, it is particularly
problematic to recover or remediate it, as many
techniques employed in temperate regions are either
unsuitable or difficult to implement in this extreme
environment. While bioremediation has been pro-
posed as the only viable management option that
can be implemented on a large scale in this area
(Snape et al., 2001), little is known about hydrocar-
bon biodegradation processes and rates in cold
environments. It has been assumed that in Antarctica,
temperatures are too low for effective biodegradation
(Morita, 1992; Nedwell, 1999). However, the metab-
olism of cold-adapted microorganisms is prepared to
function optimally at low temperature (Margesin,
2000). Such organisms have been shown to be useful
tools for bioremediation process in cold environ-
ments like in Alpine, Arctic and Antarctic areas
(Gounot, 1991; Margesin and Schinner, 1997; Delille
et al., 1997). In sub-Antarctic soils, temperature
above 20 jC is reached only during the austral
summer period. More generally air temperatures
ranges from � 2 and 15 jC. Such conditions greatly
influence soil microbial conversion and degradation
rates and demand a high level of microbial cold
adaptation. Several results concerning hydrocarbon-
degrading bacteria are available for Antarctic soils
(MacCormack and Fraile, 1997; Kerry, 1990; Delille,
2000; Aislabie et al., 2001). However, many ques-
tions raised about the feasibility of bioremediation in
cold regions are still unanswered. Some bioremedi-
ation experiments have been conducted on Antarctic
soils (Kerry, 1993; Delille, 2000; Delille et al., 2003;
Ferguson et al., 2003), but to our knowledge, not yet
in sub-Antarctic soils. In order to determine the best
bioremediation approach for hydrocarbon-contami-
nated soils in sub-Antarctic areas, a controlled field
study was initiated in December 2000 in soils of the
Grande Terre in Kerguelen Archipelago. The purpose
of the present study was to determine the effects of a
small temperature increase on the removal of crude
oil and diesel fuel contamination under various sub-
Antarctic conditions.
2. Material and methods
2.1. Study sites
The long-term experiment was conducted in
Kerguelen Archipelago near ‘‘Port aux Franc�ais ’’
research station (49j21VS, 70j13VE, Fig. 1). Two
pristine soils with no known history of hydrocarbon
contamination were selected. The first one (S1)
supported an abundant vegetal cover (Acaena
magellanica), while the second one (S2) was com-
pletely dry and desert. Temperature of the first 2 cm
of the soils was measured daily at 11:00 AM for S1
and 11:30 AM for S2 using a digital thermometer.
Both soils experienced large temperature fluctua-
tions with surface temperature frequently dropping
near 0 jC in winter and reaching over 20 jC during
D. Delille et al. / Cold Regions Science and Technology 40 (2004) 61–70 63
summer sunny afternoons. The total organic carbon
and nitrogen contents were determined by CHN
analysis. Carbon and nitrogen concentration were
158 and 14 g kg� 1 respectively for S1 and 44 and
6 g kg� 1 for S2. The sieve analysis from S1 gave
nearly 2.3% fine ground (< 40 to 63 Am), 22.6%
loam (63–250 Am), 36 % medium ground (250–
800 Am), 22.3% coarse ground (800–2000 Am) and
16.8% gravel and plant residues (2000 to >3150
Am) while the sieve analysis from S2 gave nearly
3.9% fine ground (< 40 to 63 Am), 4.7% loam (63–
250 Am), 2.4 % medium ground (250–800 Am),
15.9% coarse ground (800–2000 Am) and 73.2%
gravel (2000 to >3150 Am). Soil pH was deter-
mined with one part of soil mixed with 2.5 parts of
sterile water. S1 had a pH of 6.4 and S2 showed a
more neutral pH of 7.9.
2.2. Experimental design
Two series of five enclosures were firmly settled
in both soils in December 2000. Wood enclosures
(0.75� 0.75 m) were arranged in two rows perpen-
dicular to the dominant wind direction. Enclosures
were separated by 1 m from each others. After
removal of the superficial plant canopy in S1, both
soils were contaminated by a direct application of
diesel fuel or Arabian light crude oil on the surface
of the enclosures. The contaminant was added
uniformly to each enclosure over a surface of about
0.25 m2 leaving a 25-cm clean strip between the
enclosure wall and the oiled surface as a buffer
zone. In the first row of enclosures, the soil was
directly in contact with the atmosphere. In the
second one, the soil of the enclosures was protected
by a double plastic coating. A black plastic sheet
was placed directly onto the soil and a transparent
plastic cover was placed 10 cm above the soil and
nailed to the wood walls. During a preliminary
study using several types of covers, including spe-
cific agricultural sheets, the double plastic cover
gave the best results in terms of increasing temper-
ature. Contaminated plots received 500 ml of diesel
fuel or Arabian light crude oil and half of them
were treated with 100 ml of the slow release
fertilizer Inipol EAP-22 (Elf Atochem, C/N/P in
proportion 62:7.4:0.7). Periodic samplings allowed
a regular survey of total, heterotrophic and hydro-
carbon-degrading bacteria. Surface (first 2 cm) soil
samples were collected using 2 ml sterile plastic
cores for bacterial counting and pre-washed (hex-
ane/acetone) glass vials for chemical analysis and
toxicity assays. Samples for bacteria were treated
immediately after their arrival at the microbiological
laboratory. Samples for chemistry were frozen at
� 20 jC and sent to ‘‘Institut des Sciences de la
Mer de Rimouski’’ (Canada) for analysis.
2.3. Bacteriological counts
Soil (1 ml) was suspended into 9 ml of sterile water
(3/4 distilled water, 1/4 sea water filtered on 0.2 Am)
and homogenized for 2 min with a vortex. A series of
dilution was then carried out up to 10� 12 and used for
the whole bacteriological counts.
Total bacteria were determined by acridine orange
direct count (AODC) on black nuclepore filters
(0.2 Am) using an Olympus BHA epifluorescence
microscope according to the method of Hobbie et al.
(1977). A minimum of 500 fluorescing cells with a
clear outline and definite cell shape cells were counted
under oil immersion (� 1000) in a minimum of 10
randomly chosen fields.
The number of viable psychrotrophic aerobic het-
erotrophic microorganisms in each soil sample was
determined using the spread plate technique on Nu-
trient Agar 2216 (Oppenheimer and ZoBell, 1952)
with distilled water in place of seawater. Inoculated
plates (six replicates) were incubated for 10 days at
15 jC. The standard deviation calculated for all
results was found V 15% for CFU estimation.
Hydrocarbon-degrading bacteria were counted
using the most probable number (MPN) method
with a basal mineral medium supplemented with
‘‘Arabian Light’’ crude oil (Mills et al., 1978).
Rezasurin was used as a growth indicator. After
inoculation (three tubes per dilution) the tubes were
incubated at 12 jC for 30 days. The standard
deviation calculated for all results was found
V 20% for MPN estimation.
2.4. Hydrocarbons analysis
Dried soil samples (20 mg) were extracted in 6 ml
hexane/dichloromethane (1:1). Aliphatic hydrocar-
bons and PAHs were identified and quantified by
D. Delille et al. / Cold Regions Science and Technology 40 (2004) 61–7064
GC/MS using a ThermoFinnigan Trace GC gas chro-
matograph coupled with a ThermoFinnigan Polaris
QR mass spectrometer operated at 70 eV with a mass
range m/z 80–850. For quality control, a 1.5 ng/
Al diesel standard solution (ASTM C12–C60 quantita-
tive, Supelco) and a 0.5 ng/Al PAHs Mix Standard
solution (Supelco) were analyzed every 15 samples.
The variation of the reproducibility of extraction and
quantification of soil samples were determined by
successive injections (n = 7) of the same sample and
estimated to F 5%.
Fig. 2. Changes in surface temperature of both pristine soils during the con
grey line: uncovered soil, black line: covered soil).
3. Results
During the course of the experiment, mean soil
temperatures ranged from 0 jC in winter to more
than 20 jC in summer in both soils. Temperatures
were always warmer in covered soils than in
corresponding uncovered ones (Fig. 2). The annual
mean temperature enhancement was 2.2 jC in S1
and 2.0 jC in S2.
During the course of the survey, the total bacterial
abundance ranged from 2.0� 108 and 6.0� 108 cells
tamination experiment (data collected between 11:00 and 11:30 AM,
D. Delille et al. / Cold Regions Science and Technology 40 (2004) 61–70 65
ml� 1. Total bacterial counts did not differ signifi-
cantly between pristine and contaminated zones (data
not shown). Direct count estimations were not sensi-
tive enough to give valuable information concerning
Fig. 3. Changes in abundance of heterotrophic and hydrocarbon degrading
uncovered soil, black area: covered soil, T0: before contamination, T1: 42
of contamination, T4: 660 days of contamination). Bars indicate standard
the differences between the various treatments. In
contrast, a significant response of more specific
bacterial communities to hydrocarbon contamination
was observed.
microorganisms during the contamination experiment (white area:
days of contamination, T2: 150 days of contamination, T3: 330 days
deviations.
D. Delille et al. / Cold Regions Science and Technology 40 (2004) 61–7066
In both soils, more than one order of magnitude
increase of heterotrophic bacterial abundance oc-
curred after 5 months of crude oil or diesel contam-
ination (Fig. 3). For both kinds of contaminants,
Fig. 4. Percentage loss of hydrocarbons in the two soils during the contam
soil, T1: 42 days of contamination, T2: 210 days of contamination, T3:
indicate standard deviations.
fertilizer addition had no clear effects on heterotrophic
bacterial assemblages in vegetated soil, but a slight
positive effect on corresponding values in desert soil
was present. The maximum difference (more than one
ination experiment (white area: uncovered soil, black area: covered
330 days of contamination, T4: 660 days of contamination). Bars
D. Delille et al. / Cold Regions Science and Technology 40 (2004) 61–70 67
order of magnitude) between covered and uncovered
soils was found after 2 years of diesel bioattenuation
in S1, but the soil coverage had no clear effects on
heterotrophic bacterial assemblages in the desert soil
(S2).
Before contamination, the hydrocarbon-degrading
bacteria never represented more than 0.5% of the total
bacterial assemblage. A spectacular enrichment in oil
degrading bacteria was observed in all contaminated
plots. This contribution often exceeded 80% of total
after 1 year of contamination. However, a decrease of
hydrocarbon-degrading bacterial abundance occurred
in both untreated soils during the second year of
contamination. Inipol amendment had a much stron-
ger stimulating effect on hydrocarbon-degrading bac-
terial abundance in the desert soil than in the
vegetated one. Soil warming had a very strong stim-
ulating effect on the specific bacterial assemblage in
the vegetated soil during the first few months of crude
oil contamination. In the other case, there were no
very strong differences between the specific bacterial
numbers collected in covered and uncovered plots.
However, the numbers were generally higher in the
covered soils than in the uncovered ones.
The results of the chemical analysis are shown in
the Fig. 4. There was a drastic decrease of hydrocar-
bon concentrations in all experimental plots. Further-
more, losses of total alkanes were higher than those of
total PAHs. After 2 years, aliphatic hydrocarbons
remained only in very low concentrations in all plots,
while about 10% of the initial PAHs concentrations
were still present in both soils. The C18/Hopane and
C18/Chrysene ratio followed a constant decrease
confirming a regular oil biodegradation process (data
not shown). Alkanes degradation was generally faster
in desert soil than in the vegetated one. Degradation of
total alkanes within Inipol-amended plots was gener-
ally faster than within untreated ones. In contrast,
nutriment addition had no clear effects on degradation
rate of total PAHs. The decrease of alkanes concen-
trations was always higher in covered soils than in the
corresponding uncovered ones. After 1 year of reme-
diation in covered vegetated soil, the remaining con-
centrations of aliphatic hydrocarbons from both diesel
and crude oil were less than one half of the
corresponding values in uncovered soils. In contrast,
soil warming had no clear effects on PAHs degrada-
tion. There was generally a small positive effect in
crude oil contaminated soils, but a small negative
effect appeared in diesel contaminated one.
4. Discussion
Before contamination, heterotrophic and hydrocar-
bon-degrading bacterial counts recorded in Kerguelen
soils agreed with those previously reported for Ant-
arctic and sub-Antarctic soils (Aislabie et al., 1998;
2001; Delille, 2000; Delille and Pelletier, 2002; Delille
et al., 2003). Hydrocarbon-degrading microorganisms
accounted for 10% of the total number of heterotro-
phic bacteria in the vegetated soil and 30% in the
desert one. The values observed in vegetated soil are
consistent with those of Atlas (1981) and Wright et al.
(1997) who reported that hydrocarbon-degrading
microorganisms represented 1–10% of the total num-
ber of heterotrophic bacteria in bacterial communities.
In contrast, the relatively large abundance of specific
microorganisms recorded in the uncontaminated desert
soil may correspond to a slightly contaminated soil
rather than to a true pristine soil. A previous accidental
oil contamination in the vicinity of the experimental
site cannot be excluded after more then 50 years of
human activities in the Port aux Franc�ais region.The addition of petroleum hydrocarbons into both
sub-Antarctic soils resulted in an enrichment for
heterotrophic and hydrocarbon-degrading microor-
ganisms. Such enhancements of specific micro-
organisms have been previously reported in polar
area (Delille and Delille, 2000; Wagner-Dobler et al.,
1998). Despite the relatively low level of contami-
nation used in the enclosures, some of the hydrocar-
bon-degrading bacterial abundances observed after
contamination of untreated soils were in the same
order of magnitude than those observed after heavy
diesel contamination in Crozet Island (>108 bacteria
ml� 1, Delille and Pelletier, 2002). This could indi-
cate that 109 bacteria ml� 1 may be a maximal value
for hydrocarbon-degrading bacterial abundance in
sub-Antarctic soils.
The rate of microbial degradation of hydrocarbons
in soils is affected by several physical, chemical and
biological parameters including the abundance and
diversity of microorganisms present in the pristine
environment before the spill, the conditions for micro-
bial activity (e.g., concentration of nutrients, oxygen
D. Delille et al. / Cold Regions Science and Technology 40 (2004) 61–7068
and temperature) and the nature, the quantity and
bioavailability of the contaminants. It is well estab-
lished that nutrients are one of the major limiting
factors of hydrocarbon biodegradation. Hydrocarbon
degradation rates in soils from Arctic regions can be
enhanced with the addition of nutrients (Braddock
et al., 1997; Whyte et al., 1999; Walworth et al.,
2001). Our results confirmed the efficiency of fertilizer
addition for alkanes biodegradation in polar soils. In
contrast, fertilizer addition had no obvious effects on
PAHs degradation. The stimulating effect of fertilizer
addition in the development of hydrocarbon-degrading
assemblage was stronger in the desert soil than in the
vegetated one. The nitrogen concentrations naturally
present in S1 would have be high enough to sustain
rapid intrinsic rates of biodegradation without human
intervention.
Among the other parameters controlling hydrocar-
bon biodegradation, temperature is generally consid-
ered as one the most important in polar area, since
degradation is thought to obey to the Arrhenius
relationship. Microbial metabolism increases as tem-
perature increases (Leahy and Colwell, 1990), usually
doubling for each 10 jC increase in temperature from
10 to 40 jC (Bossert and Bartha, 1984). However, the
metabolism of cold-tolerant bacteria is adapted to
work and function at low temperature (Delille and
Perret, 1989; Gounot, 1991; Margesin and Schinner,
1997; Feller et al., 1996; Whyte et al., 1996). In
laboratory experiments, Gibb et al. (2001) demon-
strated that the hydrocarbon degradation rate was
reduced when the temperature decreased from 20 to
5 jC when the microbial population was in the log
phase of growth. In contrast, the degradation rate was
temperature independent when the population was in
the stationary phase. In field works, it has been
recently shown in Arctic soils that freezing point is
not the ultimate limit for in situ biodegradation of
hydrocarbons by cold-adapted microorganisms and
that biodegradation may proceed at subzero temper-
atures (Rike et al., 2003). Soil covering induced an
annual mean increase of temperature of only 2 jC.Furthermore, the plastic black cover may have inter-
fered with physical weathering of the contaminant in
reducing natural evaporation and photodegradation.
Despite these restrictions, soil cover allowed a sig-
nificant reduction of the time necessary for the
achievement of some bioremediation objectives. A
nearly complete biodegradation of alkanes is ob-
served after 1 year of natural exposition in amended
vegetated soil and after 2 years in all other covered
soils. It could take at least 1 year more of bioattenua-
tion to reach the same results in non-covered soils. A
higher temperature increase may induce a further
reduction of bioremediation time. The most economic
way to reach such large increase of temperature
appears to be a biopiles system. One objective of
our future work will be to access the efficiency of
such biopile installation.
Like nutrient amendment, soil covering had no
measurable impact on PAHs biodegradation. It seems
that alkanes are the preferred carbon source for soil
bacterial assemblages, while aromatics larger than
three rings are relatively neglected. These results
provide new evidence supporting the hypothesis that
the naturally occurring hydrocarbon-degrading bacte-
ria may grow on both saturated and aromatic hydro-
carbons, although the two degradation pathways are
differently regulated (Roszak and Colwell, 1987;
Atlas and Bartha, 1992; Nyman, 1999).
In conclusion, the present data set provides evi-
dence of the presence of indigenous hydrocarbon-
degrading microorganisms in sub-Antarctic soils and
their high potential for hydrocarbons biodegradation.
Our field tests revealed that, even in absence of any
specific treatment, up to 80% of the hydrocarbons
were degraded within 1 year, supporting the feasibil-
ity of developing a bioremediation protocol. The rate
of aliphatic hydrocarbons degradation was improved
by both bioremediation treatments used in this study.
Even a small increase of temperature (2 jC) had a
favorable impact on alkanes degradation. In contrast,
none of the used procedures have a significant effect
on PAHs degradation. Thus, success of bioremedia-
tion depends largely of the contaminant character-
istics. It is also interesting to note that the results
obtained under the same general climatic conditions
and with the same experimental design differ from
one soil to another. Spill management requires the
development of a quick action in response planning.
However, care must be taken in extrapolating the
results of any experimental study to more general
environmental conditions. The design of an efficient
bioremediation system always requires a careful
study of the local conditions. Considerations for the
physical chemical and microbial properties of the
D. Delille et al. / Cold Regions Science and Technology 40 (2004) 61–70 69
contaminated soil and weather conditions is essential
in establishing appropriate response and recovery
methods.
Acknowledgements
Funding for this work was provided by the French
Polar Institute ‘‘IPEV’’ and the Canadian Research
Chair in marine ecotoxicology (E.P.).
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