www.elsevier.com/locate/coldregions

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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