Assessment of the biodegradation potential of hydrocarbons

in contaminated soil from a permafrost site

Marion Børresen*, Gijsbert D. Breedveld1, Anne G. Rike2

Norwegian Geotechnical Institute, P.O. Box 3930, Ullevaal stadion, N-0806 Oslo, Norway

Received 2 July 2002; accepted 23 October 2002

Abstract

Biodegradation of hydrocarbons in the Arctic and Antarctic has shown to be very slow, due to low temperatures combined

with low contents of nutrients, organic matter and water. In this study, the degradation potential of diesel and hexadecane by

indigenous microorganisms in a contaminated soil from a permafrost site was evaluated. Soil samples were taken from two

profiles at a hydrocarbon-contaminated site at Svalbard, at depths of 0.5 m (active layer), 2.0 m (transition zone between active

layer and permafrost) and 3.5 m (permafrost). The aerobe biodegradation potential was assessed using two different laboratory

approaches: nutrient amended liquid cultures with arctic diesel as the carbon source, and soil microcosms with radiolabelled

hexadecane. The experiments were performed at 5 jC.Biodegradation of hydrocarbons at 5 jC was obtained with both methods. In the liquid cultures 18–54% of the initial diesel

concentration was degraded after 32 days. The total degradation increased with sampling depth such that the greatest

degradation was observed in cultures inoculated with soil from the permafrost. In the soil microcosm experiments, 0.9–15.8%

of the initial hexadecane concentration mineralised after 128 days, at rates from 0.4 to 6.2 mg hexadecane/kg/day. In the soil

microcosms study, greatest total degradation was observed in soil from the transition zone, followed by the samples from active

layer and permafrost, respectively.

Both liquid cultures and soil microcosms can be used to study the biodegradation potential of contaminated arctic soil at 5

jC. The methods can be regarded as complementary, with the liquid cultures being qualitative and the soil microcosms

quantitative in nature. The liquid cultures provide information regarding the indigenous microorganisms’ capability to degrade

different compounds under optimised conditions. The soil microcosm experiments result in mineralisation rates in the soil under

the prevailing conditions.

D 2003 Elsevier Science B.V. All rights reserved.

Keywords: Biodegradation; Hydrocarbons; C-14 hexadecane; Permafrost; Low temperature; Arctic

1. Introduction

Bioremediation, the use of microorganisms or

microbial processes to degrade contaminants, has

become an important method to clean up hydrocar-

bon-contaminated soil and groundwater. Successful

bioremediation is, however, dependent on the temper-

0165-232X/03/$ - see front matter D 2003 Elsevier Science B.V. All rights reserved.

doi:10.1016/S0165-232X(02)00092-7

* Corresponding author. Fax: +47-22230448.

E-mail addresses: marion.borresen@ngi.no (M. Børresen),

gijsbert.dirk.breedveld@ngi.no (G.D. Breedveld),

anne.gunn.rike@ngi.no (A. G. Rike).1 Fax: +47-22230448.2 Fax: +47-22230448.

www.elsevier.com/locate/coldregions

Cold Regions Science and Technology 37 (2003) 137–149

ature, type of contaminant and microorganisms

present, pH, and the availability of nutrients and

water. Bioremediation in the Arctic is often consid-

ered as non-optimal due to low air temperatures

(below freezing 9–10 months of the year), combined

with low levels of nutrients, organic matter and water

in the soil. Once contaminants are introduced into the

arctic environment, they may persist for decades (Rike

et al., 2001; Braddock et al., 1999; Atlas, 1981).

Biodegradation of hydrocarbons has nevertheless

been reported at low temperatures in Arctic (Whyte

et al., 2001; Braddock et al., 1999; Whyte et al.,

1999), Antarctic (Bej et al., 2000; Aislabie et al.,

1998; Kerry, 1993), and Alpine soils (Margesin,

2000). In these investigations, the hydrocarbon deg-

radation was attributed to the activity of indigenous

cold-adapted psychrophilic and psychrotrophic micro-

organisms, which are characterised by low-temper-

ature growth ranges of V 0 to 15–20 jC and V 0 to

30–35 jC, respectively (Morita, 1975). Although

bacterial activity is considered to decline with

decreasing temperatures, metabolic activity in bacteria

has been measured at temperatures as low as � 20 jC(Rivikina et al., 2000). It is believed that the active

layer is where most hydrocarbon contaminants accu-

mulate and is presumably the predominate site of

biological activity (Mohn and Stewart, 2000). Fuel

infiltration rates in frozen soils are found to decrease

as ice saturation increases (McCauley et al., 2002),

but the presence of unsaturated void spaces and

fissures in the frozen soil may provide a means for

contaminant migration into permafrost and frozen

soils. The permafrost table is therefore not an imper-

meable barrier to hydrocarbon contamination (Biggar

et al., 1998).

In recent years, methods for optimising hydro-

carbon biodegradation in cold climates has received

increased attention. Laboratory and field studies have

been performed to investigate the effect of parameters

such as temperature, nutrients, introduction of cold-

adapted microorganisms and bioventing on the deg-

radation rates of hydrocarbons (Filler et al., 2001;

Gibb et al., 2001; Walworth et al., 2001; Mohn and

Stewart, 2000; Bradley and Chapelle, 1995; Liddel et

al., 1994). Nutrients such as nitrogen and phospho-

rous are regarded to be of essential importance for

microbial growth. Still, the addition of fertilizers has

been reported to enhance, inhibit or have no effect on

the biodegradation of hydrocarbons (Braddock et al.,

1999; Roy and Greer, 2000). It is assumed that even

relatively low nutrient additions may lead to toxic

effects on microbial populations in certain soils due to

osmotic stress created by the decreasing soil–water

potential (Walworth et al., 1997). Determining soil

characteristics such as nutrient availability and mois-

ture content are regarded as important factors to

understand how optimal hydrocarbon degradation by

microorganisms can be achieved.

In this work, the biodegradation potential by indig-

enous microorganisms in two soil profiles from a

hydrocarbon-contaminated arctic soil was studied.

The soil samples were characterised for various phys-

ical, chemical and biological parameters and their

hydrocarbon degradative capacity at low temperature

was subsequently assessed. Two different methods

were used to examine the degradation potential:

nutrient amended liquid cultures utilising arctic diesel

as the carbon source, and soil microcosms with

untreated soil samples spiked with 14C-hexadecane.

Liquid cultures (Bej et al., 2000; Whyte et al., 1998)

or soil microcosms (Roper and Pfaender, 2001; Roy

and Greer, 2000; Whyte et al., 1999) are often used

for assessing microbial activity. In this study, both

approaches were used on the same soil samples in

order to compare the methods. All laboratory experi-

ments were performed at 5 jC, a temperature assumed

not to provide an optimal temperature for psychro-

philic and psychrotrophic microorganisms, but which

corresponds to representative summer air-temperature

in the Arctic.

2. Materials and methods

2.1. Soil sampling and characterisation

The sampling site is located in Longyearbyen at

Spitsbergen (78j15VN, 15j30VE), the largest island

in the Svalbard archipelago. The soil at the sampling

site consists of marine and beach materials deposited

during the last glaciation period in the area (Tol-

gensbakk et al., 2000). The sampling site was used

as a fire-fighting training site from 1974 to 2000.

The activity resulted in spills of various petroleum

products and fire-extinguishing media. Concentra-

tions up to 24000 mg mineral oil/kg where found

M. Børresen et al. / Cold Regions Science and Technology 37 (2003) 137–149138

during a site assessment in 1999 (Sørlie et al.,

2000).

To approximately determine the extent of contam-

ination, CO2 and O2 pore gas concentrations were

measured at 0.5 m depths utilising active pore gas

sampling techniques (Multiwarn II, Drager, Ger-

many). Elevated oxygen consumption rates and car-

bon dioxide production can often be related to the

presence of organic contaminants in the soil (Brad-

dock et al., 1999). Soil samples for the laboratory

experiments were taken from the most contaminated

area, corresponding to the highest concentration of

CO2 and the lowest concentration of O2 (sampling

point 1), and from a point determined to be located

outside the contaminated area (sampling point 2). Soil

samples were taken at 0.5 m depth in the active layer,

at 2.0 m depth in the transition zone or ‘‘permafrost

table’’ located between the active layer and the

permafrost and at 3.5 m depth in the permafrost at

both sampling points. The soil samples from sampling

point 1 are hereafter specified as 1-a (0.5 m), 1-b (2.0

m) and 1-c (3.5 m), and the samples from sampling

point 2 as 2-a (0.5 m), 2-b (2.0 m) and 2-c (3.5 m).

The soil sampling was performed in pre-drilled holes

with a special sampler (OD/ID 120/70 mm) designed

for sampling in frozen gravel and stony soils. Equip-

ment used in the soil sampling was washed in 70%

ethanol. The soil samples were transferred to pre-

sterilised jars and stored in the freezer prior to

analyses and experiments.

pH and conductivity were determined in a soil–

water (1:5) slurry. Total organic carbon (TOC) and

total nitrogen were determined by CHN-analyser and

moisture content was calculated from weight loss on

drying. The aromatic hydrocarbons benzene, toluene,

ethylbenzene, xylenes and naphtalene (BTEXN) and

total petroleum hydrocarbons (TPH) ranging from C12

to C32 were analysed on dichloromethane extracts of

the soil by gas chromatography–mass spectrometer

(GC–MS screening). The content of nitrate, ammo-

nium and ortho-phosphate were measured on CaCl2extractions (0.1 M) of the soil with colourimetric

methods, and sulphate by ion chromatography (IC).

Grain-size distribution curves of the soils were estab-

lished using standard sieves and the ‘‘falling drop’’

method.

The number of heterotrophic bacteria in the soil

samples was determined by plate counts as colony

forming units (CFU) on nutrient broth agar (Difco).

Most probable number (MPN) technique was used to

assay the hydrocarbon and the glucose-degrading

microbial populations in the soil samples as described

in Brown and Braddock (1990). A mineral growth

medium (MM) (1.00 g NH4Cl, 0.41 g MgSO4�H2O,

0.33 g CaCl2�2H2O, 0.5 g KCl and 1.0 g NaCl/l) with

added trace metal stock solution (Greer et al., 1990) to

a final concentration of 0.05% (vol/vol) was used. For

the carbon source, glucose or arctic diesel (Go-12,

Esso) was added to a final concentration of 0.1% (w/

vol) or 0.5% (vol/vol), respectively. MPN was calcu-

lated according to Alexander (1982). All bacterial

enumeration was performed at 5 jC and counted after

4 weeks incubation.

2.2. Degradation in liquid cultures

Degradation of diesel in liquid cultures was per-

formed under aerobe conditions in 250 ml culture

flasks containing 50 ml MM supplemented with 1%

(vol/vol) trace metal stock solution, 1% (vol/vol)

phosphate stock solution (0.3 g KH2PO4 and 1.7 g

K2HPO4/l) and 0.1% (vol/vol) arctic diesel. The final

pH of the medium was 7.3. One milliliter of a pre-

culture with 125 g soil/l MM shaken overnight at 5

jC, corresponding to cell numbers of 105 to 107 CFU/

ml, was used as inoculum. To quantify the diesel

removal caused by abiotic processes such as evapo-

ration and sorption, sodium azide (2% w/vol) was

added to control cultures. Duplicate cultures were

incubated in the dark at 5 jC for 0, 4, 8, 16 or 32

days under continuous shaking. At the end of the

incubation period, 5a-androsane was added to the

culture flasks as extraction standard and the cultures

were extracted with 2 ml hexane with ortho-terphenyl

added as an internal standard. The hydrocarbon con-

centration was determined by GC–FID. The total

biodegradation was calculated as the difference be-

tween hydrocarbon concentration in the cultures and

the abiotic controls with Na azide.

2.3. Degradation in soil microcosms

Aerobic hexadecane mineralisation in the soil

samples was determined by radiorespirometry in soil

microcosms in 250 ml biometer flasks with soil

corresponding to 40 g dry weight. The soil was spiked

M. Børresen et al. / Cold Regions Science and Technology 37 (2003) 137–149 139

with n-[1-14C]hexadecane (8.93 MBq/mg, Amer-

sham) and unlabelled hexadecane (Merck Eurolab)

to a final concentration of 5000 mg/kg. The flasks

were sealed with rubber stoppers and silicon, and the

evolved CO2 was trapped in 10 ml 1 M KOH in the

side arm of the biometer flasks. Samples of 1 ml KOH

was regularly withdrawn using a syringe and 1 ml

fresh KOH was added. The flasks were aerated to

maintain aerobic conditions during KOH sampling.

The trapped 14CO2 activity was measured as disinte-

grations per minute (dpm) by liquid scintillation

counting. The amount of mineralised hexadecane

was estimated from the dpm numbers, corrected for

background and quenching by external standards.

Duplicate flasks were prepared for each soil sample.

Control microcosms were set up to follow abiotic loss

as evaporation and degradation of the radiolabelled

compound, and to estimate the background radio-

activity. In control 1, used to quantify the evaporation

and abiotic degradation of 14C-hexadecane, the soil

was autoclaved twice on two consecutive days and

sodium azide was added to a final concentration of

1% (w/w) before 14C-hexadecane was added. In con-

trol 2, used to quantify the background radioactivity,

only unlabelled hexadecane was added. All laboratory

experiments were performed at 5 jC.

3. Results and discussion

3.1. Soil characterisation

The physical and chemical characterisation of the

soil samples is given in Table 1. The grain-size

analysis of the sampling materials showed that the

soils used in the experiments were sandy soils (48–

60% sand) with low contents of clay ( < 5%) and silt

( < 21%). The materials at the sampling site are earlier

classified as marine and beach materials (Tolgensbakk

et al., 2000). However, the specific conductivity in the

soil ranged from 107 to 250 AS/cm, indicating a low

salt content and weak marine influence. A moisture

content of 10–20% is optimal for microbial activity in

the unsaturated zone, but in arid soils and clays a

lower moisture content is capable of supporting active

populations of hydrocarbon-degrading microorgan-

isms (King et al., 1992). The moisture contents in

the soil samples, except sample 1-b, were between

Table 1

Physical and chemical characterisation of soil samples

Sampling point 1 Sampling point 2

1-a 1-b 1-c 2-a 2-b 2-c

Sampling depth (m) 0.5 2.0 3.5 0.5 2.0 3.5

Moisture (%) 11.0 6.8 13.0 12.3 10.8 13.9

Grain-size distribution

Clay (%) 2.2 0 4.9 0.4 2.7 5.3

Silt (%) 17.8 3.0 20.1 20.6 15.3 19.7

Sand (%) 52.4 59.5 51.6 53.1 50.6 48.4

Gravel (%) 27.6 37.5 23.4 25.9 31.4 26.6

Specific conductivity (AS/cm) 149 107 144 155 240 144

Hydraulic conductivity, K (m/s) 5.6� 10� 6 8.1�10� 4 5.7� 10� 7 4.2� 10� 7 9.4� 10� 7 4.2� 10� 7

Total petroleum hydrocarbons

(TPH) C12–C32 (mg/kg)

3600 21500 1310 205 430 1600

Hexadecane (mg/kg) 12.0 52.5 1.1 1.6 1.9 0.9

BTEXN (mg/kg) 3.4 5.1 4.3 3.8 6.3 0.9

pH 6.4 7.0 7.3 5.7 6.5 7.8

TOC (g/100g) 2.4 0.9 1.0 1.5 0.9 0.9

Nitrate (mg/kg) < 1.2 8.4 < 0.8 2.4 < 0.8 < 0.8

Ammonium (mg/kg) 1.4 72.7 < 0.8 < 0.8 < 0.8 < 0.8

Total N (g/100 g) 0.09 < 0.05 0.07 0.11 0.18 0.08

C/N 26.7 – 14.3 13.6 5.0 11.3

o-Phosphate (mg/kg) 16.2 < 2.0 < 2.0 < 2.0 < 2.0 < 2.0

M. Børresen et al. / Cold Regions Science and Technology 37 (2003) 137–149140

11% and 14%, indicating optimal moisture contents in

most of the samples.

The hydraulic conductivity, calculated from the d10(mm) values, was 10� 4 to 10� 7 m/s at point 1, and

10� 7 m/s at point 2. The hydraulic conductivity

values indicate that the permeability is high enough

for the migration of hydrocarbons through both soil

profiles. The presence of TPH and BTEXN in all of

the samples confirms this assumption. The highest

TPH concentrations were found in the soil samples at

point 1, ranging from 1310 to 21 500 mg TPH/kg, and

in the permafrost sample at point 2 (1600 mg TPH/

kg). The BTEXN concentrations, on the other hand,

was highest in the transition zone at both sampling

points (5.1 and 6.3 mg BTEXN/kg at point 1 and

point 2, respectively). The content of TOC was gen-

erally low and exceeded 1% in the samples from

active layer (0.5 m) only.

The main fire-extinguishing chemical used at the

site was ABC Favoritt 111 (NOHA brannteknikk,

Norway), which contains ammonium dihydrogen

phosphate (30–60%) and ammonium sulphate (30–

60%). The increased levels of ammonium, nitrate, and

phosphate in the upper portions of the soil profile at

sampling point 1 is probably due to the extended use

of this and other fire-extinguishing chemicals. The

nutrient levels at sampling point 2 were generally

under the detection limits. Nitrogen (as nitrate or

ammonia) and phosphorous (as phosphate) are some

of the major nutritional factors for microbial growth

(King et al., 1992), and low concentrations indicate

that either N or P may become the limiting factor for

biodegradation in this soils. An optimal C/N ratio for

bioremediation in soil is 10 according to Alexander

(1994). The C/N ratios in the soil samples, except

sample 2-b, were between 11 and 27 (on weight

basis), indicating optimal C/N ratios in most of the

samples. The pH in both soil profiles ranged from 6 to

8, increasing with depth. The measured pH values

correspond to the general preferable pH for maximum

rate of growth for microorganisms in soil (King et al.,

1992).

The active layer is generally considered to be the

zone of greatest microbial activity, as it often contains

the highest content of available water, nutrients and

TOC, and obtains maximum temperatures in the

summer months (Mohn and Stewart, 2000; Rike et

al., 2001). Microbial enumeration demonstrated, how-

ever, the presence of significant numbers of microbial

cells in all the soil samples investigated, including the

samples taken from the permafrost table and the

permafrost (Table 2). The number of heterotrophic

bacteria on nutrient broth agar varied between 105 and

107 CFU/g soil, with highest numbers in soil from

sampling point 1. The number of bacteria present in

the soil was lower compared to earlier enumerations

conducted on contaminated arctic soils (106 to 107

CFU/g at 5 jC (Whyte et al., 2001), 107 to 108 CFU/g

at 5 jC (Whyte et al., 1999) and 107 to 108 CFU/g at

20 jC (Rike at al., 2001)). The number of glucose-

and diesel-degraders in soil varied from 103 to 106 and

102 to 104 MPN/g, respectively. The number of

bacteria appeared to increase with increasing depths

in the soil profiles, this was observed independent of

TPH or nutrient concentrations in the soil. The diesel-

degrading organisms represented 1.4–87% of the

glucose-degraders at point 1 and from 0.1% to 0.5%

at point 2. Enrichment of hydrocarbon-degrading

organisms is a characteristic commonly observed in

hydrocarbon-contaminated soil (Atlas, 1995), and the

results indicate a stronger selective enrichment of the

diesel-degraders in point 1 than in point 2, which is

less contaminated. The high number of diesel-

degraders shows that enrichment of hydrocarbon-

degrading organisms occurs at low temperatures,

and even in the permafrost under freezing conditions.

The soil characterisation showed in brief that the

soil samples investigated have pH, moisture and C/N

Table 2

Enumeration of microorganisms in soil samples at 5 jC

Sampling point 1 Sampling point 2

1-a 1-b 1-c 2-a 2-b 2-c

Heterotrophs (CFU/g d.w.) 6.5� 107 1.5� 107 6.5� 106 3.8� 105 8.0� 106 3.0� 106

Glucose-degraders (MPN/g d.w.) 1.5� 103 1.6� 105 3.0� 106 1.7� 105 1.1�106 1.0� 106

Diesel-degraders (MPN/g d.w.) 1.3� 103 2.5� 104 4.3� 104 8.4� 102 1.7� 103 1.2� 103

M. Børresen et al. / Cold Regions Science and Technology 37 (2003) 137–149 141

ratios at or close to assumed optimal values for

biodegradation in soil. TPH and BTEXN concentra-

tions are high in all samples, and the nutrient concen-

trations are also high in some parts of the most

polluted soil profile (point 1). The microbial enumer-

ation demonstrated the presence of significant num-

bers of indigenous microorganisms in the soil, and

that diesel-degrading organisms constitute up to 87%

of glucose-degrading organisms. The soil character-

isation demonstrated that the indigenous microorgan-

isms were able to adapt to the range of different

environmental conditions in the examined soils and

suggest that hydrocarbon-contaminated arctic soils

have potential for bioremediation.

3.2. Degradation in liquid cultures

Diesel degradation was studied in liquid cultures

inoculated with soil extracts from the six soil samples

(Fig. 1). There was a lag phase of 8 days in the

cultures inoculated with bacteria from the permafrost

table and permafrost at point 1 (1-b and 1-c) and the

active layer at point 2 (2-a). In the remainder of the

cultures, there was a 16-day lag phase (active layer at

point 1 (1-a) and permafrost table and permafrost at

point 2 (2-b and 2-c)). The observed lag phase in the

liquid cultures is probably due to the low initial cell

number of diesel-degrading organisms in the inocu-

lum, and is the time needed by the population to reach

a requisite cell number for optimal diesel degradation.

The diesel degradation in the liquid cultures was

measured as percent degradation of initial diesel

concentration. After 32 days at 5 jC, the total diesel

degradation ranged from 18% to 54% at sampling

point 1 and from 28% to 32% at point 2. This

correspond to values of 3% to 39% at point 1 and

from 12% to 17% at point 2, when corrected for

abiotic loss. The total diesel degradation after 32 days

was highest in the cultures inoculated with extracts

from the permafrost samples with 54% and 32%

degraded at point 1 and point 2, respectively. Lowest

degradation (18%) was found in the sample from

active layer at point 1 (1-a). The low degradation

may be related to low initial numbers of glucose- and

diesel-degraders in the extract. Abiotic processes

caused a total diesel loss of about 15% in the liquid

cultures. The lighter diesel components in the carbon

range of C9–C12 were nearly completely removed by

abiotic processes as observed in the controls without

bacterial activity (Fig. 2A). This demonstrates that the

removal of the low molecular alkanes is affected by

abiotic loss such as evaporation and sorption to glass

walls, and not by microbial degradation. In the C12–

C18 range, the microbiological alkane reduction was

between 7% (1-a) and 56% (1-c). The reduction in the

abiotic control was below 4%, which indicates that the

reduction is mainly caused by biological degradation

in this oil fraction (Fig. 2B). Alkanes having chain

length longer than C18 represented the most recalci-

trant diesel fractions. The abiotic loss was insignif-

icant and the biotic loss was observed to be 10% or

less in all samples, with exception of sample 1-c

where 30% of the alkane fraction >C18 was biode-

graded (Fig. 2C). The diesel degradation patterns

(chromatograms) for the liquid cultures with inoculum

from the permafrost soils are given in Fig. 3. The

degradation pattern of the two samples are quite

similar, and the results revealed an important loss of

the lighter alkanes during the first 4 days of the

experiment (t0– t1), followed by the subsequent

removal of the longer chain alkanes over time (t2–

t4). After 32 days incubation, most of the linear chain

alkanes were removed by biotic or abiotic processes,

and in the most degraded sample it is likely the most

recalcitrant diesel constituents (branched alkanes and

aromatic compounds) that remain. The recalcitrant

Fig. 1. Degradation of diesel at 5 jC in liquid cultures, data not

corrected for abiotic loss (initial diesel concentration 1000 Al/l).Each point represents the mean from duplicate cultures and the error

bars represent the range of the values.

M. Børresen et al. / Cold Regions Science and Technology 37 (2003) 137–149142

constituents, also called the unresolved complex mix-

ture, are observed as the ‘‘hump’’ in the chromato-

gram (Fig. 3 (t4)).

The mineral oil/hexadecane concentrations in the

soil did not express a direct correlation to the diesel

degradation rate in the liquid cultures, but the bio-

degradation capacity can partly be correlated to the

number of hydrocarbon-degrading microorganisms

in soil, which tends to increase with increasing

depth. The degradation experiment in liquid cultures

confirms, as assumed from the soil characterisation

data, that there are microorganisms present in the

soil capable of degrading hydrocarbons at low tem-

perature. In the liquid cultures, however, the bio-

degradation potential was studied under optimised

conditions, where nutrients, trace metals and carbon

source were easily available for the microorganisms.

Under such conditions degradation potentials are

normally overestimated compared to actual degrada-

tion in soils at in situ conditions.

3.3. Degradation in soil microcosms

Hexadecane mineralisation was studied by radio-

respirometry in soil microcosms. To avoid hexadecane

limitations in the radiorespirometry experiments, sub-

strate assessment with varying concentrations of hex-

adecane in the soil microcosms was performed. Soil

samples taken from different depths in sampling point

1 (0.5, 2.0 and 3.5 m) were air-dried and mixed

together before addition of sterile water to a moisture

content of 10%. 14C-hexadecane/kg d.w. (100–50000

mg) was added to the samples and the flasks were

incubated at 5 jC for 42 days. Although low biode-

gradation was observed, hexadecane mineralisation

was demonstrated at all substrate concentrations

investigated (Fig. 4). Highest accumulated hexade-

cane mineralisation as percentage of initial concen-

tration was achieved in the microcosms with lowest

substrate concentration (45% mineralised at initial

substrate concentration of 100 mg/kg). The percent

hexadecane mineralisation decreased with increasing

substrate concentrations, and reached minimum min-

eralisation at maximum substrate concentration (1%

mineralised at substrate concentrations of 50000 mg/

kg). The results clearly show that the hexadecane

mineralisation in soil presented as percentage

degraded of initial concentration is strongly dependent

on the initial substrate concentrations, and reducing

the substrate concentration can lead to apparently

better degradation results. A possible reason for the

results observed in Fig. 4 can be that the proportion of

the radiolabelled to unlabelled hexadecane is greater

at lower concentrations and, consequently, more of the14C-hexadecane would be mineralised by the degra-

dative microorganisms.

The hexadecane mineralisation rates in the expo-

nential phase were calculated at different substrate

Fig. 2. Degradation of diesel components corresponding to different

alkane chain lengths in the liquid cultures, data not corrected for

abiotic loss (initial diesel concentration 1000 Al/l).

M. Børresen et al. / Cold Regions Science and Technology 37 (2003) 137–149 143

Fig. 3. Chromatograms of diesel oil extracted from the liquid cultures after 0 to 32 days at 5 jC (initial diesel concentration 1000 Al/l). Culture Ais inoculated with bacteria from sampling point 1 and B from sampling point 2. Both samples are taken from 3.5 m depth (permafrost). The

extraction times were t0—0 day, t1—4 days, t2—8 days, t3—16 days, t4—32 days. I.S. indicates internal standard, E.S. extraction standard, and

C-10 to C-26 the n-alkanes present in the arctic diesel.

M. Børresen et al. / Cold Regions Science and Technology 37 (2003) 137–149144

concentrations (Fig. 5). The mineralisation rate was 2

mg/kg/day at a substrate concentration of 100 mg

hexadecane/kg and increased to 20 mg/kg/day at

concentrations of 5000 mg/kg. At 50000 mg/kg, the

mineralisation rate increased to 22 mg/kg/day only.

The results indicate that the substrate availability is

limiting the mineralisation rate at substrate concentra-

tions below 5000 mg/kg. Above this concentration

other environmental factors such as population density

and nutrient availability are probably more important.

To avoid substrate limitations in the mineralisation

experiment with soil samples from various depths in

the permafrost profile, substrate concentrations of

5000 mg hexadecane/kg soil was used.

Hexadecane degradation was studied by minerali-

sation of 14C-hexadecane at 5 jC in soil microcosms

from various depths at sampling points 1 and 2 (Fig.

6). The microcosms were incubated for 128 days.

Highest hexadecane mineralisation was determined in

the soil samples from sampling point 1. In the sample

from the transition zone at point 1, 15.8% of initial

concentration was mineralised, followed by 5.1% and

4.6% degradation in the samples from the permafrost

and active layer, respectively. In soils from the less

contaminated sampling point 2, highest hexadecane

mineralisation was found in the sample from the

transition zone. 1.9% of initial concentration was

mineralised after 128 days, followed by 1.4% miner-

alisation from active layer and 0.9% from the perma-

frost. The samples from the transition zone showed

highest degradation activity in both profiles. In the

samples from active layer a 20-day lag phase was

observed. The lag phases may correspond to the time

needed by the microbial population to reach the

appropriate cell number for efficient diesel degrada-

tion or may be related to the exposure of fire-exhaust-

ing media in the uppermost part of the soil in point 1,

inhibiting bacterial growth. Cumulative levels of 14C-

hexadecane mineralisation in the controls were less

than 0.02% at the end of the incubation period.

Fig. 4. Accumulated hexadecane mineralisation (percent of initial concentration) in soil at substrate concentrations ranging from 100 to 50000

mg hexadecane/kg. Each point represents the mean from duplicate microcosms. The numbers at the right hand side of the curves represent total

mass hexadecane mineralised in the soil (mg hexadecane/kg soil).

M. Børresen et al. / Cold Regions Science and Technology 37 (2003) 137–149 145

The hexadecane mineralisation rates in soil were

calculated in the linear range of the mineralisation

curves. The rates ranged from 0.4 mg hexadecane/kg/

day in the permafrost sample at point 2 to 6.2 mg

hexadecane/kg/day in the sample from the transition

zone at point 1. The difference between the maximum

and minimum mineralisation rates is probably not

caused by the different TPH or hexadecane concen-

trations in the soil, varying from 205 to 21 500 mg

TPH/kg and from 0.9 to 52.5 mg hexadecane/kg. The

permafrost sample from point 2 has higher TPH and

hexadecane concentrations than three of the other

samples, but is still the sample with lowest hexade-

cane mineralisation. The elevated mineralisation rate

at point 1 is probably a result of the significant

concentrations of ammonium, nitrate and phosphate

in parts of the soil profile. The concentrations of

ammonium and nitrate are especially pronounced in

the sample from the transition zone at point 1 (1-b),

which also express the highest degree of hexadecane

mineralisation.

Hexadecane mineralisation rates in two untreated

soil samples at 25 jC in a non-arctic soil were reported

to be 0.17 and 4.3 mg hexadecane/kg/day by Roy and

Greer (2000). The contaminant levels in the soils were

3800 and 2000 mg TPH/kg, and the C/N ratios were 81

and 32, respectively. The mineralisation rates are in the

same range as in this study, even though the degrada-

tion experiments conducted by Roy and Greer (2000)

were performed under higher temperature and C/N

ratios. The similar mineralisation rates indicate that the

microorganisms in this study have adapted to the

contaminants, the low temperature and the low C/N

ratios in the arctic soils. The C/N ratio is often used as

a criterion to decide if N fertilizer would be required

for effective bioremediation of a contaminated soil,

and can be a useful indicator of the N limitations of a

system (Roy and Greer, 2000). The relation between

C/N ratios and total mineralisation in this experiment

indicates that the C/N ratio is not a good indicator for

predicting the outcome of biodegradation in arctic

soils.

3.4. Comparison of liquid cultures and soil micro-

cosms

The degradation experiments confirmed that both

liquid cultures and soil microcosms can be used to

determine if there are hydrocarbon degradative micro-

organisms present in the soil. In both experiments

biodegradation of hydrocarbons at 5 jC in permafrost

soils was demonstrated. The highest degree of biode-

gradation was achieved in liquid cultures, where 3–

39% of initial diesel concentration was removed bio-

logically. The biodegradation activity increased in the

samples from greater depths and reached maximum

levels in cultures with soil from the permafrost in both

profiles. The total biodegradation can partly be corre-

lated to the number of hydrocarbon-degrading micro-

organisms in soil, which tends to increase with

increasing depths. The results indicate that initial

microbial numbers play an important role in liquid

culture studies. In the soil microcosms a biological

mineralisation of 0.9–15.8% of initial hexadecane

concentration was obtained. The highest mineralisa-

tion rate was demonstrated in the transition zone

between the active layer and permafrost, and the

mineralisation seems to depend more directly to envi-

ronmental conditions in the soil, such as the nutrient

content. The microorganisms in the liquid cultures

have easy access to components essential for growth

(nutrients, trace metals, water, oxygen and carbon

source). If the microorganisms capable of degrading

Fig. 5. Hexadecane mineralisation rates in soil (mg hexadecane/kg

soil/day) at different substrate concentrations. Each point indicates

mean values of the mineralisation rates in the exponential growth

phase in duplicate microcosms and the error bars indicate the range

of the values.

M. Børresen et al. / Cold Regions Science and Technology 37 (2003) 137–149146

hydrocarbons are present in the soil, they will develop

and mineralise the carbon source available when given

sufficient time. In the microcosms the microorganisms

have to rely on the natural presence of moisture and

nutrients, often limiting the mineralisation. The diesel

fuel used as carbon source in the liquid cultures

consisted of a wide range of hydrocarbons. By adding

this mixture of components, the ability of the micro-

organisms to mineralise the different diesel constitu-

ents could be readily studied. In the microcosms only

one radiolabelled compound was used. The results

therefore provide limited information on the indige-

nous microorganisms’ degradative capabilities for dif-

ferent hydrocarbon components. The abiotic controls

revealed that the evaporation of the lighter components

(C9–C12) is quite important even at 5 jC. Abioticremoval of 14C-hexadecane in the microcosms was not

detected, although abiotic controls were used.

By introducing a radiolabelled compound as the

carbon source, the hydrocarbon mineralisation by the

indigenous microorganisms could be measured as

production of 14CO2. In this experiment radiorespir-

ometry has shown to be a more sensitive method for

determination of low mineralisation rates than meas-

urements based on chromatography. The lowest min-

eralisation rate achieved in the microcosms degrada-

tion experiment was 0.4 mg hexadecane/kg/day,

which corresponds to a total mineralisation of 46

mg hexadecane/kg in 128 days. This change in

hexadecane concentrations is hardly detectable with

the GC. Another advantage with radiolabelled com-

pounds is that CO2 produced by degradation of soil–

C and other non-labelled compounds in soil do not

interfere with the results.

The two assessment methods used can be regarded

as complementary, with the liquid cultures being

qualitative and the soil microcosms more quantitative

in nature. The liquid cultures determine if the indig-

enous microorganisms present in the soil are capable

of degrading different compounds under optimised

Fig. 6. Hexadecane mineralisation in soil microcosms (initial hexadecane concentration 5000 mg/kg). Each point represents the mean from

duplicate microcosms and the error bars indicate the range of the values.

M. Børresen et al. / Cold Regions Science and Technology 37 (2003) 137–149 147

conditions. This method appears to be applicable if

the objective of the study is to determine maximal

degradation potential of the indigenous microbial

population present in a soil sample. If the goal is to

determine the actual in situ biodegradation rates in the

soil, the use of soil microcosms is more suitable. In

the soil microcosms experiments, the hexadecane

mineralisation rate in untreated soil samples from

different depths were found. These results are prob-

ably more closely related to the actual in situ micro-

bial activity in the contaminated soil than the results

from the liquid culture experiments, even though soil

microcosms also show higher mineralisation rates due

to better oxygen access for the microorganisms than

under in situ conditions.

In both experiments, hydrocarbon degradative

populations were identified in all the soil samples

studied, including the permafrost samples. These

results indicate an auspicious degradation potential

that is important if in situ remediation is considered as

a remediation strategy in an arctic soil. When hydro-

carbon biodegradation results are to be transferred

from the laboratory to field conditions, it is important

to take into account that optimised conditions utilised

in these experiments cannot be achieved in situ. The

best approach to find realistic in situ mineralisation

rates is to use soil microcosms. By performing labo-

ratory experiments at low temperatures and using

different amendments techniques (e.g. addition of

nutrients, water, etc.), the best conditions for hydro-

carbon mineralisation of arctic soils can be established

and transferred further to achieve a more successful in

situ treatment of contaminated arctic sites.

Acknowledgements

This project is funded by the Norwegian Research

Council and the Norwegian Geotechnical Institute.

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