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Soil Biology & Biochemistry 32 (2000) 1161±1172
www.elsevier.com/locate/soilbio

Limiting factors for hydrocarbon biodegradation at low
temperature in Arctic soils
William W. Mohn*, Gordon R. Stewart
Department of Microbiology and Immunology, University of British Columbia, 300-6174 University Boulevard, Vancouver, BC, Canada V6T 1Z3
Received 24 August 1999; received in revised form 10 February 2000; accepted 3 March 2000

Abstract
Hydrocarbon fuel spills are common in the Arctic. But, little is known about hydrocarbon-degrading micro¯ora in Arctic
tundra soils or the potential for bioremediation of these soils. We examined mineralization of radiolabeled hydrocarbons in
microcosms containing soils collected from sites across the Canadian Arctic. The soils all contained psychrotolerant
microorganisms which mineralized dodecane and substantially removed total petroleum hydrocarbons (TPH) at 78C. Dodecane
mineralization was severely limited by both N and P. Dodecane mineralization kinetics varied greatly among di€erent soils.
Multiple regression analysis showed that soil N and TPH concentrations together accounted for 73% of the variability of the lag
time preceding dodecane mineralization. Soil characteristics were less e€ective as predictors of mineralization kinetic parameters
other than lag time. High total C concentrations were associated with high mineralization rate constants, and high sand contents
were associated with long times for half-maximal dodecane mineralization. Very high concentrations of TPH (100 mg gÿ1 of dry
soil) and heavy metals (e.g., 1.4 mg Pb gÿ1 of dry soil) did not prevent dodecane mineralization. Inoculation of soils with
indigenous or non-indigenous hydrocarbon-degrading microorganisms stimulated dodecane mineralization. Bioremediation of

hydrocarbon-contaminated Arctic tundra soils appears to be feasible, and various engineering strategies, such as heating or
inoculating the soil, can accelerate hydrocarbon biodegradation. 7 2000 Elsevier Science Ltd. All rights reserved.
Keywords: Arctic; Biodegradation; Bioremediation; Cold; Fuel; Hydrocarbon; Soil microcosm

1. Introduction
Hydrocarbon pollution is common throughout the
Arctic. In that region hydrocarbon fuels are used
extensively as the primary energy source for heating,
transportation and generation of electricity. Large and
small fuel spills occur frequently during transportation
and use of these fuels. Some military radar stations in
the Canadian Arctic tundra subregion (cool-Artic vegetation zone) are among those contaminated sites and
are designated for remediation. While a number of studies have examined biodegradation of hydrocarbons in
Arctic marine environments (reviewed in Swannell et

* Corresponding author. Tel.: +1-604-822-4285; fax: +1-604-8226041.
E-mail address: wmohn@interchange.ubc.ca (W.W. Mohn).

al., 1996), very few have examined biodegradation of
hydrocarbons in Arctic tundra soils (Braddock et al.,

1997; Whyte et al., 1999).
Polar soil environments di€er from other soil environments in a number of ways that might a€ect hydrocarbon biodegradation. Polar soils have unique
periglacial features, including permafrost and numerous types of patterns primarily due to freeze±thaw
e€ects (Fitzpatrick, 1997). In the Arctic tundra, an
active soil zone, above the permafrost, thaws for a
period of typically 1±2 months in the summer. Summer temperatures in the active zone are highly
dynamic and vary greatly, from near freezing at the
permafrost interface to occasionally above 208C at the
surface. Permafrost restricts water movement and
sometimes results in a saturated active zone. The active
zone is where most hydrocarbon contaminants exist

0038-0717/00/$ - see front matter 7 2000 Elsevier Science Ltd. All rights reserved.
PII: S 0 0 3 8 - 0 7 1 7 ( 0 0 ) 0 0 0 3 2 - 8

1162

W.W. Mohn, G.R. Stewart / Soil Biology & Biochemistry 32 (2000) 1161±1172

and is presumably the site of most biological activity.

Little is known about the relationships between permafrost and hydrocarbon contaminants. Many Arctic
tundra soils are low in organic content, a characteristic
which can be expected to directly a€ect sorption of
hydrocarbons to the soil and to indirectly a€ect biodegradation of hydrocarbons.
Arctic tundra soil microbial communities have not
been well characterized. There is presently no convincing evidence that the composition or structure of
these communities is unique to Arctic tundra;
although, the biomass may be low and the organisms
more cold-adapted, relative to soils in temperate
regions (Robinson and Wookey, 1997). Organic decomposition is slow in Arctic tundra soils, largely due
to low temperatures. Some studies indicate the presence of hydrocarbon-degrading microorganisms in
Arctic tundra soils (Whyte et al., 1996, 1999; Braddock
et al., 1997) and groundwater (Braddock and
McCarthy, 1996). Similarly, hydrocarbon-degrading
microorganisms have been reported in Antarctic soils
(Kerry, 1993; Aislabie et al., 1998).
Physical, chemical and biological factors have complex e€ects on hydrocarbon biodegradation in soil
(Bossert and Compeau, 1995). For this reason, experts
frequently recommend that soil bioremediation projects begin with treatability studies to empirically test
the biodegradability of the hydrocarbon contaminants

and to optimize treatment conditions. On the other
hand, it is possible that the expense of such treatability
studies could be avoided or minimized, if certain soil
characteristics could be measured and used to predict
the potential for bioremediation of a site, the kinetics
of hydrocarbon removal or the optimal values for certain controllable treatment conditions. For example,
certain co-contaminants such as heavy metals might
preclude hydrocarbon bioremediation. Or, soil particle
size distribution might partly dictate the potential rate
and extent of hydrocarbon removal.
We examined hydrocarbon biodegradation in microcosms with 18 soil samples from across the Canadian
Arctic tundra. We determined the e€ects on biodegradation kinetics of a number of factors, including (i)
intrinsic soil properties (particle size, C content, water
holding capacity), (ii) soil contaminants (petroleum
hydrocarbons, heavy metals), (iii) controllable conditions (temperature, N and P content) and (iv) inoculation with hydrocarbon-degrading microorganisms.
We addressed the question of whether measuring soil
characteristics could allow prediction of the outcome
of soil bioremediation. Also, we identi®ed treatment
options which generally appear to bene®t bioremediation of Arctic tundra soils. This is the ®rst such comprehensive
study

characterizing
hydrocarbon
biodegradation in Arctic tundra soils. Unlike most studies on the general topic of hydrocarbon biodegrada-

tion, we examined the process in soil at a low
temperature, 78C, which commonly occurs in Arctic
tundra soils.

2. Material and methods
2.1. Soil samples
Eighteen hydrocarbon-contaminated soil samples
were taken during July and August 1998 from eight
radar stations across the Canadian Arctic tundra,
BAR-1 (708N, 1408W), BAR-4 (698N, 1288W), PIN-M
(708N, 1248W), CAM-4 (688N, 898W), FOX-M (688N,
818W), FOX-B (688N, 738W), DYE-M (668N, 618W),
and LAB-2 (588N, 648W). Samples were taken from
the top 10 cm of soil with clean trowels or scoops and
placed in glass bottles. Samples were stored refrigerated or on ice (0±108C) and were shipped by air. Soil
samples were sieved (4.7 mm mesh) and well mixed

prior to use. The initial microcosm experiment with
each sample was begun within 2 months of taking the
sample.
Twenty characteristics of each soil sample were
measured (Table 1). Soil pH was determined in a
slurry with distilled water. Water holding capacity
(WHC) was determined gravimetrically (Gardner,
1965). The following measurements were performed by
Paci®c Soil Analysis (Richmond, British Columbia):
total C and organic C (Nelson and Sommers, 1982.),
total N (Bremner and Mulvaney, 1982), available P
(Olsen and Sommers, 1982) as well as % gravel, %
sand, % silt and % clay (Day, 1965). The Analytical
Services Unit, Queen's University (Kingston, Ontario)
measured total Aroclors (by gas chromatography with
electron-capture detector) and eight metals (by atomic
absorption analysis). Aroclors and Cd were not
detected in any sample.
Total petroleum hydrocarbons (TPH) were
measured by gas chromatography, as follows. In 130 

20 mm screw cap tubes with caps lined with PTFEfaced rubber, 3 g (dry weight) soil samples were shaken with 6±8 g anhydrous Na2SO4 until the mixture
¯owed freely. Then, 8 ml hexane was added to each
tube, and the tubes were shaken vigorously for 5 min,
sonicated in a water bath for 10 min at room temperature, left overnight and shaken once again. After the
soil settled, portions of the hexane phases were centrifuged (12,000  g ), and the supernatants were placed
in sample vials. The samples were analyzed with a
Hewlett-Packard HP-5890 II gas chromatograph, with
a HP-5 column (25 m, 0.32 mm bore, 0.17 mm ®lm
thickness) and a ¯ame ionization detector. The carrier
gas was hydrogen, with a ¯ow rate of 2.4 ml minÿ1.
The injector and detector temperatures were 2808 C
and 3008 C, respectively. The temperature program

Soil

BAR-1a
BAR-4a
BAR-4b
BAR-4c
BAR-4d

BAR-4e
CAM-4a
CAM-4b
CAM-4c
DYE-Ma
FOX-Ba
FOX-Bb
FOX-Ma
FOX-Mb
FOX-Mc
LAB-2a
PIN-Mb
PIN-Mc

% Composition

WHC
(g gÿ1)

Gravel

Sand

Silt

Clay

50.0
15.1
26.0
11.0
13.8
19.5
58.8
40.4
31.6
17.8
7.0
11.8
45.0

45.3
39.5
29.5
31.8
19.9

85.2
68.7
88.8
89.6
78.2
95.2
93.7
82.4
81.2
83.8
68.3
75.0
77.0
68.6

68.8
89.5
88.2
89.1

11.2
15.1
6.9
5.3
12.7
4.2
4.7
12.3
11.2
14.2
27.2
20.9
18.4
24.4
22.9
9.2
10.0
9.5

3.6
16.2
4.3
5.1
9.1
0.6
1.6
5.3
7.6
3.0
4.5
4.1
4.6
7.0
8.3
1.3
1.8
1.4

0.18
0.33
0.18
0.24
0.29
0.18
0.20
0.22
0.24
0.17
0.46
0.44
0.14
0.12
0.08
0.17
0.15
0.19

pH

7.3
7.4
7.7
7.0
7.8
7.8
6.9
6.6
6.8
6.8
5.6
5.3
7.9
7.8
7.2
5.9
8.2
7.8

C%
(total)

2.7
7.4
2.4
4.2
2.1
0.6
1.0
1.0
1.4
0.2
1.5
1.5
11.7
12.7
15.3
0.2
8.9
8.6

C%
(organic)

0.73
6.83
2.25
4.25
1.47
0.59
0.95
1.00
1.40
0.15
1.50
1.50
1.96
4.30
6.21
0.15
0.30
0.81

Total N
(mg gÿ1)

400
1000
300
300
800
200
500
600
1000
200
800
900
300
400
300
200
200
200

Avail P
(mg gÿ1)

1.3
2.6
4.6
9.5
5.6
12.0
2.0
2.0
2.3
2.6
6.1
6.1
0.0
0.0
0.3
3.8
1.0
6.9

Metals
(mg gÿ1)
Cu

Ni

Co

Pb

Zn

Cr

As

20.2
15.9
9.6
11.4
20.2
16.2
5.9
8.0
11.6
24.8
34.6
27.0
5.1
5.0
8.4
18.2
7.1
14.2

21.2
21.9
14.3
22.0
22.6
13.7
5.9
5.8
8.7
27.2
28.5
27.9
5.7
11.9
6.2
27.2
0.0
0.0

0.0
8.5
6.4
0.0
7.8
5.7
7.8
8.7
8.7
7.8
10.0
9.2
0.0
0.0
0.0
6.8
0.0
0.0

16
21
11
1443
23
0
135
10
0
10
10
0
10
40
36
20
0
103

60
108
59
514
92
33
51
60
53
49
92
89
22
90
68
33
35
67

27
0
24
30
0
0
32
27
42
87
65
64
0
26
0
72
0
0

5.1
12.9
14.8
7.5
12.8
18.7
0.9
1.0
0.9
0.4
18.8
16.6
1.7
2.0
1.7
0.8
2.2
1.7

TPH
(mg gÿ1)

Lag
(day)

Tmid
(day)

k
(dayÿ1)

Ymax
(%)

11500
25200
8020
24100
1230
115
9830
4400
2020
250
2060
9120
14400
12600
46000
5370
195
6390

5.0
2.0
7.4
6.0
4.0
11.4
5.6
4.2
2.6
7.5
5.0
4.0
4.9
7.7
2.3
11.0
13.2
7.0

35
11
22
29
17
27
31
28
23
12
15
21
16
20
12
49
25
13

0.07
0.34
0.31
0.17
0.21
0.15
0.12
0.14
0.15
0.60
0.27
0.06
0.51
0.37
0.38
0.08
0.13
0.47

66
43
44
51
30
59
56
60
61
40
39
64
37
41
55
61
34
29

W.W. Mohn, G.R. Stewart / Soil Biology & Biochemistry 32 (2000) 1161±1172

Table 1
Characterization of soil samples and kinetic parameters for dodecane mineralization in those soil samples

1163

1164

W.W. Mohn, G.R. Stewart / Soil Biology & Biochemistry 32 (2000) 1161±1172

started at 408 C for 2 min, increased at 308 C minÿ1 to
3008 C, and remained at that temperature for 20 min.
The peak area sum for the hydrocarbon range was
quanti®ed using a standard curve for Jet-A1 fuel.
2.2. Microcosms
Soil microcosms were prepared in 130  20 mm
screw cap culture tubes with caps lined with PTFEfaced rubber. To each tube, 0.45 mCi of dodecane1-14C (Sigma-Aldrich Canada, Oakville, Ontario), dissolved in 4.3 ml of ®lter sterilized Jet-A1 fuel, was
added. Then, 3.0 g (dry weight) of moist soil was
added and the tube was rolled and gently shaken to
mix the soil and label. Then, a solution containing N
and P salts was added in order to bring the water content of the soil to between 50% and 90% (usually 50±
60%) water-holding capacity, the pH 6.5±7.5. Unless
otherwise indicated, N and P were added as ammonium chloride and sodium phosphate at respective
concentrations in soil water of 200 and 23 mM, which
were equivalent in the soils tested to from 130 to 900
mg N and from 30 to 210 mg P gÿ1 of dry soil. Because
the soils di€ered greatly in water-holding capacity, this
method of N and P addition was chosen to avoid very
di€erent salt concentrations in the water phase which
would have resulted if constant amounts of N and P
gÿ1 soil were added. A plastic 10  75 mm tube containing 0.5 ml of 0.5 M NaOH was placed inside the
culture tube to trap CO2. The culture tubes were then
sealed with screw caps and incubated, unless otherwise
speci®ed, at 78 C. The NaOH solution in the inner
tube was replaced periodically. The solution removed
was added to 5 ml of Beckman Ready Gel scintillation
cocktail and counted in a Beckman LS6000IC counter.
All microcosms with 14C-labeled hydrocarbons were in
triplicate, and the data shown are means of the triplicates. Where mentioned, ``signi®cant'' di€erences
between treatments were determined using Student's ttest …P < 0:05).

measure …w 2 † above 0.2. Three kinetic parameters were
determined for the logistic phase, the rate constant
(k ), the time of half-maximal mineralization …tmid † and
the maximal extent of mineralization …Ymax ). The ®nal
phase was a slow, linear rate of 14CO2 production that
was interpreted to primarily involve ``recycling'', mineralization of 14C which had previously been incorporated into biomass. This ®nal phase was not analyzed.
When comparing treatments with the same TPH
concentration, mineralization was expressed as % conversion of 14C to CO2. When the TPH concentration
was manipulated (see Section 2.5), the mass of dodecane mineralized was calculated by assuming that
dodecane constitutes 3.3% of TPH in the fuel added.
When comparing soils with di€erent concentrations of
weathered hydrocarbons, no assumptions were made
regarding how weathered dodecane would a€ect mineralization of added labeled dodecane. The mixing of
labeled dodecane with variable and unknown amounts
of weathered dodecane would a€ect Ymax but would
not a€ect values for the kinetic parameters k and tmid :
Thus, only the latter kinetic parameters were used in
comparing soils with di€erent concentrations of weathered hydrocarbons.
Since only one C atom was labeled, 14CO2 production only proved that there was partial mineralization of the hydrocarbons. However, what is currently
known of the biochemistry of aerobic hydrocarbon

2.3. Mineralization kinetic model
Three distinct phases of hydrocarbon mineralization
(production of 14CO2) in microcosms were identi®ed.
The lag phase (lag) was de®ned as the initial time until
1% of the added 14C was detected as 14CO2. The logistic phase was de®ned by ®tting the data to a logistic
equation using Microcal Origin software (Microcal
Software, Inc., Northampton, MA):
ÿ


Y ˆ Ymax … ÿ Ymax ÿ Yinit †= 1 ‡ ek…tÿtmid †
Where Y is the extent of mineralization. Data points
were included in the logistic phase until the time when
adding further points increased the goodness-of-®t

Fig. 1. E€ect of added ammonium and phosphate on dodecane mineralization in three soils at 78C …n ˆ 3).

W.W. Mohn, G.R. Stewart / Soil Biology & Biochemistry 32 (2000) 1161±1172

1165

biodegradation suggests that complete mineralization
of linear hydrocarbons will occur when one C atom is
mineralized, and we assumed that this was the case.
Hydrocarbon mineralization was always negligible in
autoclaved control microcosms, indicating that
measured mineralization was biologically catalyzed.

SAS statistical software (SAS Institute.). In an additional experiment, the e€ect of TPH concentration
was further tested by incubating three di€erent soils
under the above standard conditions with three or
four concentrations (see Table 3) of added Jet A-1
fuel.

2.4. Experiments investigating N and P limitation

2.6. Experiment investigating temperature

Three sources of N plus P were examined in three
di€erent soils. First, we examined NH4Cl, NaH2PO4
plus Na2HPO4 as sources of N plus P in three di€erent
soils. These salts were formulated for an N to P ratio
of 13:3 g and a neutral pH. In preliminary experiments, N plus P consistently stimulated dodecane
mineralization, but this activity was inhibited by high
concentrations in soil water of 800 mM ammonium
chloride plus 92 mM sodium phosphate, which were
equivalent to 2.2 mg N and 520 mg P gÿ1 of dry soil.
Therefore, throughout this study, ammonium chloride
and sodium phosphate were added at respective concentrations in soil water of 200 and 23 mM (as
described in Section 2.2). In the ®rst experiment presented (Fig. 1), we tested whether both N and P were
required by adding ammonium chloride and sodium
phosphate individually or together, and we tested
whether dodecane mineralization was biologically catalyzed with autoclaved controls.
In a second experiment, we examined two additional
sources of N plus P, (i) urea plus diaminophosphate
(DAP) and (ii) the commercial product Inipol EAP22
(Elf Atochem, Philadelphia, PA). On the basis of soil
TPH concentration, we used recommended amounts of
these fertilizers (von Fahnestock et al., 1998). Urea
plus DAP was added in order to achieve a 100:15:1
mass ratio of C:N:P. Inipol EAP22 was added as 10%
of the mass of TPH, according to the manufacturer's
instructions. Additionally, we con®rmed the need for
addition of N plus P with controls having no added N
or P, and we con®rmed that mineralization was biologically catalyzed with autoclaved controls with FOXMa soil.

The e€ect of temperature on dodecane mineralization was determined by incubating three soils in
microcosms under the standard conditions at 78C,
158C, 228C and 308C.

2.5. Experiments investigating soil characteristics
The e€ects of soil characteristics on dodecane mineralization were examined by incubating 18 di€erent
soils (Table 1) under standardized conditions. To
make any e€ects of soil characteristics apparent, the
standard conditions were designed not to otherwise
limit dodecane mineralization. Thus, N and P were
added, the water content was adjusted and the pH was
bu€ered (Section 2.2). The relationships between 18
soil characteristics (Table 1) and three dodecane mineralization kinetic parameters (lag time, tmid , k ) were
examined by stepwise multiple regression analysis with

2.7. Experiments investigating inoculation
In experiments testing inoculation, microcosms were
inoculated with enrichment cultures grown on Jet A-1
fuel. Inocula were from soil samples used in this study.
A standard mineral medium (Bedard et al., 1986) was
used with 1 g Jet A-1 fuel lÿ1 as the sole organic substrate. The enrichment cultures were incubated on a
tube roller at 78C, and all manipulations of the cultures were done at 78C or lower. The cultures were
serially transferred three times with 1% transfers and
incubation periods of 4, 2 and 2 weeks. Then, the cultures were frozen at ÿ708C and lyophilized, with no
cryoprotectant added, to select for organisms resistant
to freezing. The enrichment cultures were revived and
maintained on the above medium. Cultures for use in
inoculating soil were frozen at ÿ708C and lyophilized,
with 6.7% skim milk plus 6.7% honey as cryoprotectants, to maximize the viability of lyophilized cells.
To test the e€ect of inoculation, three soils were
incubated in microcosms under the standard conditions after inoculation with the enrichment cultures.
Maximum inocula were added at ®nal densities of 110
mg of protein gÿ1 of dry soil for DYE-Ma and LAB2a enrichment cultures and 190 mg of protein gÿ1 of
dry soil for the FOX-MC enrichment culture. These
protein concentrations correspond to approximately
109 cells gÿ1 of dry soil, assuming that cell dry weight
is 55% protein and that individual cells have a dry
weight of 280 fg. However, it is likely that a signi®cant
fraction of the cells were killed during lyophilization,
so this cell density should be considered a maximum
value. Each inoculum was also tested at 100- and
10,000-fold lower concentrations, diluted in cryoprotectant. The amount of cryoprotectant was kept constant in each treatment, 2.0 mg of skim milk plus 2.0
mg of honey gÿ1 of dry soil. Control treatments had
cryoprotectant without cells added. A second set of
control treatments had nothing added.
To test whether the source of an enrichment culture
a€ected the kinetics of dodecane mineralization, a second experiment was done in which each of three

1166

W.W. Mohn, G.R. Stewart / Soil Biology & Biochemistry 32 (2000) 1161±1172

enrichment cultures were separately used to inoculate
two soils which were incubated under the standard
conditions. Relative to the above experiments with
inocula, the inoculum concentration in these experiments was intermediate, approximately 107 cells gÿ1
of dry soil, and the cryprotectant concentration was
100-fold lower, 20 mg skim milk plus 20 mg honey gÿ1
of dry soil.
2.8. Experiment investigating mineralization of
hexadecane and phenanthrene
To compare mineralization of hexadecane and phenanthrene to that of dodecane, microcosms were prepared as in Section 2.2, except that dodecane-1-14C,
hexadecane-1-14C or phenanthrene-9-14C were used
(Sigma-Aldrich Canada, Oakville, Ontario). Each of
three soils was tested with two or three labeled hydrocarbons. To con®rm that mineralization was biologically catalyzed, controls with each soil had labeled
dodecane added to autoclaved microcosms.
2.9. Experiment investigating TPH removal

stimulatory e€ect. Mineralization was biological since
it was consistently inhibited by autoclaving (Figs. 1
and 2) and since it was stimulated by N plus P.
Other forms of N plus P, potentially less toxic than
ammonium plus phosphate, were also tested. Urea
plus DAP was most e€ective in one soil; while, only
Inipol was e€ective in the other two soils tested
(Fig. 2). Urea plus DAP was not e€ective in the two
soils with the highest TPH concentrations, suggesting
that this mixture was toxic at the concentrations used
in those soils, equivalent to > 790 mg N and > 50 mg P
gÿ1 of dry soil. Consistent with such a toxic e€ect,
treatments with those two soils with no fertilizer added
mineralized slightly more dodecane than those with
urea plus DAP added. Neither urea plus DAP nor Inipol appeared to be appreciably more e€ective as fertilizers than ammonium chloride plus sodium phosphate;
although, this comparison is not based upon parallel
treatments.
3.2. Soil characteristics
The kinetics of dodecane mineralization varied
greatly in soils with di€erent characteristics (Fig. 3,

To compare dodecane mineralization and TPH
removal, both were simultaneously measured in parallel microcosms. This comparison was made with three
soils. Dodecane mineralization was measured in triplicate microcosms (Section 2.2). To measure TPH
removal, 12 replicate microcosms were prepared as
described in Section 2.2, except without the 14Clabelled substrate and the CO2 traps. The tubes for
measuring TPH removal were opened at least once per
week during incubation to replenish oxygen (since they
were not opened to replace CO2 trapping solution).
Periodically, single tubes for TPH removal were frozen
to stop TPH removal and stored frozen until TPH
analysis. At the end of the experiment, the frozen
tubes were thawed, and each tube (microcosm) was
extracted in its entirety for TPH analysis (Section 2.1).
Thus, there was no sub-sampling of microcosms, and
there were no replicates for TPH analysis at individual
sampling times.

3. Results
3.1. N and P limitation
Both N and P limited biological mineralization of
dodecane in Arctic tundra soils. Individually, both ammonium chloride and sodium phosphate consistently
stimulated dodecane mineralization in three uninoculated soils (Fig. 1). Mineralization rates were barely
detectable when neither N nor P were added to soils
(see Fig. 2), so ammonium plus phosphate had a large

Fig. 2. E€ect of di€erent N plus P fertilizers on dodecane mineralization in three soils at 78C …n ˆ 3).

1167

ns
0.016 (2 0.0073)
ns
ÿ0.0001 (2 0.0000)
ns
ns
S.E.: standard error of estimate; ns, not signi®cant.
a

11 (2 0.89)
0.17 (2 0.048)
ÿ29 (2 18)
Lag time
Rate
tmid

ÿ76 (2 14)
ns
ns

% Sand estimate (2 S.E.)
Total C estimate (2 S.E.)
TPH estimate (2 S.E.)
N estimate (2 S.E.)

ns
ns
0.63 (2 0.22)

0.73
0.24
0.34

0.0001
0.04
0.01

Pr > F
R2
Soil variables …x n )
Intercept (b ) estimate (2 S.E.)

Fig. 3. Dodecane mineralization in ®ve di€erent soils at 78C; curves
show best ®t of the logistic function …n ˆ 3).

Kinetic parameter ( y )

Table 1). In most cases, the kinetics of dodecane mineralization closely ®t the logistic model used. The most
salient observation from comparison of the di€erent
soils is that very great variation in the soil characteristics did not severely or consistently a€ect dodecane
mineralization. In particular, extremely high concentrations of TPH and of heavy metals had no apparent
negative e€ect on dodecane mineralization (Table 1).
Non-linear relationships between the soil characteristics and mineralization kinetics, such as optimum
values and thresholds, were not apparent in graphs of
the data (not shown).
Stepwise multiple regression analysis indicated that
certain soil characteristics could account for substantial variability in dodecane mineralization kinetic parameters (Table 2). Following are the only signi®cant
…P < 0:05† relationships found. Soil N accounted for
48% of the variability of the lag time preceding mineralization. The N value measured was the original N
concentration before ammonium was added to the
microcosms. Soil TPH accounted for an additional
25% of the variability of the lag time, and longer lag
times were associated with higher TPH values. Total C
accounted for 24% of the variability of k, and % sand
accounted for 34% of the variability of tmid :
High concentrations of added fuel inhibited dodecane mineralization in three soils, as indicated by
accompanying decreases of k and increases of tmid
(Table 3). However, TPH concentrations as high as
100 mg gÿ1 did not completely inhibit dodecane mineralization. At higher TPH concentrations, N and P
probably limited the ®nal extent of dodecane mineralization …Ymax ). In all soils with 30 mg TPH or more
gÿ1, the relative amount of C exceeded 1000:13:3
(C:N:P).

Table 2
Statistics for the regressions …y ˆ mx ‡ b or y ˆ m1 x 1 ‡ m2 x 2 ‡ b† of kinetic parameters for dodecane mineralization ( y ) as a function of one or two soil characteristics …x n † in soil microcosmsa

W.W. Mohn, G.R. Stewart / Soil Biology & Biochemistry 32 (2000) 1161±1172

1168

W.W. Mohn, G.R. Stewart / Soil Biology & Biochemistry 32 (2000) 1161±1172

Table 3
Kinetic constants for dodecane mineralization in three soils, each with di€erent amounts of Jet A-1 fuel added …n ˆ 3)
Soil

TPH (mg gÿ1)

w2

lag (day)

k (dayÿ1)

tmid (day)

Ymax (mg gÿ1)

FOX-MA

14.4
30
100
1
3
30
100
5.4
10
30
100

3.70Eÿ04
3.00Eÿ05
1.70Eÿ04
3.00Eÿ06
2.00Eÿ06
2.00Eÿ06
8.00Eÿ06
2.00Eÿ05
1.00Eÿ05
4.00Eÿ05
5.00Eÿ05

5.1
9.4
26.7
7.9
13.6
32.7
50.0
9.9
15.0
44.4
66.8

0.156
0.055
0.015
0.192
0.065
0.051
0.061
0.063
0.056
0.042
0.031

21.4
65.1
65.1
21.3
45.1
84.0
78.1
47.9
90.3
114
115

0.208
0.724
0.148
0.016
0.064
0.124
0.207
0.117
0.221
0.178
0.210

DYE-MA

LAB2-A

3.3. Temperature
The low temperature used in this study, 78C, was
clearly below the optimum for dodecane mineralization. The e€ect of temperature was consistent in three
soils (Table 4). Temperature greatly a€ected the lag
period preceding, and the rate of, mineralization.
Increasing the temperature from 78C to 158C had the
greatest e€ect; while increasing the temperature above
228C had relatively little additional e€ect. Temperature
had little e€ect on the ®nal extent of mineralization.
3.4. Inoculation of soil
Inoculation of soil with enrichment cultures (mixed
consortia) of indigenous hydrocarbon-degrading
microorganisms consistently stimulated dodecane mineralization in three soils (Fig. 4). The e€ects of inoculation were dicult to quantify, because the largest
inocula caused dodecane mineralization which did not
®t the kinetic model well. However, large inocula
clearly stimulated dodecane mineralization. With each
soil, at every timepoint, accumulation of 14CO2 was
signi®cantly greater in the treatments with the largest
inocula than in the corresponding uninoculated controls. Inocula appear to have reduced the lag time
prior to mineralization and increased the rate of mineralization. In DYE-Ma and LAB-2a soils, those with
the lowest TPH concentrations, the largest inocula

were required for a substantial e€ect. While inoculation reduced the time required for dodecane mineralization, it had less e€ect on the ®nal extent of
mineralization.
The skim milk plus honey used as cryoprotectant
for the inocula was inhibitory to dodecane mineralization (Fig. 4). With LAB-2a and FOX-Mc soils, treatments
without
cryoprotectant
accumulated
signi®cantly more 14CO2 than those with cryoprotectant during the ®rst 32 and 21 days, respectively. This
di€erence was less pronounced in DYE-Ma soil, but
from day 18 to 28, the treatment without cryoprotectant accumulated signi®cantly more 14CO2 than that
with cryoprotectant. One possibility is that these organic compounds caused rapid microbial growth
which depleted N and P required by the dodecanemineralizing micro¯ora. Thus, the smaller inocula
might have had a greater e€ect than observed, had
they contained proportionally lower amounts of cryoprotectants.
The source of an enrichment culture a€ected the
kinetics of dodecane mineralization. In LAB-2a soil,
the native consortium (i.e., the consortium enriched
from that sample) mineralized dodecane faster than
two consortia enriched from other soil samples, as
indicated by the k and tmid values (Fig. 5B, Table 5).
With LAB-2a soil, during days 18±34, the treatment
inoculated with the LAB-2a consortium accumulated
signi®cantly more 14CO2 than the closest treatment,

Table 4
Kinetic constants for dodecane mineralization in three soils incubated at four temperatures …n ˆ 3)
8C

7
15
22
30

DYE-Ma soil

FOX-Mc soil

LAB-2a soil

lag (day)

k(dayÿ1)

tmid (day)

Ymax (%)

lag (day)

k (day)

tmid (day)

Ymax (%)

lag (day)

k (dayÿ1)

tmid (day)

Ymax (%)

8.0
3.5
2.1
1.8

0.51
1.42
2.54
2.68

18.8
8.8
4.7
3.9

39
43
39
40

3.0
0.8
0.4
0.2

0.36
0.99
1.53
1.64

13.9
4.1
2.4
2.2

54
52
50
54

9.7
4.0
2.8
2.6

0.08
0.28
0.47
0.59

42.4
18.2
10.9
9.9

57
52
49
47

1169

W.W. Mohn, G.R. Stewart / Soil Biology & Biochemistry 32 (2000) 1161±1172

Fig. 4. E€ect of inocula on dodecane mineralization in three soils at
78C; approximate concentrations of inocula are given as cells gÿ1 of
dry soil; cryoprot, cryprotectant of skim milk plus honey …n ˆ 3).

Fig. 5. E€ect of inocula (approximately 107 cells gÿ1 of dry soil)
enriched from three di€erent sources on dodecane mineralization in
two soils at 78C …n ˆ 3).

that inoculated with the FOX-Mc consortium. In
DYE-Ma soil, the native consortium mineralized dodecane slower than two consortia enriched from other
soil samples (Fig. 5A, Table 5). In DYE-Ma soil, mineralization did not ®t the model well, confounding
comparisons of kinetic parameters. With DYE-Ma
soil, from day 12 onward, the treatment inoculated
with the DYE-Ma consortium accumulated signi®cantly less 14CO2 than the closest inoculated treatment,
that inoculated with the FOX-Mc consortium. In both
soils, inoculation consistently stimulated dodecane
mineralization, as in the previous experiment.

two soils, dodecane was mineralized at a higher rate
and to a greater extent than was the longer n-alkane,
hexadecane (Fig. 6B and C). The magnitude of these
di€erences varied greatly between the two soils. There
was no consistent relationship between mineralization
kinetics of dodecane and of the polyaromatic hydrocarbon, phenanthrene (Fig. 6). Thus, in di€erent soils,
the mineralization kinetics of di€erent hydrocarbons
found in Arctic diesel fuel varied independently.

3.5. Mineralization of hexadecane and phenanthrene
Throughout this study, dodecane mineralization was
used as a measure of hydrocarbon biodegradation. In

3.6. Removal of TPH
Despite the above variability in relative mineralization rates of di€erent hydrocarbons, there was a general correlation between kinetics of dodecane
mineralization and biological removal of TPH (Fig. 7).
The large variability apparent in the TPH measure-

Table 5
Kinetic constants for dodecane mineralization in two soils, each inoculated separately with three enrichment cultures …n ˆ 3)
Inoculum

DYE-Ma
FOX-Mc
LAB-2a
uninoc

DYE-Ma soil

LAB-2a soil

w2

lag (day)

k (dayÿ1)

tmid (day)

Ymax (%)

w2

lag (day)

k (dayÿ1)

tmid (day)

Ymax (%)

±
±
0.10
0.18

4.0
4.5
3.8
6.5

±
±
0.13
0.23

025
022
21.1
21.0

055
060
67
45

0.10
0.50
0.18
0.84

3.2
3.6
3.2
4.8

0.065
0.091
0.11
0.10

34.4
33.0
27.8
39.0

59
56
54
51

1170

W.W. Mohn, G.R. Stewart / Soil Biology & Biochemistry 32 (2000) 1161±1172

ments precludes detailed comparison with dodecane
mineralization. The merit of mineralization measurements is evident from this variability. The amount of
hydrocarbon initially present in, and removed from,
each soil varied greatly. It should be noted that TPH
removal did not clearly stop during tests with any of
the three soils. Thus, TPH removal may have continued after dodecane mineralization stopped. With the
higher initial TPH concentrations, N and P would
likely have eventually limited TPH removal. The above
results show that dodecane mineralization is an indicator of total hydrocarbon biodegradation, but the
kinetics of dodecane mineralization cannot be extrapolated to other hydrocarbons.

4. Discussion

Fig. 6. Mineralization of di€erent hydrocarbons in three soils at 78C
…n ˆ 3).

Fig. 7. Mineralization of dodecane …n ˆ 3† and removal of TPH
…n ˆ 1† in three soils at 78C.

We and other investigators (Agosti and Agosti,
1972; Cundell and Traxler, 1974; Bradley and Chapelle, 1995; Braddock et al., 1997; Aislabie et al., 1998)
found that polar regions typically have soil micro¯ora
capable of hydrocarbon biodegradation, as do boreal
(Westlake et al., 1978) and alpine regions (Margesin
and Schinner, 1997). We (Table 4) and other investigators (Margesin and Schinner, 1997; Whyte et al.,
1999) found evidence that these hydrocarbon-degrading soil communities are collectively psychrotolerant,
quite active at low temperatures (4±78C), but with
higher optimal temperatures (15±308C). This observation is consistent with reports that microbial populations in cold environments include mainly
psychrotolerant, as opposed to mainly psychrophilic,
organisms (reviewed in Gounot, 1991). Accordingly, a
number of psychrotolerant hydrocarbon-degrading
bacterial isolates have been reported (Kolenc et al.,
1988; Kotturi et al., 1991; Whyte et al., 1996; Master
and Mohn, 1998). From an applied perspective, heating appears to be an e€ective means to accelerate bioremediation of hydrocarbon-contaminated Arctic
tundra soils. However, heating to a moderate temperature (158C), rather than to common laboratory incubation temperatures (> 258C), may provide the
greatest bene®t relative to the expense of heating.
The hydrocarbon-degrading soil micro¯ora of polar
regions are limited by N and P, as are such micro¯ora
in warmer regions. Addition of N plus P stimulated
hydrocarbon degradation in this and another study
(Braddock et al., 1997) with Arctic tundra soils and in
a study with Antarctic tundra soils (Aislabie et al.,
1998). In this study, addition of both N and P was
necessary for optimal dodecane mineralization, and
omission of either resulted in greatly reduced dodecane
mineralization (Figs. 1 and 2). In our study and the
study of Braddock et al. (1997), high concentrations of
fertilizers inhibited hydrocarbon biodegradation.

W.W. Mohn, G.R. Stewart / Soil Biology & Biochemistry 32 (2000) 1161±1172

Degradation of the high TPH concentrations that we
found at some Arctic sites, greater than 50 mg gÿ1 of
dry soil, probably requires more N and P than can be
added at once without toxic e€ects.
The soil characteristics we examined had limited predictive value for hydrocarbon bioremediation. In particular, N and TPH values together accounted for
73% of the variability of lag time prior to dodecane
mineralization (Table 2). Soil characteristics had much
less value in predicting mineralization kinetics following the lag period. Thus, an empirical test appears
necessary to predict the kinetics of hydrocarbon
removal from a particular soil. On the other hand, no
soil characteristics that we examined precluded biodegradation. In particular, high TPH concentration and
extensive heavy metal co-contamination did not prevent hydrocarbon biodegradation (Table 1). Aislabie et
al. (1998). concluded that 50 mg of organic lead gÿ1 of
soil as a co-contaminant prevented development of
fuel-degrading micro¯ora in one Antarctic tundra soil.
However, we observed that up to 1440 mg of total lead
gÿ1 of soil did not prevent dodecane mineralization.
We found that one intrinsic soil property, high sand
content, was associated with high tmid values (slower
mineralization) but did not prevent biodegradation
(Table 2). All the soils tested were extremely sandy.
Our study indicates that native micro¯ora are able to
adapt to the range of conditions in the soils examined
and suggests that hydrocarbon-contaminated Arctic
tundra soils can generally be bioremediated.
It is of interest that higher soil N content values,
prior to addition of ammonium, were related to
shorter lag times preceding dodecane mineralization
(Table 2). A plausible explanation is that the original
N content is an indicator of biomass. Thus, higher N
contents may have been associated with higher populations of hydrocarbon-degrading microorganisms.
Because of the wide range of TPH concentration,
ratios of C:N:P varied greatly, from 4000:13:3 to
8.8:13:3. In many cases, the amounts of N and P available would not be expected to allow complete mineralization of the TPH. However, the extent of
mineralization of labeled hydrocarbons …Ymax † was
usually near the maximum expected value of 50%
(Table 1). This expected value assumes that aerobic
chemoorganotrophs will convert approximately equal
amounts of organic C consumed to CO2 and biomass.
One possible explanation for the apparently complete
mineralization of dodecane in these experiments is that
n-dodecane is less recalcitrant than other hydrocarbons
present and was mineralized before N and P limitation
had an e€ect. Branched hydrocarbons were shown to
be more recalcitrant than linear hydrocarbons in a
mixed microbial culture (Geerdink et al., 1996). It is
also possible that the labeled dodecane was more bioavailable than weathered hydrocarbons, further increas-

1171

ing its mineralization rate relative to the other
hydrocarbons present.
The relative degradability of labeled dodecane added
to soil is an important consideration in interpreting
results from our study. Obviously, the kinetics of
labeled dodecane mineralization cannot be directly extrapolated to the complex, weathered mixture of
hydrocarbons in the soil. In fact, our results showed
that the mineralization kinetics of di€erent labeled
hydrocarbons varied relative to each other in di€erent
soils (Fig. 6). However, it is reasonable to conclude
that the population mineralizing labeled dodecane will
respond to environmental factors in a way similar to
the population degrading the other hydrocarbons. This
assumption is supported by the similar trends of dodecane mineralization and TPH removal observed
(Fig. 7). Bioavailability of weathered hydrocarbons is
an important factor potentially limiting their biodegradation which cannot be directly studied using added
labeled substrates. Thus, it is possible that we did not
detect the e€ect of a factor which a€ects bioavailability
of weathered hydrocarbons, but does not a€ect the
bioavailability of the added labeled dodecane.
Our study clearly indicated that inoculation accelerated dodecane mineralization in the soil systems tested.
In some cases, a large inoculum more than halved the
time required for maximum dodecane mineralization
(Fig. 4A and B). In contrast, other studies have shown
that inoculation did not further stimulate hydrocarbon
degradation in Arctic tundra soils (Whyte et al., 1999)
or alpine soils (Margesin and Schinner, 1997) to which
N plus P had been added. In general, evidence indicating that inocula stimulate hydrocarbon biodegradation
in soils or water bodies is extremely rare. We conclude
that inoculation can reduce the time required for bioremediation of particular soils, including hydrocarboncontaminated soils typical of radar stations on the
Arctic tundra. However, the value of inoculation
requires consideration of several factors, including
logistic and economic ones. The very short summer
period when Arctic tundra soils are unfrozen may
make bioremediation rate increases of practical importance. However, alternative means, such as heating,
may be more cost-e€ective ways to accelerate bioremediation.

Acknowledgements
We thank the Environmental Science Group, Royal
Military College, for collecting and transporting soil
samples. We thank Dr. Marie-Claude Fortin for assistance with statistical analyses. This work was supported
by a Strategic Grant from the National Science and
Engineering Research Council of Canada.

1172

W.W. Mohn, G.R. Stewart / Soil Biology & Biochemistry 32 (2000) 1161±1172

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