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 dierent 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 eective 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 gÿ1of 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.72000 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 veg-etation zone) are among those contaminated sites and are designated for remediation. While a number of stu-dies have examined biodegradation of hydrocarbons in Arctic marine environments (reviewed in Swannell et
al., 1996), very few have examined biodegradation of hydrocarbons in Arctic tundra soils (Braddock et al., 1997; Whyte et al., 1999).
Polar soil environments dier from other soil en-vironments in a number of ways that might aect hy-drocarbon biodegradation. Polar soils have unique periglacial features, including permafrost and numer-ous types of patterns primarily due to freeze±thaw eects (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. Sum-mer 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
Soil Biology & Biochemistry 32 (2000) 1161±1172
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* Corresponding author. Tel.: 4285; fax: +1-604-822-6041.
and is presumably the site of most biological activity. Little is known about the relationships between perma-frost and hydrocarbon contaminants. Many Arctic tundra soils are low in organic content, a characteristic which can be expected to directly aect sorption of hydrocarbons to the soil and to indirectly aect biode-gradation of hydrocarbons.
Arctic tundra soil microbial communities have not been well characterized. There is presently no convin-cing 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 de-composition is slow in Arctic tundra soils, largely due to low temperatures. Some studies indicate the pre-sence 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 com-plex eects on hydrocarbon biodegradation in soil (Bossert and Compeau, 1995). For this reason, experts frequently recommend that soil bioremediation pro-jects 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 cer-tain 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 micro-cosms with 18 soil samples from across the Canadian Arctic tundra. We determined the eects on biodegra-dation 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 con-ditions (temperature, N and P content) and (iv) inocu-lation 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 bioremedia-tion of Arctic tundra soils. This is the ®rst such
com-prehensive study characterizing hydrocarbon
biodegradation in Arctic tundra soils. Unlike most stu-dies 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 refriger-ated 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 PTFE-faced rubber, 3 g (dry weight) soil samples were sha-ken 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 tempera-ture, left overnight and shaken once again. After the soil settled, portions of the hexane phases were centri-fuged (12,000g), 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
Table 1
Characterization of soil samples and kinetic parameters for dodecane mineralization in those soil samples
Soil % Composition WHC
(g gÿ1)
pH C%
(total) C% (organic)
Total N (mg gÿ1)
Avail P (mg gÿ1)
Metals (mg gÿ1)
TPH (mg gÿ1)
Lag (day)
Tmid
(day)
k
(dayÿ1)
Ymax
(%)
Gravel Sand Silt Clay Cu Ni Co Pb Zn Cr As
BAR-1a 50.0 85.2 11.2 3.6 0.18 7.3 2.7 0.73 400 1.3 20.2 21.2 0.0 16 60 27 5.1 11500 5.0 35 0.07 66
BAR-4a 15.1 68.7 15.1 16.2 0.33 7.4 7.4 6.83 1000 2.6 15.9 21.9 8.5 21 108 0 12.9 25200 2.0 11 0.34 43
BAR-4b 26.0 88.8 6.9 4.3 0.18 7.7 2.4 2.25 300 4.6 9.6 14.3 6.4 11 59 24 14.8 8020 7.4 22 0.31 44
BAR-4c 11.0 89.6 5.3 5.1 0.24 7.0 4.2 4.25 300 9.5 11.4 22.0 0.0 1443 514 30 7.5 24100 6.0 29 0.17 51
BAR-4d 13.8 78.2 12.7 9.1 0.29 7.8 2.1 1.47 800 5.6 20.2 22.6 7.8 23 92 0 12.8 1230 4.0 17 0.21 30
BAR-4e 19.5 95.2 4.2 0.6 0.18 7.8 0.6 0.59 200 12.0 16.2 13.7 5.7 0 33 0 18.7 115 11.4 27 0.15 59
CAM-4a 58.8 93.7 4.7 1.6 0.20 6.9 1.0 0.95 500 2.0 5.9 5.9 7.8 135 51 32 0.9 9830 5.6 31 0.12 56
CAM-4b 40.4 82.4 12.3 5.3 0.22 6.6 1.0 1.00 600 2.0 8.0 5.8 8.7 10 60 27 1.0 4400 4.2 28 0.14 60
CAM-4c 31.6 81.2 11.2 7.6 0.24 6.8 1.4 1.40 1000 2.3 11.6 8.7 8.7 0 53 42 0.9 2020 2.6 23 0.15 61
DYE-Ma 17.8 83.8 14.2 3.0 0.17 6.8 0.2 0.15 200 2.6 24.8 27.2 7.8 10 49 87 0.4 250 7.5 12 0.60 40
FOX-Ba 7.0 68.3 27.2 4.5 0.46 5.6 1.5 1.50 800 6.1 34.6 28.5 10.0 10 92 65 18.8 2060 5.0 15 0.27 39
FOX-Bb 11.8 75.0 20.9 4.1 0.44 5.3 1.5 1.50 900 6.1 27.0 27.9 9.2 0 89 64 16.6 9120 4.0 21 0.06 64
FOX-Ma 45.0 77.0 18.4 4.6 0.14 7.9 11.7 1.96 300 0.0 5.1 5.7 0.0 10 22 0 1.7 14400 4.9 16 0.51 37
FOX-Mb 45.3 68.6 24.4 7.0 0.12 7.8 12.7 4.30 400 0.0 5.0 11.9 0.0 40 90 26 2.0 12600 7.7 20 0.37 41
FOX-Mc 39.5 68.8 22.9 8.3 0.08 7.2 15.3 6.21 300 0.3 8.4 6.2 0.0 36 68 0 1.7 46000 2.3 12 0.38 55
LAB-2a 29.5 89.5 9.2 1.3 0.17 5.9 0.2 0.15 200 3.8 18.2 27.2 6.8 20 33 72 0.8 5370 11.0 49 0.08 61
PIN-Mb 31.8 88.2 10.0 1.8 0.15 8.2 8.9 0.30 200 1.0 7.1 0.0 0.0 0 35 0 2.2 195 13.2 25 0.13 34
PIN-Mc 19.9 89.1 9.5 1.4 0.19 7.8 8.6 0.81 200 6.9 14.2 0.0 0.0 103 67 0 1.7 6390 7.0 13 0.47 29
W.W.
Mohn,
G.R.
Stewart
/
Soil
Biology
&
Biochemis
try
32
(2000)
1161±1172
started at 408C for 2 min, increased at 308C minÿ1to 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
PTFE-faced rubber. To each tube, 0.45 mCi of
dodecane-1-14C (Sigma-Aldrich Canada, Oakville, Ontario), dis-solved 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 con-tent 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 am-monium 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 210mg P gÿ1of dry soil. Because the soils diered greatly in water-holding capacity, this method of N and P addition was chosen to avoid very dierent salt concentrations in the water phase which would have resulted if constant amounts of N and P gÿ1 soil were added. A plastic 1075 mm tube con-taining 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 with14C-labeled hydrocarbons were in triplicate, and the data shown are means of the
tripli-cates. Where mentioned, ``signi®cant'' dierences
between treatments were determined using Student's t-test P<0:05).
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 added14C was detected as14CO2. The logis-tic phase was de®ned by ®tting the data to a logislogis-tic equation using Microcal Origin software (Microcal Software, Inc., Northampton, MA):
YYmax ÿYmaxÿYinit=
ÿ
1ek 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
measure w2above 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 of14CO2production that was interpreted to primarily involve ``recycling'', min-eralization of 14C which had previously been incorpor-ated into biomass. This ®nal phase was not analyzed.
When comparing treatments with the same TPH concentration, mineralization was expressed as % con-version of 14C to CO2. When the TPH concentration was manipulated (see Section 2.5), the mass of do-decane mineralized was calculated by assuming that dodecane constitutes 3.3% of TPH in the fuel added. When comparing soils with dierent concentrations of weathered hydrocarbons, no assumptions were made regarding how weathered dodecane would aect min-eralization of added labeled dodecane. The mixing of labeled dodecane with variable and unknown amounts
of weathered dodecane would aect Ymax but would
not aect values for the kinetic parameters k and tmid:
Thus, only the latter kinetic parameters were used in comparing soils with dierent concentrations of weath-ered hydrocarbons.
Since only one C atom was labeled, 14CO2
pro-duction only proved that there was partial mineraliz-ation of the hydrocarbons. However, what is currently known of the biochemistry of aerobic hydrocarbon
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.
2.4. Experiments investigating N and P limitation
Three sources of N plus P were examined in three dierent soils. First, we examined NH4Cl, NaH2PO4 plus Na2HPO4as sources of N plus P in three dierent soils. These salts were formulated for an N to P ratio of 13:3 g and a neutral pH. In preliminary exper-iments, 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 con-centrations in soil water of 200 and 23 mM (as described in Section 2.2). In the ®rst experiment pre-sented (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 cata-lyzed 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 bio-logically catalyzed with autoclaved controls with FOX-Ma soil.
2.5. Experiments investigating soil characteristics
The eects of soil characteristics on dodecane miner-alization were examined by incubating 18 dierent soils (Table 1) under standardized conditions. To make any eects 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 buered (Section 2.2). The relationships between 18 soil characteristics (Table 1) and three dodecane miner-alization kinetic parameters (lag time, tmid, k) were
examined by stepwise multiple regression analysis with
SAS statistical software (SAS Institute.). In an ad-ditional experiment, the eect of TPH concentration was further tested by incubating three dierent soils under the above standard conditions with three or four concentrations (see Table 3) of added Jet A-1 fuel.
2.6. Experiment investigating temperature
The eect of temperature on dodecane mineraliz-ation was determined by incubating three soils in
microcosms under the standard conditions at 78C,
158C, 228C and 308C.
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 sub-strate. The enrichment cultures were incubated on a tube roller at 78C, and all manipulations of the cul-tures 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 cul-tures 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 cryoprotec-tants, to maximize the viability of lyophilized cells.
To test the eect of inoculation, three soils were incubated in microcosms under the standard con-ditions 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 LAB-2a 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 gÿ1of 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 cryopro-tectant. The amount of cryoprotectant was kept con-stant 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 aected the kinetics of dodecane mineralization, a sec-ond experiment was done in which each of three
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 exper-iments 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 phe-nanthrene to that of dodecane, microcosms were pre-pared 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 hydro-carbons. To con®rm that mineralization was biologi-cally catalyzed, controls with each soil had labeled dodecane added to autoclaved microcosms.
2.9. Experiment investigating TPH removal
To compare dodecane mineralization and TPH removal, both were simultaneously measured in paral-lel microcosms. This comparison was made with three soils. Dodecane mineralization was measured in tripli-cate microcosms (Section 2.2). To measure TPH removal, 12 replicate microcosms were prepared as
described in Section 2.2, except without the 14
C-labelled 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 am-monium chloride and sodium phosphate consistently stimulated dodecane mineralization in three uninocu-lated 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
stimulatory eect. 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 eective in one soil; while, only Inipol was eective in the other two soils tested (Fig. 2). Urea plus DAP was not eective in the two soils with the highest TPH concentrations, suggesting that this mixture was toxic at the concentrations used in those soils, equivalent to > 790mg N and > 50mg P gÿ1 of dry soil. Consistent with such a toxic eect, 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 Ini-pol appeared to be appreciably more eective as fertili-zers 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 dierent characteristics (Fig. 3,
Table 1). In most cases, the kinetics of dodecane min-eralization closely ®t the logistic model used. The most salient observation from comparison of the dierent soils is that very great variation in the soil character-istics did not severely or consistently aect dodecane mineralization. In particular, extremely high concen-trations of TPH and of heavy metals had no apparent negative eect on dodecane mineralization (Table 1). Non-linear relationships between the soil character-istics 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 substan-tial variability in dodecane mineralization kinetic par-ameters (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 miner-alization. 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 ofk, and % sand accounted for 34% of the variability oftmid:
High concentrations of added fuel inhibited do-decane 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 gÿ1did not completely inhibit dodecane miner-alization. At higher TPH concentrations, N and P probably limited the ®nal extent of dodecane mineral-ization Ymax). In all soils with 30 mg TPH or more
gÿ1, the relative amount of C exceeded 1000:13:3
(C:N:P).
Fig. 3. Dodecane mineralization in ®ve dierent soils at 78C; curves show best ®t of the logistic function n3).
3.3. Temperature
The low temperature used in this study, 78C, was clearly below the optimum for dodecane mineraliz-ation. The eect of temperature was consistent in three soils (Table 4). Temperature greatly aected the lag period preceding, and the rate of, mineralization. Increasing the temperature from 78C to 158C had the greatest eect; while increasing the temperature above 228C had relatively little additional eect. Temperature had little eect 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 min-eralization in three soils (Fig. 4). The eects of inocu-lation were dicult 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 con-trols. Inocula appear to have reduced the lag time prior to mineralization and increased the rate of min-eralization. In DYE-Ma and LAB-2a soils, those with the lowest TPH concentrations, the largest inocula
were required for a substantial eect. While inocu-lation reduced the time required for dodecane mineral-ization, it had less eect on the ®nal extent of mineralization.
The skim milk plus honey used as cryoprotectant for the inocula was inhibitory to dodecane mineraliz-ation (Fig. 4). With LAB-2a and FOX-Mc soils,
treat-ments without cryoprotectant accumulated
signi®cantly more 14CO2 than those with cryoprotec-tant during the ®rst 32 and 21 days, respectively. This dierence was less pronounced in DYE-Ma soil, but from day 18 to 28, the treatment without cryoprotec-tant accumulated signi®cantly more 14CO2 than that with cryoprotectant. One possibility is that these or-ganic compounds caused rapid microbial growth which depleted N and P required by the dodecane-mineralizing micro¯ora. Thus, the smaller inocula might have had a greater eect than observed, had they contained proportionally lower amounts of cryo-protectants.
The source of an enrichment culture aected 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 3
Kinetic constants for dodecane mineralization in three soils, each with dierent amounts of Jet A-1 fuel added n3)
Soil TPH (mg gÿ1) w2 lag (day) k(dayÿ1) tmid(day) Ymax(mg gÿ1)
FOX-MA 14.4 3.70Eÿ04 5.1 0.156 21.4 0.208
30 3.00Eÿ05 9.4 0.055 65.1 0.724
100 1.70Eÿ04 26.7 0.015 65.1 0.148
DYE-MA 1 3.00Eÿ06 7.9 0.192 21.3 0.016
3 2.00Eÿ06 13.6 0.065 45.1 0.064
30 2.00Eÿ06 32.7 0.051 84.0 0.124
100 8.00Eÿ06 50.0 0.061 78.1 0.207
LAB2-A 5.4 2.00Eÿ05 9.9 0.063 47.9 0.117
10 1.00Eÿ05 15.0 0.056 90.3 0.221
30 4.00Eÿ05 44.4 0.042 114 0.178
100 5.00Eÿ05 66.8 0.031 115 0.210
Table 4
Kinetic constants for dodecane mineralization in three soils incubated at four temperatures n3)
8C DYE-Ma soil FOX-Mc soil LAB-2a soil
lag (day) k(dayÿ1) t
mid(day) Ymax(%) lag (day) k(day) tmid(day) Ymax(%) lag (day) k(dayÿ1) tmid(day) Ymax(%)
7 8.0 0.51 18.8 39 3.0 0.36 13.9 54 9.7 0.08 42.4 57
15 3.5 1.42 8.8 43 0.8 0.99 4.1 52 4.0 0.28 18.2 52
22 2.1 2.54 4.7 39 0.4 1.53 2.4 50 2.8 0.47 10.9 49
that inoculated with the FOX-Mc consortium. In DYE-Ma soil, the native consortium mineralized do-decane slower than two consortia enriched from other soil samples (Fig. 5A, Table 5). In DYE-Ma soil, min-eralization 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 less14CO2than the closest inoculated treatment, that inoculated with the FOX-Mc consortium. In both soils, inoculation consistently stimulated dodecane mineralization, as in the previous experiment.
3.5. Mineralization of hexadecane and phenanthrene
Throughout this study, dodecane mineralization was used as a measure of hydrocarbon biodegradation. In
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 dierences varied greatly between the two soils. There was no consistent relationship between mineralization kinetics of dodecane and of the polyaromatic hydro-carbon, phenanthrene (Fig. 6). Thus, in dierent soils, the mineralization kinetics of dierent hydrocarbons found in Arctic diesel fuel varied independently.
3.6. Removal of TPH
Despite the above variability in relative mineraliz-ation rates of dierent hydrocarbons, there was a
gen-eral correlation between kinetics of dodecane
mineralization and biological removal of TPH (Fig. 7). The large variability apparent in the TPH
measure-Fig. 4. Eect of inocula on dodecane mineralization in three soils at 78C; approximate concentrations of inocula are given as cells gÿ1of dry soil; cryoprot, cryprotectant of skim milk plus honey n3).
Fig. 5. Eect of inocula (approximately 107 cells gÿ1 of dry soil) enriched from three dierent sources on dodecane mineralization in two soils at 78C n3).
Table 5
Kinetic constants for dodecane mineralization in two soils, each inoculated separately with three enrichment cultures n3)
Inoculum DYE-Ma soil LAB-2a soil
w2 lag (day) k(dayÿ1) tmid(day) Ymax(%) w2 lag (day) k(dayÿ1) tmid(day) Ymax(%)
DYE-Ma ± 4.0 ± 025 055 0.10 3.2 0.065 34.4 59
FOX-Mc ± 4.5 ± 022 060 0.50 3.6 0.091 33.0 56
LAB-2a 0.10 3.8 0.13 21.1 67 0.18 3.2 0.11 27.8 54
uninoc 0.18 6.5 0.23 21.0 45 0.84 4.8 0.10 39.0 51
ments precludes detailed comparison with dodecane mineralization. The merit of mineralization measure-ments 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 contin-ued 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 indi-cator of total hydrocarbon biodegradation, but the kinetics of dodecane mineralization cannot be extrapo-lated to other hydrocarbons.
4. Discussion
We and other investigators (Agosti and Agosti, 1972; Cundell and Traxler, 1974; Bradley and Cha-pelle, 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 investi-gators (Margesin and Schinner, 1997; Whyte et al., 1999) found evidence that these hydrocarbon-degrad-ing soil communities are collectively psychrotolerant, quite active at low temperatures (4±78C), but with higher optimal temperatures (15±308C). This obser-vation is consistent with reports that microbial
popu-lations 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, heat-ing appears to be an eective means to accelerate
bior-emediation of hydrocarbon-contaminated Arctic
tundra soils. However, heating to a moderate tempera-ture (158C), rather than to common laboratory
incu-bation 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.
Fig. 6. Mineralization of dierent hydrocarbons in three soils at 78C
n3).
Fig. 7. Mineralization of dodecane n3 and removal of TPH
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 eects.
The soil characteristics we examined had limited pre-dictive value for hydrocarbon bioremediation. In par-ticular, 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 follow-ing 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 biode-gradation. In particular, high TPH concentration and extensive heavy metal co-contamination did not pre-vent hydrocarbon biodegradation (Table 1). Aislabie et al. (1998). concluded that 50 mg of organic lead gÿ1of soil as a co-contaminant prevented development of fuel-degrading micro¯ora in one Antarctic tundra soil. However, we observed that up to 1440mg 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 popu-lations 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 avail-able would not be expected to allow complete
mineral-ization 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 eect. 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 bioa-vailable than weathered hydrocarbons, further
increas-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 ex-trapolated to the complex, weathered mixture of hydrocarbons in the soil. In fact, our results showed that the mineralization kinetics of dierent labeled hydrocarbons varied relative to each other in dierent 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 do-decane mineralization and TPH removal observed (Fig. 7). Bioavailability of weathered hydrocarbons is an important factor potentially limiting their biodegra-dation which cannot be directly studied using added labeled substrates. Thus, it is possible that we did not detect the eect of a factor which aects bioavailability of weathered hydrocarbons, but does not aect the bioavailability of the added labeled dodecane.
Our study clearly indicated that inoculation acceler-ated 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 indicat-ing that inocula stimulate hydrocarbon biodegradation in soils or water bodies is extremely rare. We conclude that inoculation can reduce the time required for bior-emediation of particular soils, including hydrocarbon-contaminated 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 im-portance. However, alternative means, such as heating, may be more cost-eective ways to accelerate bioreme-diation.
Acknowledgements
We thank the Environmental Science Group, Royal Military College, for collecting and transporting soil samples. We thank Dr. Marie-Claude Fortin for assist-ance with statistical analyses. This work was supported by a Strategic Grant from the National Science and Engineering Research Council of Canada.
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