Cold Regions Science and Technology 96 (2013) 122–128

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Cold Regions Science and Technology

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A feasibility study on the bioremediation of hydrocarbon-contaminated

soil from an Alpine former military site: Effects of
temperature and biostimulation
J. Mair, F. Schinner, R. Margesin ⁎
Institute of Microbiology, University of Innsbruck, Technikerstrasse 25, A-6020 Innsbruck, Austria

a r t i c l e i n f o a b s t r a c t

Article history: A laboratory feasibility study on the bioremediation of hydrocarbon-contaminated soil from an Alpine former
Received 11 November 2012 military site was conducted over a period of 30 weeks. We determined the effects of temperature (10 °C and
Revised 4 July 2013 20 °C) and of various biostimulation treatments (inorganic nitrogen–phosphorus–potassium fertilization and
Accepted 20 July 2013
the two commercial products Inipol EAP22 and Terramend) versus natural attenuation on the loss of total petro-
leum hydrocarbons (TPH), microbial activity (soil respiration) and community composition (phospholipid fatty
Keywords:
Soil
acids, PLFA). The hydrocarbon contamination was removed almost completely (up to 92.7%) at 20 °C, while at
Hydrocarbons 10 °C losses up to 69% were obtained. Biostimulation by the addition of nutrients had a significantly stimulating
Alpine effect on the biodegradation activity of the indigenous soil microorganisms; however, a considerable amount of
Bacteria hydrocarbon loss could be attributed to natural attenuation. Shifts in microbial community composition during
Fungi bioremediation included the significant increase of soil fungi at 10 °C and of Gram-negative soil bacteria at
Fertilization 20 °C. Significantly positive correlations between hydrocarbon loss, soil respiration and patterns of phos-
pholipid fatty acids demonstrated the involvement of a wide range of soil microorganisms (Gram-positive
and Gram-negative bacteria, fungi) in the bioremediation of the investigated soil.
© 2013 Elsevier B.V. All rights reserved.

1. Introduction this strategy generally underperformed or gave no better results than

Petroleum hydrocarbons are the most widespread contaminants in
the environment. A number of methods, including physical, chemical
and biological treatments, are available for the treatment of petroleum
contamination (Ma and Jiao, 2012; Singh and Ward, 2004; van Hamme
et al., 2003). Contaminants can be degraded or otherwise be made
harmless by natural processes (natural attenuation) that include bio-
degradation by indigenous microorganisms and a number of abiotic
processes, such as dispersion, sorption and transformations (Hinchee,
1998). Bioremediation attempts to accelerate the natural biodegrada-
tion rates through the optimization of limiting environmental condi-
tions and is an ecologically and economically effective method. The
basis for the development of biological remediation methods is the ca-
pacity of a broad spectrum of microorganisms to utilize hydrocarbons
as the sole source of carbon and energy (biodegradation) and has
been described already very early (ZoBell, 1946). The most widely
used bioremediation procedure in cold soils is biostimulation of the in-
digenous microorganisms by supplementation of appropriate nutrients
and optimization of other limiting factors, such as oxygen content, pH
and temperature control. Bioaugmentation by inoculating allochtho-
nous hydrocarbon degraders has been used as a bioremediation option
to treat petroleum-contaminated cold and temperate sites. However,
⁎ Corresponding author. Tel.: +43 512 5076021; fax: +43 512 5072929.
E-mail address: rosa.margesin@uibk.ac.at (R. Margesin).
fertilization (Filler et al., 2009; Margesin, 2004; Tyagi et al., 2011).
Cold environments are increasingly exposed to petroleum explora-
tion, production and transport, and these activities increase the risk of
accidental oil release. Such environments include polar and alpine
soils, permafrost, sediments, sea, and also subsoils and groundwaters
of temperate climates where temperatures rarely exceed 10 °C. In soils,
deep penetration of hydrocarbons from the topsoils into the subsoils
represents a direct risk of groundwater contamination. Successful
bioremediation of hydrocarbon-contaminated soils in various cold
environments, including the Arctic, Antarctic and Alpine areas, has
been reported (Bej et al., 2010; Brakstad et al., 2008; Filler et al.,
2008; Greer et al., 2010). Several remediation schemes, such as
biopiles, landfarming and engineered bioremediation have been
implemented successfully at petroleum-contaminated cold sites
(e.g. Filler et al., 2006, 2009; Paudyn et al., 2008; Walworth and
Ferguson, 2008).
To evaluate the performance of microorganisms during hydrocarbon
bioremediation, monitoring methods are applied. Soil biological activi-
ties, e.g. soil respiration and enzyme activities, give information on the
impact of the effects of environmental stress, such as contamination,
on soil metabolic activity (Chakraborty et al., 2012; Margesin and
Schinner, 2005). Direct, non-culture-based, molecular methods, e.g. soil
DNA or phospholipid fatty acid (PLFA) profiling, provide information
on the microbial community structure (Chakraborty et al., 2012;
Macnaughton et al., 1999; Zelles, 1999). Specific PLFA patterns re-
flect the physiological stress, the nutritional status and the viable

0165-232X/$ – see front matter © 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.coldregions.2013.07.006
 J. Mair et al. / Cold Regions Science and Technology 96 (2013) 122–128
biomass of the microbial population (Macnaughton et al., 1999;
Schinner et al., 1996; Zelles, 1999).
It was the objective of this study to investigate the feasibility of
bioremediation of petroleum–hydrocarbon-contaminated soil collected
from an Alpine former military site. In a laboratory study, we investigat-
ed the natural attenuation and determined the effect of biostimulation
by applying three different fertilizers. Changes in the hydrocarbon con-
centration, microbial activity (respiration) and community composition
(PLFA patterns) were monitored over a period of 30 weeks at 10 °C and
at 20 °C. These two temperatures correspond to the mean annual soil
temperature in the sampling area (10 °C) and to the temperature that
the soil would be manipulated to as part of the bioremediation treat-
ment (20 °C).
2. Materials and methods
2.1. Sampling site and soil sampling
Soil samples were collected from a former military site in October
2010. The site was located in the European Alpine region in Welsberg-
Taisten (46° 45′ 10.57″ N; 12° 06′ 47.39″ E), South Tyrol, Italy. The
area used to be a military site until 1990 . It had a size of 20,000 m2
and hosted 21 buildings (caserns, storage rooms, shooting stand,
garages, etc.). After the removal of 11 underground storage tanks
for heating oil, diesel oil and gasoline, a preliminary examination of
soil samples pointed to petroleum hydrocarbon contamination.
Since the area is within a drinking water protection area and thus
in an especially environmentally sensitive area, the authorities de-
cided to examine the feasibility of bioremediation treatment. The
contaminated soil material was excavated with the help of a bucket
to a depth of 3–5 m deep. Five soil samples (15 kg each) of the exca-
vated material were collected and immediately transported to the
laboratory. All soil samples were gently crumbled and sieved through
a 6.3 mm screen in order to eliminate rough materials, thoroughly
mixed, and stored at field humidity at 4 °C until processing. Immediately
before the setup of the bioremediation experiment, a composite sample
of the five collected samples was produced.
2.2. Biostimulation experiment
Eight 2-L pans containing 1.5 kg (fresh mass) of the composite soil
sample were prepared. To test the effect of four treatments,
(1) Two pans remained unfertilized to evaluate natural attenuation.
(2) Two pans were amended with an agricultural inorganic NPK
fertilizer (containing 9.5% NH3-N, 5.5% NO3-N, 6.6% P2O5-P
and 12.2% K2O-K; N:P = 2.3:1, K:P = 1.8:1) using a C:N ratio of
20:1 for the initial hydrocarbon concentration. Pre-investigations
on the effect of various C:N ratios (10:1, 20:1, 30:1, 40:1; 50:1)
showed that a C:N ratio of 20:1 was optimal for the bioremedia-
tion treatment of the studied soil (data not shown). The N con-
centration in soil solution as calculated according to Walworth
et al. (2007) was 2230 mg N kg−1 soil H2O, which is in agree-
ment with the recommendation by Walworth et al. (2007),
according to which microbial activity is reduced at NH2O above
2500 mg N kg−1 soil H2O.
(3) Two pans were amended with the oleophilic fertilizer Inipol
EAP22® (Elf Atochem North America, Inc., Philadelphia)
using hydrocarbons:EAP ratio of 10:1 (w/w) as recommended
(Bruchon et al., 1996). The product is an oil-in-water emulsion
containing organic nutrients: N in the form of urea emulsified
with oleic acid, P in the form of lauryl phosphate, and trace
quantities of an emulsion stabilizer (Churchill et al., 1995). In-
formation on the N:P content of this fertilizer is contradictory
and ranges from 2.6:1 (Pritchard et al., 1992) to 18.5:1, as calcu-
lated from a C:N:P ratio of 62:7.4:0.7 (Coulon et al., 2005).
123
(4) Two pans were amended with the commercial product
Terramend® hydrocarbons (FMC Environmental Solutions)
at a final concentration of 1% (w/w) as recommended by the
manufacturer for initial TPH (total petroleum hydrocarbons)
contents of 4000–10,000 mg kg− 1 soil dry mass. This prod-
uct consists of major, minor and micronutrients (35–45%
monoammonium phosphate, 20–25% calcium carbonate, 0–2%
ammonium nitrate, 0–2% monobasic ammonium phosphate,
0–2% potassium nitrate, 20–30% organic amendment not
further specified).
The soil water content was adjusted in all pans with sterile water to
ca. 50% of the maximum water holding capacity. The pans were closed
with covers that contained six holes (diameter 2 cm) closed with cotton
wool stoppers. One pan per treatment was incubated in the dark at
20 °C and 50% relative humidity, while the second pan of each treat-
ment was incubated at 10 °C. The soil in the pans was mixed thoroughly
twice a week, whereby water loss during incubation was compensated
for by the addition of sterile water. Immediately after the setup of the
experiment, weekly (up to 8 weeks) and afterwards in longer time in-
tervals (10, 12, 15, 20, 25 and 30 weeks), soil samples (ca. 50 g) were
removed from each pan to determine the residual TPH concentration,
soil dry mass and soil pH. After 0, 15 and 30 weeks, ca. 20 g of soil sam-
ples was collected for PLFA analysis.
2.3. Physical and chemical soil properties
Physical and chemical soil properties were examined with 3 repli-
cates. Soil dry mass content was determined from mass loss after 24 h
at 105 °C. Soil pH was determined in 10 mM CaCl2. Soil nutrient con-
tents (nitrate, nitrite, phosphorus) of the soil sample at the beginning
of the experiment were determined spectrophotometrically (Schinner
et al., 1996).
Hydrocarbon content in the range of C10 to C40 was determined by
gas chromatography and a flame ionization detector after extraction
(three replicates) with heptane (DIN ISO 16703, modified according to
ÖNORM EN 14309).
2.4. Soil respiration
The setup for the measurement of soil respiration occurred simulta-
neously with the setup of the decontamination experiment. Soil respira-
tion was determined by using the system OxiTop® Control (WTW).
Twenty grams of soil fresh mass was weighed into each of 24 100-ml
vessels. Six vessels each were treated as described for the biostimulation
experiment (see Section 2.2). The vessels were provided with the corre-
sponding measuring heads that were filled with NaOH platelets. After-
wards the vessels were tightly closed and three vessels per treatment
were incubated at 10 °C and at 20 °C. The consumption of oxygen due
to soil respiration resulted in a negative pressure that was constantly
monitored via the OxiTop® Control measuring heads. NaOH platelets
were regularly replaced.
2.5. PLFA
Phospholipids were determined with three replicates as previously
described (Margesin et al., 2007, 2009) and were extracted, fractionated
and quantified using the procedures described (Bardgett et al., 1996;
Frostegard et al., 1993). Separated fatty acid methyl-esters were identi-
fied using gas chromatography and a flame ionization detector. Fatty
acid nomenclature was used as described (Frostegard et al., 1993). The
fatty acids i15:0, a15:0, 15:0, i16:0, 16:1ω7c, 17:0, i17:0, cy17:0,
18:1ω7c and cy19:0 were chosen to represent bacterial biomass
(bacterial PLFA), and 18:2ω6,9c (fungal PLFA) was taken to indicate
fungal biomass (Zelles, 1999). The Gram-positive specific fatty acids
i15:0, a15:0, i16:0 and i17:0 and the Gram-negative specific fatty
124 J. Mair et al. / Cold Regions Science and Technology 96 (2013) 122–128

acids cy17:0 and cy19:0 (Haubert et al., 2006) were taken as a mea- 7,000

TPH content (mg kg-1 soil dry mass)

sure of the ratio between Gram-positive and Gram-negative bacteria.
6,000

2.6. Statistical data treatment 5,000

A three-factorial analysis of variance was used to attribute the vari- 4,000

ance of the data (TPH removal, soil respiration, PLFA patterns) to the
factors temperature, fertilization, incubation time, and their interac- 3,000 10°C
tions. TPH removal, soil respiration and PLFA data were further analyzed unfertilized
by multiple range analysis (Fisher LSD test, P ≤ 0.05). Data obtained 2,000
NPK
at 10 °C and 20 °C were further compared for equality of means by
1,000 EAP
an independent two-sample T-test. Significance was accepted at the
Terramend
P ≤ 0.05 level of probability. Correlations between the measured pa-
0
rameters were analyzed by the Pearson product–moment correlation 0 5 10 15 20 25 30
technique. Weeks

3. Results and discussion 7,000

TPH content (mg kg-1 soil dry mass)

20°C
6,000
3.1. Soil properties
unfertilized
5,000
The soil investigated was a mixture of gravel, sand and clay; the NPK
C-horizon was silicate (gneiss and schist). The soil pH (CaCl2) was 4,000 EAP
6.1–6.2. Contents of soil nutrients were low (2.2 mg NH4-N kg− 1 Terramend
soil, 2.5 mg NO3-N kg− 1 soil, b 1 mg P kg− 1 soil) and carbonate con- 3,000
tent was 3–4%. The soil contained 6220 mg hydrocarbons kg− 1 soil
dry mass at the beginning of the experiment. The contamination 2,000
could be attributed to diesel oil; the presence of heavy oil was not de-
tected. At the time of sampling, the mean soil temperature in the 1,000
sampling area was 10–18 °C. Annual air temperature in the sampling
area ranges from − 15 °C to + 25 °C, and the mean annual soil tem- 0
0 5 10 15 20 25 30
perature is 7–9 °C.
Weeks

3.2. TPH removal Fig. 1. Effect of biostimulation treatments on TPH removal at 10 °C and 20 °C. Data are
mean values of three replicates; standard deviations were ≤10%.
A three-factorial analysis of variance showed that all three factors
determined in this study (temperature, biostimulation treatments, In this study, TPH loss occurred significantly faster at 20 °C than at
incubation time) and their interactions had a statistically highly signifi- 10 °C during the whole incubation period, and also the effect of the var-
cant (P ≤ 0.001) influence on TPH loss. Over the whole incubation peri- ious fertilizers became evident sooner at 20 °C (after 6 weeks at 20 °C,
od, TPH content decreased with time at both incubation temperatures, but only after 20 weeks at 10 °C; Fig. 1). At 20 °C, inorganic NPK
however, TPH removal was influenced by fertilization treatment and nutrients accelerated bioremediation to the highest extent and
was significantly (P ≤ 0.05) higher at 20 °C compared to 10 °C (Fig. 1, much earlier (after 6 weeks) than other fertilization treatments:
Table 1), which has been reported by a number of bioremediation stud- Significantly biostimulating effects of Terramend and oleophilic
ies on soils from cold regions (Coulon et al., 2005; Margesin and fertilization (compared to natural attenuation) became only visible
Schinner, 1997; Walworth et al., 2001, 2003). In our study, Q10 values after 10 weeks. After 30 weeks of incubation, i.e. at the end of the
of 1.2–1.6 and 1.1–1.5 were calculated after 15 and 30 weeks of treat- experiment, natural attenuation had resulted in relative TPH losses of
ment, respectively, which reflect the adaptation of the indigenous 57.5% and 82.5% at 10 °C and 20 °C, respectively (residual contamination
soil microbial community to the low temperature and confirm earlier 2643 and 1089 mg kg−1 soil dry mass, respectively). Comparable values
reported results (Margesin and Schinner, 1997). (59.8%; residual contamination 2499 mg kg−1 soil dry mass) were
A remarkable TPH loss was obtained at both temperatures. At 20 °C,
the TPH contamination was removed almost completely (up to 92.7%,
residual contamination 454 mg kg−1 soil dry mass) after 30 weeks,
while at 10 °C losses up to 69% (residual contamination 1906 mg kg−1 Table 1
soil dry mass) were obtained. Natural attenuation played a major role: Effect of biostimulation on relative TPH loss (6220 mg TPH/kg dry soil = 100%) after 8, 15
Regardless of the temperature, TPH loss in the absence of nutrients and 30 weeks at 10 °C and 20 °C. Different letters in a column denote statistically
(P ≤ 0.05) significant differences between treatments.
was only lower by ca. 10–12% compared to biostimulation treatments.
Natural attenuation is a sum of the natural biodegradation activity of Temperature Treatment Relative TPH loss (%) after
the indigenous soil microbial population and abiotic loss due to physical 8 weeks 15 weeks 30 weeks
and chemical processes and is a cost-effective remediation alternative
10 °C Unfertilized 34.5a 47.5a 57.5a
for low-risk petroleum-contaminated sites (Hinchee, 1998). Our earlier NPK 36.3a 46.5a 69.3b
studies demonstrated that hydrocarbon loss in alpine soils with an aged EAP 30.0a 46.5a 59.8a
contamination can be mainly attributed to microbial activity, since Terramend 29.6a 50.4a 69.0b
abiotic losses (due to dispersion, sorption, evaporation and abiotic 20 °C Unfertilized 45.4a 55.2a 82.5a
transformations) are already largely completed, while abiotic processes NPK 58.2b 74.7d 92.7c
are the major factors contributing to natural attenuation in freshly con- EAP 43.4a 63.9b 87.5b
Terramend 45.0a 68.4c 88.6b
taminated alpine soils (Margesin, 2007).
 J. Mair et al. / Cold Regions Science and Technology 96 (2013) 122–128 125

Respiration (mg O2 g-1 soil dry mass)

obtained at 10 °C when the oleophilic fertilizer was applied. Inorganic 18
NPK nutrients and Terramend resulted at both temperatures in sig- 16
10°C
nificantly higher TPH losses, whereby a significant difference be-
14
tween Terramend (88.6% loss, residual contamination 707 mg kg− 1 unfertilized
NPK
soil dry mass) and NPK nutrients (92.7% loss, residual contamination 12 EAP
Terramend
454 mg kg− 1 soil dry mass) could be detected at 20 °C but not at 10
10 °C (69–69.3% TPH loss; residual contamination 1906–1928 mg kg−1
8
soil dry mass). Biodegradation rates were in the range of 17–22
and 24–28 mg hydrocarbons kg− 1 soil day− 1 at 10 °C and 20 °C, 6
respectively. Altogether, relative TPH losses were generally higher 4
by ca. 30% at 20 °C compared to losses obtained at 10 °C. A similar
2
trend was observed in other soil bioremediation studies in cold
areas (Coulon et al., 2005; Margesin, 2007; Walworth et al., 2001, 0
0 2 4 6 8 10 12 14
2003) and can be explained by the effect of temperature on bioavail-
ability and solubility of hydrophobic substances, such as some aliphatic Weeks
and polyaromatic hydrocarbons. Besides, the decreased volatilization

Respiration (mg O2 g-1 soil dry mass)

and solubility of some hydrocarbons at low temperature affect toxicity.
A temperature decrease also results in a decrease in diffusion rates of
organic compounds and in an increase in viscosity, which affects the
degree of distribution (Rojo, 2009; Whyte et al., 1998).
The impact of N in fertilizers on soil water potential is greater in dry
than in moist soils (Walworth et al., 1997). NPK fertilization resulted in
an N concentration in a soil solution of 2230 mg N kg−1 soil H2O, which
is in good agreement with the recommendation by Walworth et al.
(2007). Oleophilic fertilization, applied as recommended by the
manufacturer, resulted in ca. a 6-fold higher N concentration (based on
nutrient levels; Bruchon et al., 1996) compared to NPK fertilization
(see also 2.2.); accordingly the N concentration in soil solution as calcu-
lated according to Walworth et al. (2007) was ca. 13,500 mg N kg−1 soil
H2O, thus well above the recommended value of 2500 mg N kg−1 soil
H2O (Walworth et al., 2007), above which microbial activity is reduced.
This may have influenced the TPH loss and microbial activity measured
in our study. The weak performance of the oleophilic fertilizer at 10 °C
may be attributed to a lower solubility of some components at 10 °C
compared to higher temperatures, and also to an increased level of tox-
icity resulting in the inhibition of microbial activity. This is supported by
reports of high toxic levels after long-term treatment with Inipol EAP22
(Coulon et al., 2005) and by studies showing high toxicity of emulsified
oil (Richmond et al., 2001). The application of this fertilizer can increase
the solubility of toxic PAH compounds in soil, which enhances their
bioavailability and thus increases the adverse effect on soil microorgan-
isms (Churchill et al., 1995). Due to unspecified components in the
Terramend fertilizer, the exact nutrient contents cannot be calculated.
3.3. Soil respiration
Soil respiration was measured up to an incubation period of 15 weeks.
Similar to TPH loss, temperature, fertilization, incubation time, and their
interactions had a statistically highly significant (P ≤ 0.001) influ-
ence on soil respiration during bioremediation. Soil respiration was
generally higher at 20 °C compared to 10 °C; bioremediation studies
of Arctic and subarctic soils showed the same trend (Walworth et al.,
2001). After 6 weeks of incubation a clear trend became visible at
both temperatures (Fig. 2): NPK fertilization was the best treatment
to stimulate soil microbial respiration both at 10 °C and 20 °C,
followed by Terramend (52% and 57% of the respiration measured
with NPK fertilization at 10 and 20 °C, respectively, after 8 weeks)
and oleophilic fertilization (51% and 49% of the respiration measured
with NPK fertilization at 10 and 20 °C, respectively, after 8 weeks),
while soil respiration was lowest in soil subjected to natural attenuation
(26% and 32% of the respiration measured with NPK fertilization at 10
and 20 °C, respectively, after 8 weeks). In the unfertilized soil, respira-
tion increased slowly during the first 10 weeks at 10 °C, but only during
the first 6 weeks at 20 °C, and remained constant afterwards. This could
be explained by the depletion of available nutrients after 6 weeks.
18 20°C
16 unfertilized
NPK
14 EAP
Terramend
12
10
8
6
4
2
0
0 2 4 6 8 10 12 14
Weeks
Fig. 2. Effect of biostimulation treatments on soil respiration at 10 °C and 20 °C. Data are
mean values of three replicates; standard deviations were ≤10%.
When comparing respiration at the two temperatures tested, Q10
values of 1.7–2.3 and 1.5–1.9 were calculated after 8 and 15 weeks of
treatment, respectively. Q10 values were highest in soil subjected to
natural attenuation or oleophilic fertilization, which indicates a more
pronounced effect of NPK and Terramend nutrients on soil respiration
at 10 °C. As already observed with TPH removal, oleophilic fertilization
had a significantly lower biostimulating effect than Terramend after 15
and 30 weeks of treatment at 10 °C but not at 20 °C. However, during
the first 4 weeks oleophilic fertilization resulted in respiration compa-
rable to that obtained with NPK fertilization, which again (see above)
points to the accumulation of toxic effects with time.
Soil respiration reflected microbial activity during bioremediation
and followed the trend observed with TPH loss, which was further
reflected by a significantly positive correlation between the residual
TPH content and soil respiration (see 3.5.). However, the effect of the
various treatments was much more pronounced on soil respiration
than on TPH loss and became earlier visible than TPH loss at 10 °C.
This could be explained by the fact that nutrients obviously stimulated
soil microbial activity, however, this enhanced activity might not have
been fully used for hydrocarbon biodegradation. The data obtained in
our study demonstrate the suitability and sensitivity of soil respiration
to evaluate soil microbial activity during bioremediation.
3.4. PLFA profiles
In this study we determined PLFA patterns at the beginning,
in the middle (15 weeks) and at the end of the experiment (30 weeks).
As observed with TPH loss and soil respiration, temperature, fertilization,
incubation time, and their interactions had also a statistically highly
significant (P ≤ 0.001) influence on PLFA patterns.
126 J. Mair et al. / Cold Regions Science and Technology 96 (2013) 122–128

soil bacteria Amounts of PLFA specific for total soil bacteria, Gram-positive and
40 10°C Gram-negative bacteria as well for soil fungi increased significantly
PLFA (nmol g-1 soil dry mass)

with time. Nonetheless, the effect of temperature on PLFA pattern

20°C
30 differed according to the various microbial groups. Both after 15
and 30 weeks of incubation (Fig. 3), comparable amounts of PLFA
20 specific for total soil bacteria were found at 10 °C and at 20 °C,
regardless of the treatment. The amount of PLFA specific for soil
10
fungi, however, was significantly higher at 10 °C compared to 20 °C
in the presence of two of the three fertilizers tested (inorganic NPK,
0
Terramend). This can be explained by the fact that fungi, especially
Terramend

Terramend
unfertilized

unfertilized
NPK

NPK
EAP

EAP
yeasts, in cold alpine environments are characterized by lower opti-
mum and maximum temperatures for growth and activity compared
to bacteria (Margesin, 2009; Margesin et al., 2003a) and are thus well
15 weeks 30 weeks adapted to cold climate conditions (Margesin et al., 2009).
Among soil bacteria, PLFA specific for the Gram-negative bacterial
1.0
PLFA (nmol g-1 soil dry mass)

soil fungi population were, in all treatments after 15 weeks (exception:

* unfertilized soil) as well as after 30 weeks, significantly higher (by a
0.8 10°C factor of 1.7–1.9 after 30 weeks) at 20 °C than at 10 °C, while the
* 20°C Gram-positive soil bacterial population was not significantly influenced
0.6 * by temperature. The ratio between PLFA specific for Gram-positive and
Gram-negative soil bacteria was slightly b1 at the beginning of the
0.4 study and remained unchanged or increased only slightly at 20 °C dur-
* ing bioremediation, while at 10 °C often significantly higher ratios were
0.2 noted (Fig. 3) which points to a significantly higher enrichment of the
Gram-negative hydrocarbon-degrading population at the higher tem-
0.0 perature. Distinct shifts in bacterial community composition towards
Terramend

Terramend
unfertilized

unfertilized
NPK

NPK
EAP

EAP

an enrichment of Gram-negative bacteria, especially Proteobacteria,

following hydrocarbon contamination in Alpine (Labbé et al., 2007;
Margesin et al., 2007; Zhang et al., 2012) and in Arctic soils (Juck
et al., 2000) have been observed. This was corroborated in our study,
15 weeks 30 weeks
however, we additionally noted a significantly higher enrichment
of Gram-negative bacteria at 20 °C than at 10 °C, which could be
7
PLFA (nmol g-1 soil dry mass)

Gram-negative bacteria explained by a better and more efficient stimulation of hydrocarbon

* *
6 degraders under more favorable temperature conditions. Shi et al.
10°C (2002) recognized a relation between the soil TPH content and the
5 20°C * variation in PLFA; however, low TPH contents resulted in a signifi-
* *
4 cant increase in PLFA specific for Gram-negative and Gram-positive
bacteria. The enrichment of Gram-negative bacteria after a contami-
3 * *
nation event was explained with the r–K scheme (Labbé et al., 2007;
2 Margesin et al., 2003b, 2007), according to which Gram-negative
1 bacteria are r strategists and grow rapidly under favorable condi-
tions (Atlas and Bartha, 1998). Temperature-dependent shifts in mi-
0 crobial community composition (towards an increase in the relative
Terramend

Terramend
unfertilized

unfertilized
NPK

NPK
EAP

EAP

amount of culturable psychrophilic heterotrophic bacteria, in the

relative amount of the fungal population, and in the relative amount
of Gram-negative bacteria) have also been observed in pristine alpine
soils (Lipson, 2007; Margesin et al., 2009).
15 weeks 30 weeks
With regard to the effect of biostimulation treatments on PLFA pat-
terns after 30 weeks (Fig. 3, Table 2), NPK nutrients had the highest
2.5 ratio G+/G- bacteria stimulating effect on the amount of PLFA specific for total soil bacteria,
* regardless of the incubation temperature. A significant stimulation of
2.0 10°C * the Gram-negative bacterial population occurred only in the presence
* 20°C * of Terramend and was present at both temperatures. On the other
1.5 * *
PLFA

* hand, all fertilization treatments had a significantly stimulating effect

on PLFA specific for Gram-positive bacteria compared to the absence
1.0 of nutrients (Table 2).
These data demonstrate the suitability of PLFA analysis for profiling
0.5 microbial communities in hydrocarbon-contaminated soils, as already
demonstrated in a number of studies (Bundy et al., 2004; Chakraborty
0.0 et al., 2012; Haines et al., 2002; Margesin et al., 2007).
Terramend

Terramend
unfertilized

unfertilized
NPK

NPK
EAP

EAP

Fig. 3. Effect of temperature on PLFA specific for total soil bacteria, soil fungi,
Gram-negative soil bacteria and the ratio between Gram-positive and Gram-negative
15 weeks 30 weeks soil bacteria in relation to various biostimulation treatments and incubation time. Values
at t0 are shown in Table 2. Asterisks indicate significant differences (P ≤ 0.05) between
results obtained at 10 and 20 °C.
 J. Mair et al. / Cold Regions Science and Technology 96 (2013) 122–128 127

Table 2
Effect of biostimulation on PLFA pattern specific for soil microorganisms (total soil bacteria, Gram-negative bacteria (G −), Gram-positive bacteria (G +), ratio between G + and
G − bacteria, soil fungi) at the beginning and after 30 weeks of bioremediation at 10 °C and 20 °C. Different letters in a column denote statistically (P ≤ 0.05) significant
differences between treatments.

Temperature Treatment PLFA (nmol g−1 soil dry mass) specific for

Total bacteria G+ bacteria G− bacteria Ratio G+/G− bacteria Total fungi

t0 7.7 1.1 1.2 0.9 0.08

10 °C Unfertilized 12.2 a 2.4 a 1.9 a 1.3 a 0.08 a
NPK 31.1 c 4.0 c 2.2 a 1.8 b 0.89 c
EAP 16.8 b 3.3 b 1.8 a 1.9 b 0.10 a
Terramend 17.6 b 3.9 c 3.3 b 1.2 a 0.56 b
20 °C Unfertilized 14.6 a 3.1 a 3.3 a 0.9 b 0.08 a
NPK 27.1 c 4.7 b 4.1 a 1.2 c 0.33 b
EAP 16.1 a 4.2 b 3.5 a 1.2 c 0.14 a
Terramend 22.3 b 4.6 b 5.7 b 0.8 a 0.25 b

3.5. Correlations between TPH content, soil respiration and PLFA profiles
Significantly (P ≤ 0.05) positive correlations were obtained be-
tween residual TPH content and soil respiration. Both TPH content and
soil respiration correlated significantly positively with PLFA specific
for Gram-negative and Gram-positive soil bacteria; soil respiration cor-
related additionally with PLFA specific for total soil bacteria, which also
correlated with PLFA reflecting soil fungi (data not shown). These posi-
tive correlations between TPH loss, soil microbial activity (respiration)
and community structure (PLFA) demonstrate the involvement of a
wide range of soil microorganisms (Gram-positive and Gram-negative
bacteria, fungi) in the bioremediation of the investigated soil. These cor-
relations also demonstrate the suitability of the applied methods to
monitor soil bioremediation. Evidence for the biodegradation activity
of indigenous bacteria and fungi in contaminated cold environments
has also been provided by high numbers and activities of hydrocarbon
degraders, high mineralization potentials and the prevalence of geno-
types with catabolic pathways for the degradation of a wide range of
hydrocarbons (Filler et al., 2008; Juck et al., 2000; Yergeau et al., 2009).
3.6. Summary
In this study, we determined the effect of various biostimulation
(fertilization) treatments versus natural attenuation on the TPH loss,
microbial activity and community composition of a hydrocarbon-
contaminated soil from a former military site at 10 °C and at 20 °C
over a period of 30 weeks. Both temperature and biostimulation had a
significant effect on TPH loss, microbial activity and microbial com-
munity structure during bioremediation. The initial TPH loss could
be clearly reduced by up to 93% and 69% at 20 °C and 10 °C, respec-
tively, however, natural attenuation played a major role and contrib-
uted to a TPH loss of 83% and 58% at 20 °C and 10 °C, respectively.
Biostimulation treatments resulted in shifts in microbial community
composition, which included the enrichment of soil fungi at 10 °C
and the enrichment of Gram-negative bacteria at 20 °C. Significantly
positive correlations between TPH content, soil respiration and PLA
specific for various groups of soil microorganisms demonstrated the
suitability of the applied methods to monitor soil bioremediation.
Acknowledgments
This study was supported by a grant from the “Autonome Provinz
Bozen, Südtirol”, Amt für Geologie und Baustoffprüfung. We thank
P. Thurnbichler for technical assistance and S. Rudolph (University
of Hohenheim) for PLFA analysis. We also thank C.R. Comeceau
(Elf Atochem, USA) for providing us with Inipol EAP22, and Mike
Mueller (FMC Environmental Solutions) for providing us with Terramend.
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