R E S EA RCH L E T T E R
Biodegradation of phenanthrene by indigenous microorganismsin soils from Livingstone Island, Antarctica
Uchechukwu V. Okere1, Ana Cabrerizo2, Jordi Dachs2, Kevin C. Jones1 & Kirk T. Semple1
1Lancaster Environment Centre, Lancaster University, Lancaster, UK; and 2Department of Environmental Chemistry, IDAEA-CSIC, Barcelona,
Catalonia, Spain
Correspondence: Kirk T. Semple, Lancaster
Environment Centre, Lancaster University,
Lancaster, LA1 4YQ, UK. Tel.: +44 1524
510554; fax: +44 1524 510217;
e-mail: [email protected]
Received 23 August 2011; revised 20
December 2011; accepted 10 January 2012.
Final version published online 23 February
2012.
DOI: 10.1111/j.1574-6968.2012.02501.x
Editor: Elizabeth Baggs
Keywords
Livingstone Island; sub-Antarctica; microbial
degradation; phenanthrene; soil.
Abstract
Biodegradation of polycyclic aromatic hydrocarbons (PAHs) in soils has been
linked to history of exposure to PAHs and prevailing environmental condi-
tions. This work assessed the capacity of indigenous microorganisms in soils
collected in Livingstone Island (South Shetlands Islands, Antarctica) with no
history of pollution (∑PAHs: 0.14–1.47 ng g�1 dw) to degrade 14C-phenanhth-
rene at 4, 12 and 22 °C. The study provides evidence of the presence of phen-
anthrene-degrading microorganisms in all studied soils. Generally, the
percentage of 14C-phenanhthrene mineralized increased with increasing tem-
perature. The highest extent of 14C-phenanhthrene mineralization (47.93%)
was observed in the slurried system at 22 °C. This work supports findings of
the presence of PAH-degrading microorganisms in uncontaminated soils and
suggests the case is the same for uncontaminated Antarctic remote soils.
Introduction
The role of indigenous microbial communities in the
removal of hydrocarbons from the environment has been
widely investigated showing that a small fraction of all
natural microbial communities irrespective of location or
prevailing environmental conditions can grow on both
aromatic and aliphatic hydrocarbons (Sepic et al., 1995;
Solano-Serena et al., 2000; Ruberto et al., 2003). The size
of these populations of degrading microorganisms often
reflects the historical exposure of the environment to
either biogenic or anthropogenic hydrocarbon sources. In
general, while hydrocarbon degraders may constitute
< 0.1% of the microbial community in unpolluted envi-
ronments, in oil-polluted ecosystems they can constitute
up to 100% of the culturable microorganisms (Atlas,
1981). Several studies (Spain et al., 1980; Carmichael &
Pfaender, 1997; Chen & Aitken, 1998; Macleod & Semple,
2006; McLoughlin et al., 2009) have shown an increase
in hydrocarbon-degrading microorganisms in different
soil environments, following exposure to aromatic
hydrocarbons.
Where biodegradation of polycyclic aromatic hydrocar-
bons (PAHs) has been observed in cold environments, it
has been attributed to cold adapted psychrotrophs and
psychrophiles, which are widely distributed in nature
because a large part of the earth’s biosphere is at temper-
atures below 5 °C (Margesin & Schinner, 1999; Ferguson
et al., 2003a, b). A significant increase in numbers of psy-
chrotrophic bacteria following contamination in cold
environments has been reported leading to suggestions of
their potential for rapid adaptation and their predomi-
nance over psychrophiles in cold environments (Delille
et al., 1998; Margesin & Schinner, 1999; Delille, 2000).
Hydrocarbon-degrading bacteria isolated from contami-
nated Antarctic soils have been identified and include the
genera Rhodococcus, Acinobacter, Pseudomonas and Sphingo-
monas (Aislabie et al., 2004, 2006; Ma et al., 2006). Many
of these microorganisms were psychrotrophic rather than
psychrophilic; while they could grow at low temperatures,
optimum growth was at temperatures > 15 °C (Aislabie
et al., 2004).
Livingstone Island is one of the South Shetland Islands
and it is separated from the Antarctica Peninsula by the
Bransfield Strait. Its temperatures are relatively constant,
rarely exceeding 3 °C in summer or falling below �11 °Cin winter, with wind chill temperatures up to 5–10 °Clower. It hosts some summer scientific stations established
FEMS Microbiol Lett 329 (2012) 69–77 ª 2012 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved
MIC
ROBI
OLO
GY
LET
TER
S
from 1988 and benefits from the Antarctic Treaty which
regulates both human presence and activities on the con-
tinent (Quesada et al., 2009). Although the region has
seen an increase in human activities in recent years result-
ing from the increased popularity of Antarctic tourism
and the opening of more research stations by national
Antarctic operators, it is still considered one of the earth’s
last pristine wildernesses, with minimal human impact
resulting in some part of it being proposed as a reference
site for comparative terrestrial and coastal studies (Ques-
ada et al., 2009). Its relative pristine status makes it an
interesting site for investigating the biodegradation of
PAHs by indigenous microorganisms in these soils with-
out any history of exposure to lignin, PAHs or similar
compounds. Indigenous PAHs have been previously
investigated (Aislabie et al., 2000, 2006; Ferguson et al.,
2003a, b; Coulon et al., 2005) in Antarctic and sub-Ant-
arctic soils, but these studies have been performed on
potentially contaminated soils with high levels of soil
PAHs concentration, from areas impacted by Antarctic
settlements and scientific stations. To our knowledge, no
direct biodegradation measurements have been carried
out in soils with extremely low amounts of PAHs, such
as those collected from different sites of Livingstone
Island and used in this study.
In the present paper we investigate the degradation of
14C-phenanthrene by indigenous soil microorganism in
soil samples from Livingstone Island at different tempera-
tures.
Materials and methods
Materials
Phenanthrene (> 99.6%), and [9-14C] phenanthrene (spe-
cific activity = 50 mCi mmol�1, radiochemical purity
> 95%) standards were obtained from Sigma Aldrich,
UK. Chemicals for the minimal basal salts (MBS) solution
were obtained from BDH Laboratory Supplies and Fisher
Chemicals. The liquid scintillation cocktail (Ultima Gold)
and glass scintillation vials (7 mL) were obtained from
Canberra Packard, UK. Sodium hydroxide was obtained
from Sigma Aldrich. Dichloromethane, hexane and meth-
anol were supplied by Merck, Darmstad, Germany. Agar-
agar and plate count agar were obtained from Oxoid Ltd,
UK.
Soils sampling and bulk characterization
Soil samples were collected from background areas of Liv-
ingstone Island. A map with the sampling sites is provided
in Fig. 1. The top 5 cm were taken using a stainless steel
corer. Samples were frozen (�20 °C) in sterile glass jars
for transportation to Lancaster University. Soil physico-
chemical properties are shown in Table 1. Soil redox, soil
pH and soil moisture content were measured by standard
methods described elsewhere(Cabrerizo et al., 2011). Par-
ticle size analysis was determined according to the method
by Gee and Bauder (1979) and calculations according to
Gee and Bauder (1986). Total carbon and nitrogen were
determined by analysing 4 mg of oven-dried (105 °C) andsieved (2 mm) soil samples on a Carlo Erba CHNS-OEA
1108 CN-Elemental analyser. For total organic carbon
(TOC) analysis, soils were heated to 430 °C to remove all
organic carbon, the ash containing inorganic carbon alone
was measured on the analyser and the TOC determined
by mass balance (Rhodes et al., 2007).
PAH concentrations in soil
Extraction and quantification: Briefly, 30 g of soil samples
were homogenized and dried by mixing with anhydrous
sodium sulphate and ground using a mortar and a pestle.
The whole sample was transferred into a soxhlet cellulose
thimble (Whatman) and extracted in soxhlet apparatus
over 24 h, using dichloromethane:methanol (2 : 1 v/v).
Prior to the extraction, samples were spiked with per-
deuterated PAHs standards (phenanthrene-d10, crysene-
d12 and perylene-d12), which were used as surrogate
standards. The extracts were reduced in a rotary evapora-
tor to 1 mL and then solvent-exchanged into isooctane.
All samples were then fractionated on a 3% deactivated
alumina column (3 g) with a top layer of anhydrous
sodium sulphate. Each column was eluted with 12 mL of
dichloromethane/hexane (2 : 1 v/v). The PAH fraction
was concentrated in a rotary evaporator and solvent-
exchanged to isooctane under a gentle stream of nitrogen.
After concentration, the samples were transferred to injec-
tion vials and 25 lL of anthracene-D10 and benzo(a)
anthracene-D12 were added as injection standards. All the
samples were analysed by GC-MS using a Thermo Elec-
tron (San Jose, CA; model Trace 2000 operating in
selected ion monitoring (SIM) mode. Details of tempera-
ture programs and monitored ions are given elsewhere
(Cabrerizo et al., 2009, 2011).
Quality assurance/control
All analytical procedures were monitored using strict
quality assurance and control measures. One field and
laboratory blanks were introduced every three soil sam-
ples. Phenanthrene, fluoranthene and pyrene were
detected in blanks, but they accounted for < 3% of the
total sample concentrations. Samples, therefore, were not
blank corrected. The surrogate per cent recoveries from
the soil samples reported here were (mean ± SD) 70% ±
ª 2012 Federation of European Microbiological Societies FEMS Microbiol Lett 329 (2012) 69–77Published by Blackwell Publishing Ltd. All rights reserved
70 U.V. Okere et al.
11 for phenanthrene-d10, 105% ± 17 for crysene-d12 and
90% ± 13 for perylene-d12.
Catabolism of 14C-phenanthrene in soil
Catabolic degradation of 14C-phenanthrene was deter-
mined in 250-mL screwcap Erlenmeyer flasks (Reid et al.,
2001). The respirometers contained 10 g of soil rehydrat-
ed to 40–60% water-holding capacity and spiked with
unlabelled and 14C-phenanthrene (80 Bq 14C-phenan-
threne g�1 soil) using toluene as a carrier solvent. A
7-mL scintillation vial containing 1 M NaOH was
attached to the screwcap to serve as a CO2 trap. Respi-
rometers were stored in the dark at 4, 12 and 22 °C. Aslurry system was also set-up containing 30 mL minimal
basal salts (MBS) medium and securely placed on a
SANYO® Gallenkamp orbital incubator set at 100 r.p.m.
and 22 °C to agitate and ensure adequate mixing over the
period of the incubation. NaOH traps were replaced every
24 h, after which 6 mL of Ultima Gold scintillation cock-
tail was added to each spent trap and the contents analy-
sed on a Packard Canberra Tri-Carb 2250CA liquid
scintillation counter. The incubation lasted for 35 days.
Lag phases were measured as the time before 14C-phen-
anthrene mineralization reached 5%. Analytical blanks
containing no 14C-phenanthrene was used for the deter-
mination of levels of background radioactivity.
Microbiological analysis
Colony-forming units (CFUs) of heterotrophic bacteria
were enumerated on plate count agar (PCA) using a via-
ble plate counts technique (Lorch et al., 1995). 12C-Phen-
anthrene was used as a sole carbon source on agar-agar
for the measurement of 14C-phenanthrene-degrading
bacteria following standard microbiological techniques
(Foght & Aislabie, 2005).
Statistical analysis
Levels of 14C-phenanthrene detected by the liquid scintil-
lation counter were corrected for background radioactiv-
ity. All samples were analysed in triplicate and errors bars
presented in graphs are standard error mean for n = 3.
SIGMA STAT version 2.03 software package was used for the
analysis of the data. Significance of 14C-phenanthrene
ByersPeninsula
Livingston Island
Decep onIsland
Juan Carlos I sta on
Soil sampling point
Soil 3
Ereby Pt.
Soil 5
Soil 1Hannan Pt.
L P l C
JhonsonGlacier
Soil 2
Sally Rocks
Las Palmas Cove
Hurd Ice Cap
Soil 4
Fig. 1. Map showing Livingstone Island and sampling sites.
FEMS Microbiol Lett 329 (2012) 69–77 ª 2012 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved
Biodegradation of phenanthrene 71
degradation between soils and temperatures were assessed
by implementing ANOVA and Tukey’s tests.
Results
Soil characteristics
Selected soils in different sections of Livingstone Island
were found to have similar physicochemical properties.
The soils are mostly sandy and slightly alkaline, with low
TOC and N contents. The sum of 23 PAH (ΣPAHs) con-
centrations was low, with values ranging between 0.14
and 1.47 ng g�1 dw soil with higher contribution of low
molecular weight PAHs (see Table 1).
Catabolism of 14C-phenanthrene in soil at
different temperatures
The catabolism of 14C-phenanthrene in Antarctica soils at
4, 12 and 22 °C (nonslurried and slurried) as determined
by the mineralization of 14C-phenanthrene to 14CO2 by
indigenous microbial communities is shown in Fig. 2.
Lag phases decreased as temperatures increased (see
Table 2). The longest lag phase (26.92 ± 0.06 days) was
observed in soil 5 at 12 °C and the shortest (1.13 ±0.16 days) was in soil 2 at 22 °C. At 4 °C, < 5% 14C-
phenanthrene was mineralized in all the five soils after a
period of 35 days. Only at 22 °C did 14C-phenanthrene
mineralize in all five soils exceed 5%.
Lowest rates of 14C-phenanthrene mineralization were
observed for all soils at 4 °C, the fastest rate observed for
all five soils at this temperature being 0.002 ± 0.001% h�1.
The rates of 14C-phenanthrene mineralization were fastest
at 22 °C under slurry conditions (0.56 ± 0.01% h�1 for soil
5). Though rates increased with increasing temperature, a
significant increase in rates of 14C-phenanthrene minerali-
zation (P < 0.05) was only observed when the rates of14C-phenanthrene mineralization at 4, 12 and 22 °C were
compared with those of the slurry system at 22 °C. Increas-ing the temperature from 4 to12 °C, 12 to 22 °C and 4 to
22 °C did not significantly increase fastest rates of mineral-
ization (P > 0.05).
Generally, 14C-phenanthrene was mineralized at higher
rates and to greater extents as temperatures increased.
At 4 °C, maximum 14C-phenanthrene mineralized was
1.14% in soil 2. Increasing the temperature to 12 °Cresulted in a maximum of 17.85% (soil 5) and a signifi-
cant increase (P < 0.05) in the amount of 14C-phenan-
threne mineralized only in soils 2 and 5. A further
increase to 22 °C resulted in a significant increase in the
amount of 14C-phenanthrene mineralized in all five soils
(P < 0.05). The maximum amount of 14C-phenanthrene
mineralized at 22 °C was 39.09% and was significantlyTable
1.Ph
ysicochem
ical
properties
offive
Antarcticsoils
Soil
PAHs
M.phe
DBT
DMP
Phe
Ant.
Flu
Pyr
B(a)ant
Cry.
Per.
Ind(1,2,
3,cd)
B(b
jk)flu
D(ah)ant.
B(ghi)
pery.
∑PA
Hs
TNTC
TOC
Sand
Clay
Silt
H2O
(%)
10.07
0.01
0.03
0.15
0.006
0.05
0.03
0.1
0.09
0.002
0.01
0.09
<LO
Q0.16
0.96
0.02
0.01
0.15
91.14
3.54
5.31
8.0
20.02
0.001
0.01
0.02
0.001
0.02
0.01
0.01
0.01
0.01
0.01
0.01
<LO
Q0.01
0.14
0.28
0.25
0.22
91.59
05.31
8.0
30.07
0.12
0.03
0.26
0.01
0.12
0.09
0.11
0.18
0.01
0.12
0.18
0.06
0.11
1.47
0.004*
0.004*
0.02*
97.81
02.18
6.25
40.05
0.001
0.02
0.06
0.003
0.024
0.02
0.01
0.01
0.001
0.02
<LO
Q<LO
Q0.04
0.26
0.001*
0.029*
0.01*
94.55
1.81
3.63
4.86
50.08
0.02
0.03
0.1
0.007
0.04
0.01
0.02
0.03
0.001
0.03
0.2
<LO
Q0.08
0.65
0.007*
0.09*
–87.38
8.40
4.2
1.15
M.phe,
Methylphen
anthrene;
DBT,
Diben
zothiophen
e;Ph
e,Ph
enan
threne;
Ant.,Anthracene;
Flu.,
Fluoranthen
e;Pyr.,Pyrene;
B(a)ant.,Ben
zo(a)anthracene;
Cry,Crysene;
Per.,Perylene;
Ind
(1,2,3,cd),Inden
o(1,2,3,cd)pyren
e;B(b
jk)flu.,Ben
zo(b
jk)fluoranthen
e;D(ah)ant.,Diben
zo(ah)anthracene;
B(ghi)p
ery.,Ben
zo(ghi)p
erylen
e.TN
,To
talnitrogen
(%);TC
,To
talcarbon(%
).
*<detectionlim
it(N:0.03–2
mgab
s;C
0.03–2
0mgab
s).
ª 2012 Federation of European Microbiological Societies FEMS Microbiol Lett 329 (2012) 69–77Published by Blackwell Publishing Ltd. All rights reserved
72 U.V. Okere et al.
greater (P < 0.05) than that mineralized at both 4 and
12 °C for all the soils.
Slurrying the soils (at 22 °C) led to further increases in
the extent of 14C-phenanthrene mineralization in all of
the soils, with a minimum of 21.98% (soil 1) and a maxi-
mum of 47.97% (soil 2) 14C-phenanthrene mineralized
over the 35 days incubation period. 14C-phenanthrene
mineralization was significantly greater in the slurried sys-
tem than at 22 °C for all the soils apart from soil 2.
Heterotrophic and 14C-phenanthrene-degrading
bacteria
CFU of phenanthrene degraders and total heterotrophs
present in the soils ranged between 104–106 and 103–104 CFU g�1. Results are shown in Fig. 3. The highest
counts of phenanthrene degraders (1.53 9 104) were
observed in soil 3 and the lowest (8.6 9 103) in soil 4.
Only incubation in slurried conditions gave increases in
both phenanthrene-degrading bacteria and total hetero-
trophs.
Discussion
Although the soils used in this study are from Livingstone
Island, a sub-Antarctic Island, far from industrialized
regions and limited human activity, PAHs were found in
all the five soils at levels similar to those reported in
uncontaminated/pristine soils (Johnsen & Karlson, 2005;
Cabrerizo et al., 2012). The higher presence of low mole-
cular weight PAHs in the soils may represent the sum of
different contributions firstly, long-range transport of semi
volatile organic pollutants to the Antarctic ecosystem.
Wania & Mackay (1996) hypothesized that as PAHs are
globally distributed, they fractionate according to the vola-
tility of the individual compounds. Secondly, PAH frac-
tionation can also occur locally (Wilcke et al., 1996). In the
case of Livingstone Island, ships and human settlements
could have served as local/regional PAH sources. Thirdly,
potential autochthonous biogenic formation of PAHs from
the degradation of organic matter (Aislabie et al., 1999;
Wilcke, 2007; Cabrerizo et al., 2011). The presence of
PAHs, especially low molecular weight biodegradable frac-
tions, justify the generalized occurrence of phenanthrene
degradable bacteria in these soils (Aislabie et al., 1998).
Respirometric assays, such as the one used in this
study for the determination of indigenous microbial
degradation of 14C-labelled organic compounds, have
been employed in numerous studies (Macleod & Semple,
2006; Swindell & Reid, 2006). The results described in
this current study show that 14C-phenanthrene degrada-
tion was evident in all selected soils and generally
increase with increasing temperature, as other studies
have already pointed out (Atlas, 1975; Ferguson et al.,
2003a, b).
Biodegradation of hydrocarbons in contaminated Ant-
arctic and sub-Antarctic soils has been found to be lim-
ited by low microbial activity, cold temperatures, nutrient
availability, low water content and alkaline pH (Foght
et al., 1999; Margesin & Schinner, 1999; Delille, 2000; Delille
Time (days)
0 10 20 30 40
0 10 20 30 40
0 10 20 30 40
0 10 20 30 40
0 10 20 30 40
0
10
20
30
40
50
0
10
20
30
40
50
0
10
20
30
40
50
0
10
20
30
40
50
0
10
20
30
40
5014
C-P
hena
nthr
ene
min
eral
ised
(%)
Fig. 2. Degradation (%) of 10 mg kg�1 14C-phenanthrene by indigenous soil microbial communities in soils 1, 2, 3, 4 and 5 from Antarctica at
4 °C (○), 12 °C (●), 22 °C (□) and 22 °C (■) slurry after 35 days incubation time.
FEMS Microbiol Lett 329 (2012) 69–77 ª 2012 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved
Biodegradation of phenanthrene 73
et al., 2004). Characterization of the five Livingstone
Island soils used in this study revealed physicochemical
properties consistent with those by which Antarctica soils
are generally defined (Bockheim, 1997; Campbell & Cla-
ridge, 2009). Poor water-holding capacity resulting from
highly coarse/sandy soils is a limiting factor for microbial
activity (Aislabie et al., 2000) and is expected to limit the
extent of 14C-phenanthrene biodegradation in the soils;
low TOC in soils can be an indication of low microbiolog-
ical activity (Margesin & Schinner, 2001). Samples taken
in the selected sites were mostly bare of vegetation and
plate counts revealed very low CFUs (Fig. 3) for both
total heterotrophs and 14C phenanthrene-degrading bac-
teria. The presence of only small numbers of PAH-
degrading bacteria can be explained by the absence of
degradation inducing chemicals from both biogenic and
anthropogenic sources. Sufficient concentrations of bio-
genic volatile organic chemicals (VOCs) from plants (Wil-
cke, 2007; McLoughlin et al., 2009) and anthropogenic
compounds have been identified as carbon sources for
microbial activity, growth and the induction of appropriate
genes for PAH degradation in indigenous microorganisms
(Macleod & Semple, 2002; Johnsen & Karlson, 2005).
Hydrocarbon degraders have been cultivated at levels
> 105 cell g�1 from contaminated polar soils and have
increased following oil spillage by 1–2 orders of magnitude
in hydrocarbon contaminated soil compared with pristine
soils (Aislabie et al., 2000). In this study, CFUs of 14C-
phenanthrene-degrading bacteria increased in all five soils
and by one order of magnitude in soils 1, 3 and 5 after
mineralization in slurry conditions (Fig. 3).
Of the three temperatures used in this study, 4 °Cwas the most representative of prevailing temperatures
at Livingstone Island hence appropriate for optimum
microbial activity. However, no significant amount of14C-phenanthrene was mineralized in any of the five
soils (Table 2).
Reduced bioavailability of PAHs at low temperatures
has also been reported as a possible reason for low levels
of microbial degradation (Eriksson et al., 2003). At low
temperatures, the solubility and bioavailability of less sol-
uble hydrophobic organic compounds, such as PAHs,
decrease because of an increase in viscosity in the physical
nature of the compounds and because of stronger sorp-
tion to the soil organic matter. Increased viscosity will
decrease the degree of organic compound distribution
(less surface area for microbial action) and subsequent
diffusion rates to sites of biological action leading to
reduced extents of degradation (Nam & Kim, 2002). Fer-
guson et al. (2003a, b)obtained similar results when they
found that mineralization of 14C-labelled octadecane was
virtually absent at temperatures below or near the freez-
ing point of water.Table
2.Lagphases
andfastestratesan
dextents
of14C
phen
anthrenemineralizationin
five
Antarcticsoils
at4,12an
d22°C
Soil
Lag(days)
Fastestrate
(%h�1)
Extents
(%)
4°C
12°C
22°C
22°C
Slurry
4°C
12°C
22°C
22°C
Slurry
4°C
12°C
22°C
22°C
Slurry
1Not
reached
Notreached
Not
reached
14.74±6.779
10�2
1.889
10�3±
3.649
10�4
1.689
10�3±
1.089
10�4
5.119
10�3±
6.899
10�4
2.459
10�1±
2.119
10�3
0.61±1.849
10�2
0.78±1.739
10�2
1.86±3.359
10�1
21.98±3.189
10�1
2Not
reached
25.97±2.739
10�1
9.51
11.13±1.619
10�1
4.489
10�3±
9.509
10�4
5.329
10�2±
9.399
10�4
1.739
10�1±
1.249
10�3
2.7
910�1±
5.679
10�3
1.14±2.729
10�1
15.23±1.8
9
10�1
39.09±6.11
47.93±4.449
10�1
3Not
reached
Notreached
25.36
20.75±2.339
10�2
1.339
10�3±
1.729
10�04
2.449
10�2±
2.459
10�18
1.239
10�1±
6.839
10�1
1.839
10�1±
2.669
10�3
0.55±6.399
10�2
3.77±4.099
10�2
15.50±2.949
10�1
26.85±1.97
4Not
reached
Notreached
Not
reached
20.39±6.619
10�1
1.999
10�3±
1.999
10�3
8.069
10�3±
8.069
10�3
5.669
10�3±
5.669
10�3
2.389
10�1±
2.389
10�1
0.63±9.069
10�2
2.28±6.749
10�2
2.41±7.019
10�1
24.18±1.13
5Not
reached
26.92±5.629
10�2
14.64±9.629
10�2
17.13±7.209
10�2
2.549
10�3±
2.069
10�4
1.279
10�1±
2.639
10�3
1.499
10�1±
4.699
10�3
5.259
10�1±
2.849
10�3
0.64±5.329
10�2
35.15±3.24
14.76±1.769
10�1
30.70±9.399
10�1
ª 2012 Federation of European Microbiological Societies FEMS Microbiol Lett 329 (2012) 69–77Published by Blackwell Publishing Ltd. All rights reserved
74 U.V. Okere et al.
At 12 °C, the extents of 14C-phenanthrene mineralized
increased significantly in two of the five soils after a long
lag phase. 14C-Phenanthrene was mineralized to a greater
extent at 22 °C than at 4 and 12 °C for all the soils. The
increasing solubility of phenanthrene with increasing tem-
perature would mean that the amount of phenanthrene
in solution (and therefore available for degradation)
would have been higher at 22 °C that at 4 and 12 °C.Despite temperatures at Livinstone Island never reaching
22 °C, degradation of 14C-phenanthrene at this tempera-
ture suggests the presence of psychrotrophic microorgan-
isms. The lag phase was shorter at 22 °C than those at 4
and 12 °C for all the soils. Results of microbiological
counts show an increase in phenanthrene degraders after
the 35 day mineralization assay. Slurrying the system
increased both the rates and extents of mineralization in
all soils. Previous studies (Labare & Alexander, 1995;
Doick & Semple, 2003) suggest that increased mineraliza-
tion as a result of slurrying a system can be as a result of
increased surface area at the contaminant-water interface
as the soil particles separate and move into suspension,
leading to rapid partitioning of the substrate into the
aqueous phase and stimulated microbial activity.
Conclusion
This study further supports claims of the ubiquitous
nature of PAH-degrading microorganisms by providing
evidence for the presence of 14C-phenanthrene-degrading
microorganisms in soils from Livingstone Island, an
uncontaminated Antarctic Island not previously studied.
Considering the unique characteristics of these soils and
the clear effect of temperature on microbial degradation,
the identification of specific phenanthrene degraders
active at different temperatures will be useful for poten-
tial bioremediation of contaminated Antarctic soils
because the introduction of foreign microbial species
into Antarctica is prohibited by the Antarctic treaty.
Also, the effect of temperature on the sequestration of
PAHs and the development of PAH catabolic properties
by indigenous Antarctic microorganisms should be
investigated.
Soil 1 Soil 2
Heterotrophs Phe. degraders
Heterotrophs Phe. degraders
Heterotrophs Phe. degraders
Heterotrophs Phe. degraders
CFU
g–1
soi
l dw
CFU
g–1
soi
l dw
CFU
g–1
soi
l dw
CFU
g–1
soi
l dw
1e+2
1e+3
1e+4
1e+5
1e+6
1e+3
1e+4
1e+5
1e+6
1e+7
1e+3
1e+4
1e+5
1e+6
1e+7
1e+3
1e+4
1e+5
1e+6
1e+7
Soil 3
Heterotrophs Phe. degradersC
FU g
–1 s
oil d
w
1e+3
1e+2
1e+4
1e+5
1e+6
1e+7 Soil 4
Soil 5Total heterotrophs before 14 °C phenanthrene degradationTotal heterotrophs after 14 °C phenanthrene degradation at 4 °CTotal heterotrophs after 14 °C phenanthrene degradation at 12 °CTotal heterotrophs after 14 °C phenanthrene degradation at 22 °CTotal heterotrophs after 14 °C phenanthrene degradation at 22 °C slurryPAH degraders before 14 °C phenanthrene degradationPAH degraders after 14 °C phenanthrene degradation at 4 °CPAH degraders after 14 °C phenanthrene degradation at 12 °CPAH degraders after 14 °C phenanthrene degradation at 22 °CPAH degraders after 14 °C phenanthrene degradation at 22 °C slurry
Fig. 3. CFUs of total PAH degraders and total heterotrophs before and after degradation at 4, 12, 22 and 22 °C slurry.
FEMS Microbiol Lett 329 (2012) 69–77 ª 2012 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved
Biodegradation of phenanthrene 75
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