Biodegradation of phenanthrene by indigenous microorganisms in soils from Livingstone Island, Antarctica

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.


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).



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.



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



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




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.



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Biodegradation of phenanthrene by indigenous microorganisms in soils from Livingstone Island, Antarctica