Effects of temperature and fertilization on the structure of total versus active bacterial communities from sub-Antarctic seawater exposed to crude oil and diesel fuel

RESEARCH/REVIEW ARTICLE
E ec s o empe a u e and e iliza ion on he s uc u e o o al
e sus ac i e bac e ial communi ies om sub-An a c ic seawa e
exposed o c ude oil and diesel uel
A u o Rod ı´guez-Blanco,
1,2,3
Aud ey Du al,
1,2
Emilien Pelle ie ,
4
Daniel Delille
1,2
& Jean-F anc
¸ois
Ghiglione
1,2
1
Mic obial Oceanog aphy Labo a o y, Pie e and Ma ie Cu ie Uni e si y Pa is 6, UMR 7621, A enue Fon aule
´, BP44, FR-66650 Banyuls-su -Me , F ance
2
Na ional Cen e o Scien i ic Resea ch, Mixed Resea ch Uni 7621, A enue Fon aule
´, BP44, FR-66650 Banyuls-su -Me , F ance
3
Depa men o Mic obiology, Ins i u e o Aquacul u e, Uni e si y o San iago de Compos ela, ES-15782 San iago de Compos ela, Spain
4
Ins i u e o Ma ine Science, Uni e si y o Quebec a Rimouski, 310 alle
´des U sulines, Rimouski, Quebec G5L 3A1, Canada
Keywo ds
Oil hyd oca bons; Inipol EAP 22;
empe a u e; 16S DNA/ RNA;
sub-An a c ic seawa e .
Co espondence
Jean-F anc
¸ois Ghiglione, Na ional Cen e o
Scien i ic Resea ch, Mixed Resea ch Uni
7621, A enue Fon aule
´, BP44, FR-66650
Banyuls-su -Me , F ance.
E-mail: [email p o ec ed]
Abs ac
Pola en i onmen s a e exposed o he isk o oil pollu ion. Howe e , he e is
limi ed knowledge ega ding how he a ia ion o physicochemical ac o s
in luencing biodeg ada ion may a ec bac e ial communi y s uc u e. The
e ec s o empe a u e (4, 10 and 208C) and o ganic e iliza ion (Inipol EAP
22) on communi y s uc u e and di e si y o bac e ia inhabi ing Ke guelen
sub-An a c ic wa e s we e s udied in c ude- and diesel-amended mic ocosms.
Dynamics o o al (i.e., 16S DNA-based) and me abolically ac i e (i.e., 16S
RNA-based) bac e ial communi y s uc u e and di e si y we e moni o ed
using capilla y-elec opho esis single-s and con o ma ion polymo phism.
Resul s showed ha o al and ac i e communi y s uc u es we e di e en ly
in luenced by empe a u e and e iliza ion in he p esence o hyd oca bons.
Bo h e iliza ion and empe a u e induced changes in o al communi y
s uc u e in he p esence o c ude oil and diesel. Howe e , empe a u e
showed a limi ed in luence on ac i e communi y s uc u e, and e iliza ion
induced changes in he p esence o c ude oil only. Simpson’s index dec eased
o o al bac e ial communi ies a all empe a u es in he p esence o c ude oil
and diesel, whe eas a lowe educ ion was obse ed o ac i e bac e ial
popula ions. In he p esence o e ilize , he di e si y o he whole communi y
app oached con ol alues a e se en incuba ion weeks; his was no obse ed
o he ac i e bac e ial communi y. This s udy e idenced quali a i e di e ences
in o al and ac i e bac e ial communi y s uc u es o Ke guelen seawa e s in
he p esence o hyd oca bons and di e en esponses ela i e o a ia ion in
empe a u e and e iliza ion. These ac o s and hyd oca bons composi ion
ha e o be aken in o accoun o unde s and bac e ial communi y dynamics
a e an oil spill.
Inc eased ship a ic esul ing om scien i ic, ou is
and oil explo a ion ac i i ies in pola a eas may ha e
impo an en i onmen al impac s, as shown by he
shipw eck o he supply ship Bahia Pa aiso nea Palme
s a ion in An a c ica (Ka l 1992). Hyd oca bon deg ada-
ion in na u al en i onmen s is mainly achie ed by
mic oo ganisms (Leahy & Colwell 1990). In An a c ic
en i onmen s, e idence o oil biodeg ada ion by mic o-
o ganisms has been shown in soils (Wa dell 1995),
sedimen s (Tumeo & Wolk 1994), sea ice (Delille e al.
1997) and seawa e (Delille e al. 1998). Di e en
en i onmen al ac o s de e mine he ex en o mic obial
deg ada ion o hyd oca bons, nu ien a ailabili y and
empe a u e being hose conside ed o ha e a majo
(page numbe no o ci a ion pu pose)
Pola Resea ch 2013. #2013 A. Rod ı
´guez-Blanco e al. This is an open-access a icle dis ibu ed unde he e ms o he C ea i e Commons A ibu ion-Noncomme cial
3.0 Unpo ed License (h p://c ea i ecommons.o g/licenses/by-nc/3.0/), pe mi ing all non-comme cial use, dis ibu ion, and ep oduc ion in any medium, p o ided he
o iginal wo k is p ope ly ci ed.
1
Ci a ion: Pola Resea ch 2013, 32, 18521, h p://dx.doi.o g/10.3402/pola . 32i0.18521
impac in his p ocess (A las & Ba ha 1972; B addock
e al. 1997; Xia e al. 2006). Nu ien a ailabili y is a
limi ing ac o since hyd oca bons a e cha ac e ized
by high ca bon o ni ogen and phospho ous a ios
ha do no suppo mic obial g ow h, unless a ailable
nu ien s co ec his imbalance (Head e al. 2006).
The addi ion o e ilize s a e oil pollu ion e en s
has shown inc eased hyd oca bon deg ada ion a es in
cold en i onmen s (B agg e al. 1994) by enhancing
bo h sap ophy ic and hyd oca bon-u ilizing mic obio a
(Delille e al. 1998).
Tempe a u e in luences mic obial communi y s uc-
u e and me abolism (Nedwell 1999), especially in
high-la i ude egions (Ge des e al. 2005), bu i also
de e mines hyd oca bon physical s a e and bioa ailabil-
i y (Leahy & Colwell 1990). Low empe a u es inc ease
oil iscosi y and educe ola iliza ion o oxic sho -chain
alkanes, which delays biodeg ada ion (Leahy & Colwell
1990). Enzyma ic ac i i y is gene ally educed a low
empe a u e, esul ing in a dec ease in hyd oca bon
deg ada ion a es (Leahy & Colwell 1990).
Changes in bac e ial communi y s uc u e can be
moni o ed using cul u e-independen echniques, since
hyd oca bon-deg ading bac e ia equen ly do no g ow
on common cul u e media (Deppe e al. 2005). Commu-
ni y s uc u e has been moni o ed in cold en i onmen s
a e an oil exposu e, bu di e en hyd oca bon mix u es
ha e a ely been es ed unde he same condi ions
(B aks ad & Lødeng 2004; Ge des e al. 2005; B aks ad
e al. 2008). S udies ha e moni o ed changes in oil-
exposed communi y s uc u e o A c ic soil a di e en
empe a u es (E iksson e al. 2001) and in di e en
e iliza ion condi ions (Ye geau e al. 2009). Howe e ,
he simul aneous s udy o hese wo pa ame e s in
seawa e communi y s uc u e has been made in empe-
a e en i onmen s only (Coulon e al. 2007; Rod ı
´guez-
Blanco e al. 2010).
In a ecen wo k, we s udied he e ec o empe a u e
and e iliza ion on mic obial g ow h and hyd oca bon
deg ada ion in sub-An a c ic seawa e s o he Ke guelen
A chipelago exposed o c ude and diesel oil mix u es
(Delille e al. 2009). The p esen s udy analyses he
s uc u al changes occu ing in he bac e ial commu-
ni ies o he Ke guelen expe imen , using a cul u e-
independen echnique named capilla y-elec opho esis
single s and con o ma ion polymo phism (CE-SSCP;
Lee e al. 1996), a ge ing 16S DNA and 16S RNA.
The use o a high- h oughpu inge p in ing echnique,
such as he CE-SSCP, pe mi s he compa ison o a la ge
numbe o samples (Ghiglione e al. 2005), p o iding
an accu a e in e -sample analysis (Hong e al. 2007).
Recen ly, CE-SSCP has been shown o p o ide simila
esul s compa ed o o he inge p in ing echniques
(Ducklow e al. 2011) and o nex gene a ion sequencing
echniques (Ghiglione & Mu ay 2012).
Me abolically ac i e bac e ia con ain mo e RNA han
es ing o s a ed cells, and 16S RNA-based inge p in -
ing has been sugges ed as an indica o o he me aboli-
cally ac i e bac e ial communi y s uc u e (DeLong e al.
1989; Kemp e al. 1993; Poulsen e al. 1993). The
impo ance o ocusing no only on he o al bac e ial
communi y (based on 16S DNA) bu also on he
me abolically ac i e ac ion (based on 16S RNA) o
e alua e he in luence o en i onmen al ac o s on
bac e ial popula ions has been unde lined in p e ious
s udies (Scha e e al. 2001; T oussellie e al. 2002;
Moesenede e al. 2005; Gen ile e al. 2006; Lami e al.
2009; Rod ı
´guez-Blanco e al. 2009). To ou knowledge,
only one s udy used a combined 16S DNA/16S RNA
app oach o compa e dynamics o o al e sus me aboli-
cally ac i e bac e ial communi y s uc u e o hype saline
mic obial ma s in he con ex o hyd oca bons pollu ion
(Bo dena e e al. 2007). He e, we p esen he i s s udy
es ing he e ec s o empe a u e and e iliza ion on
bo h o al (DNA-based) and ac i e (RNA-based) bac e ial
communi ies inhabi ing sub-An a c ic seawa e s when
subjec ed o c ude oil and diesel uel.
Ma e ials and me hods
Expe imen al se -up
Seawa e was collec ed on 13 Ap il 2006 a he su ace, in
he Mo bihan Bay o he Ke guelen A chipelago (49822?
S, 70812?E), 2 km om he Po -au-F anc
¸ais scien i ic
s a ion. Samples we e immedia ely ca ied o he biolo-
gical labo a o y o his s a ion, whe e he expe imen s
we e conduc ed. Seawa e samples (600 mL) we e added
o s e ile 1L-E lenmeye lasks, oge he wi h 0.5 mL o
ei he A abian ligh c ude oil o diesel uel. Oil was added
alone o seawa e o wi h Inipol EAP 22 o ganic e ilize
(10% V/V; El A ochem, Colombes, F ance). The A abian
ligh c ude used in his expe imen was liquid pe oleum
wi h a high Ame ican Pe oleum Ins i u e g a i y (328),
a low pou poin o 288C and a low dynamic iscosi y
o 31 cP a 08C. The Inipol EAP22 is a mic o-emulsion
con aining bu oxye hanol, oleic acid and su ac an s wi h
a dynamic iscosi y o 250 cP a 208C and a pou poin
a 118C.
One lask o each ea men (i.e., non-amended sea-
wa e ; seawa e amended wi h c ude oil only; seawa e
amended wi h c ude oil and Inipol EAP 22; seawa e
amended wi h diesel uel only; seawa e amended wi h
S uc u e o o al e sus ac i e bac e ial communi ies exposed o hyd oca bons A. Rod ı
´guez-Blanco e al.
2
(page numbe no o ci a ion pu pose) Ci a ion: Pola Resea ch 2013, 32, 18521, h p://dx.doi.o g/10.3402/pola . 32i0.18521
diesel uel and Inipol EAP 22) was incuba ed a h ee
di e en empe a u es (20, 10 and 48C) o 7 weeks
unde o bi al s i ing.
DNA ex ac ion and pu i ica ion
A minimum o 50 mL o seawa e was p e- il e ed
h ough a 3 mm-po e il e (47 mm, Polyca bona e
Nuclepo e†, Wha man, Ken , UK) in o de o emo e
la ge o ganisms and hen il e ed h ough a 0.22-mm po e
il e (47 mm, Polyca bona e Nuclepo e) be o e being
s o ed a 808C un il analysis. Cellula lysis was
pe o med in wo s eps on a lysis bu e , con aining
50 mM T izma base (Sigma-Ald ich, S . Louis, MO, USA),
40 mM e hylenediamine e aace ic acid (Sigma-Ald ich)
and 0.75 M Suc ose (Sigma-Ald ich). Fi s , a eshly
p epa ed lysozyme solu ion ( inal concen a ion 1 mg
mL
1
) was added o he 0.22-mm po e il e and samples
we e incuba ed a 378C o 45 min. Second, sodium
dodecyl sulpha e ( inal concen a ion 1%) and p o ei-
nase K ( inal concen a ion 0.2 mg mL
1
) we e added
and ubes we e incuba ed a 508C o 1 h.
A olume o 600 mL o he esul ing cellula lysa e was
ea ed wi h 10 mL o a 100 mg mL
1
RNase A solu ion
(Qiagen, Hilden, Ge many) be o e DNA ex ac ion which
was ca ied ou using he DNeasy Tissue ki (Qiagen),
as desc ibed by he manu ac u e ins uc ions.
RNA ex ac ion and cDNA syn hesis
Samples o RNA analysis we e p ocessed in pa allel o
DNA ex ac ion as p e iously desc ibed (Ghiglione e al.
2009). B ie ly, he emaining 400 mL o cellula lysa e
was ea ed wi h DNase I o RNA ex ac ion using
an SV To al RNA Isola ion ki (P omega, Madison,
WI, USA). E ec i e DNA diges ion was e i ied be o e
cDNA syn hesis by nega i e 16S DNA polyme ase
chain eac ion (PCR) ampli ica ion (PCR condi ions as
epo ed below). RNA was e e se ansc ibed (RT)
in o single-s and cDNA using Moloney Mu ine Leuke-
mia Vi us (M-MLV) e e se ansc ip ase (P omega),
acco ding o he manu ac u e ’s ins uc ions. PCR am-
pli ica ion o cDNA was ca ied ou as o DNA (see
below).
PCR-CE-SSCP inge p in ing
The highly a iable V3 egion (E. coli 16S RNA gene
posi ions 329-533; B osius e al. 1981) o he 16S
ibosomal RNA gene (16S DNA) was ampli ied o
bo h DNA and cDNA ex ac s. The bac e ial uni e sal
p ime se w49 o wa d (5’ACG GTC CAG ACT CCT ACG
GG-3’; Delbe
`s e al. 2001) and w34 e e se (5’-TTA
CCG CGG CTG CTG GCA C-3’; Lee e al. 1996) we e
used. The e e se p ime was luo escen ly labelled wi h
5’- e achlo o- luo escein-CE phospho amidi e (TET,
Applied Biosys emsLi e Technologies, No walk, CT,
USA) a he 5’-end posi ion. PCR ampli ica ion o 50-mL
eac ions con aining empla e, p ime s ( inal concen a-
ion 0.25 mM), dNTPs (Eu ogen ec, Se aing, Belgium;
inal concen a ion 0.6 mM), P u DNA polyme ase (1 U,
P omega) and P u eac ion bu e was un in a Robocycle
he mocycle (S a agene, Agilen Technologies, San a
Cla a, CA, USA). PCR s eps we e ini ial dena u a ion a
948C o 1 min, ollowed by 25 cycles o dena u a ion a
948C o 1 min, annealing a 618C o 15 s and ex ending
a 728C o 30 s wi h a inal 10 min ex ension a 728C.
Size (ca. 200 bp leng h) and concen a ion o PCR
p oduc s we e de e mined by aga ose gel elec opho esis
(2%) wi h a DNA size s anda d (Low DNA Mass Ladde ,
GibcoLi e Technologies, Ca lsbad, CA, USA).
Successi e dilu ions in nuclease- ee s e ile wa e
we e made o ob ain 10 ng mL
1
o PCR p oduc . One
mL o he dilu ion was mixed wi h 18.9 mL o o mamide
and 0.1 mL o he in e nal size s anda d Gene-Scan-400
Rox (Applied Biosys ems), dena u ed a 948C o 5 min,
and immedia ely cooled on ice o 5 min be o e elec o-
kine ical injec ion (5 s, 12 kV) in o a capilla y ube (47
cm50 mm) illed wi h 5.6% o Genescan polyme in
a ABI P ism 310 Gene ic analyse (Applied Biosys ems).
Elec opho esis was ca ied ou a 15 kV o 30 min
sample
1
a 308C, and da a we e collec ed wi h ABI
P ism 310 collec ion so wa e (Applied Biosys ems).
TET-labelled agmen s we e de ec ed by a lase wi h a
i ual C il e (de ec ion wa eleng hs: 532, 537 and
584 nm).
CE-SSCP inge p in s compa a i e analysis
To al inge p in ing a ea was no malized among all he
aligned CE-SSCP p o iles ob ained using he in e nal
size s anda d Gene-Scan-400 Rox h oughou he S a is-
ical Analysis o Finge p in s Using Molecula Biology
(SAFUM) so wa e (Zemb e al. 2007). Simila i y ma-
ices based on B ay-Cu is dis ances and unweigh ed
pai g oup me hod wi h a i hme ic mean (UPGMA)
dend og ams we e gene a ed using he P ime -E5 so -
wa e package (Cla ke & Wa wich 2001). One-way
Anosim es s o signi ican di e ences in bac e ial com-
muni y s uc u e we e also pe o med using P ime -E5.
Peak de ec ion and Simpson index alues we e au o-
ma ically ob ained om each inge p in using he
SAFUM so wa e.
A. Rod ı
´guez-Blanco e al. S uc u e o o al e sus ac i e bac e ial communi ies exposed o hyd oca bons
Ci a ion: Pola Resea ch 2013, 32, 18521, h p://dx.doi.o g/10.3402/pola . 32i0.18521 3
(page numbe no o ci a ion pu pose)
Resul s
Compa a i e analysis o he s uc u e o o al
bac e ial communi ies (16S DNA) in samples
collec ed du ing mic ocosm expe imen s
16S DNA-based inge p in s o samples incuba ed wi h
he oleophilic e ilize clus e ed sepa a ely om hose
incuba ed only wi h c ude o diesel oil (Fig. 1a, clus e s
3 and 7, and Fig. 1b, clus e s 6 and 7), sugges ing
nu ien s addi ion o induce changes in o al bac e ial
communi y s uc u e. Howe e , hese changes occu ed
mos ly a high empe a u es (i.e., 108C and 208C), since
e ilize -en iched incuba ions made a low empe a u e
(i.e., 48C) induced mos ly no a ia ions in s uc u e (Fig.
1a, clus e s 3 and 4, and Fig. 1b, clus e s 4 and 5). In he
p esence o c ude oil, e ilize -induced bac e ial com-
muni y s uc u e changed be ween he i s and he
se en h week o incuba ion, wi h simila changes
obse ed a 10 and 208C (Fig. 1a, clus e s 3 and 7).
Con e sely, e ilize -induced s uc u e emained s able
o e ime in he p esence o diesel ( om he hi d week
a 108C), bu wi h sepa a ed clus e s ob ained a 10 and
208C (Fig. 1b, clus e s 6 and 7). Compiling all da a,
Fig. 1 Unweigh ed pai g oup me hod wi h a i hme ic mean dend og ams based on B ay-Cu is simila i ies among 16S DNA capilla y-elec opho esis
single s and con o ma ion polymo phism p o iles in he p esence o (a) A abian ligh c ude oil and (b) diesel uel incuba ions. The ini ial communi y
(TO); non-amended con ols (squa es); samples amended wi h c ude oil (ci cles) o c ude oil wi h Inipol EAP 22 (in e ed iangles) and samples
amended wi h diesel oil ( hombs) o diesel oil wi h Inipol EAP 22 ( iangles) a e shown o incuba ion empe a u es o 48C (whi e), 108C (g ey) and 208C
(black). Dashed lines indica e cu -o simila i y alue, which we e assigned o 5060% o adequa e clus e de ini ion.
S uc u e o o al e sus ac i e bac e ial communi ies exposed o hyd oca bons A. Rod ı
´guez-Blanco e al.
4
(page numbe no o ci a ion pu pose) Ci a ion: Pola Resea ch 2013, 32, 18521, h p://dx.doi.o g/10.3402/pola . 32i0.18521
Anosim es s showed weak s uc u al di e ences induced
by Inipol EAP 22 in diesel incuba ions (Global R0.145;
signi icance le el o sample s a is ic3.7%), and no
di e ences in c ude oil incuba ions. (Global R0.067;
signi icance le el o sample s a is ic16.4%).
Clus e s con aining mainly samples incuba ed a he
same empe a u e we e mo e equen ly obse ed in he
diesel- ela ed dend og am (Fig. 1b, clus e s 26) han in
he c ude oil- ela ed dend og am (Fig. 1a, clus e 6),
indica ing he in luence o empe a u e on o al commu-
ni y s uc u e o be mo e impo an in he p esence o
diesel. Anosim es s con i med hese empe a u e- ela ed
s uc u al di e ences in all diesel incuba ions (Global
R0.1; signi icance le el o sample s a is ic12.3%),
and o c ude oil incuba ions he absence o such
di e ences (Global R0.024; signi icance le el o sample
s a is ic33.3%).
In ac , a sligh di e en impac o oil mix u e alone
was obse ed o he o al bac e ial communi y s uc u e.
C ude oil-addi ion induced s uc u al changes a all
empe a u es, as shown by sepa a e clus e s con aining
con ols and c ude oil-amended samples (Fig. 1a, clus e s
1 and 2). Howe e , a simple diesel addi ion caused no
sys ema ic changes in he s uc u e o o al bac e ial
communi ies (Fig. 1b, clus e s 2 and 3).
The s uc u e o ini ial bac e ial communi ies was
simila o ha o non-amended con ols incuba ed
a 48C, om he i s o he se en h week o incuba ion
(Fig. 1a, b, clus e 1). This indica es a low impac o
con inemen in bac e ial communi y s uc u e along he
s udy.
Compa a i e analysis o he s uc u e o ac i e
bac e ial communi ies (16S RNA) in samples
collec ed du ing mic ocosm expe imen s
The 16S RNA-based dend og am p o ided di e en
esul s in he ongoing dynamics o he ac i e bac e ial
ac ion. Samples collec ed a e 1 and 3 weeks, including
non-amended con ols, s ongly esembled one ano he ,
cons i u ing well sepa a ed clus e s in bo h c ude oil
and diesel- ela ed dend og ams (Fig. 2a, clus e 1; Fig.
2b, clus e 2). The sample co esponding o he ini ial
communi ies clus e ed sepa a ely om hese clus e s
(Fig. 2a, b). Di e ences in ac i e communi y s uc u e
ela ed o ime o incuba ion we e signi ican in bo h
c ude oil (Global R0.228; signi icance le el o sample
s a is ic0.2%) and diesel incuba ions (Global R
0.498; signi icance le el o sample s a is ic0.1%).
Samples incuba ed in he p esence o e ilize p i-
ma ily clus e ed sepa a ely om hose incuba ed in
he p esence o c ude oil only (Fig. 2a, clus e s 1, 2, 5
and 6), whe eas hey mos ly clus e ed oge he wi h
hose incuba ed wi h diesel only (Fig. 2b, clus e s 2, 3
and 5). Thus, e iliza ion induced changes in ac i e
communi y s uc u e depending on he oil mix u e
es ed, bu empe a u e had a low in luence on s uc u e:
no empe a u e- ela ed clus e s we e ound ei he in
c ude oil o in diesel uel- ela ed 16S RNA-based
dend og ams (Fig. 2).
Compa a i e analysis o Simpson’s index alues
ob ained om CE-SSCP inge p in s o o al
(16S DNA) and ac i e (16S RNA) bac e ial
communi ies
Simpson’s index alues o 16S DNA and 16S RNA CE-
SSCP inge p in s ob ained om c ude oil and diesel
amended cul u es we e ela i ized o alues ob ained
om non-amended con ols (Fig. 3).
Rela i e Simpson’s index alues o o al bac e ial
communi ies (16S DNA) ini ially dec eased in he
p esence o bo h c ude oil and diesel a 108C and 208C
(Fig. 3a, c), whe eas his educ ion occu ed la e on a
48C (Fig. 3a, c), indica ing some in luence o empe a u e
in di e si y dynamics along he incuba ion pe iod.
Di e si y emained low a e se en incuba ion weeks
excep a 108C in he p esence o bo h c ude oil and
diesel. In he p esence o e ilize , he di e si y o he
whole communi y app oached con ol alues a e se en
incuba ion weeks, showing mino di e ences among
empe a u es (Fig. 3b, d).
Rega ding alues o ac i e bac e ial popula ions,
lowe di e si y educ ion was gene ally obse ed a e
c ude and diesel oil addi ion (Fig. 3e, g). Fe ilize
addi ion ended o educe di e si y, and his educ ion
occu ed ea lie a 48C (Fig. 3 , h).
Discussion
In a p e ious a icle based on he same expe imen as
desc ibed he e, we obse ed ha bac e ial g ow h and
hyd oca bon deg ada ion inc eased in he p esence o
e ilize Inipol EAP 22, whe eas empe a u e a ia ion
induced mino changes in g ow h and oil deg ada ion
(Delille e al. 2009). The p esen s udy analysed changes
in bac e ial communi y s uc u e du ing he same
expe imen , o unde s and whe he he obse ed quan-
i a i e changes in g ow h and biodeg ada ion also
co esponded o some quali a i e changes in communi y
s uc u e. Al hough empe a u e ange and subs a e
concen a ion ha e a c ucial ole o play in bac e ial
g ow h (Pome oy & Wiebe 2001; Ki chman e al. 2009),
bac e ial species ole a e di e en empe a u e anges.
A. Rod ı
´guez-Blanco e al. S uc u e o o al e sus ac i e bac e ial communi ies exposed o hyd oca bons
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Lowe g ow h empe a u es a e mos ly de ined by he
loss o memb ane unc ion, and uppe g ow h empe a-
u es a e de e mined by dena u a ion o key cellula
componen s (Nedwell 1999). In he p esence o hyd o-
ca bons, only some species a e esponsible o a signi i-
can pa o hyd oca bon emo al (Head e al. 2006),
sugges ing ha e iliza ion would s imula e he g ow h
o a limi ed ac ion o he bac e ial communi y, a leas
ini ially. Conside ing empe a u e a ia ion and subs a e
concen a ion oge he seems o be o high in e es ,
since in e ac ions be ween hese wo pa ame e s may
exis . Fo ins ance, subs a e up ake o assimila ion can
be al e ed by empe a u e (Pome oy & Wiebe 2001).
Inipol EAP 22 is an oleophilic e ilize ha supplies
o ganic nu ien s in a slow elease o m, bu i also
has su ac an cha ac e is ics educing oil iscosi y and
p e en ing he o ma ion o wa e -in-oil emulsions
(Ladousse & T amie 1991).
Ou esul s showed ha e ilize addi ion induced
s uc u al changes o he o al bac e ial communi y a
highe empe a u es (108C and 208C) in bo h c ude o
diesel oil condi ions, whe eas hey induced changes in
he ac i e bac e ial ac ion only in he p esence o c ude
oil. Tempe a u e had a mo e ema kable e ec on o al
communi y s uc u e in he p esence o diesel, while
no e ec was de ec ed in he s uc u e o he ac i e
Fig. 2 Unweigh ed pai g oup me hod wi h a i hme ic mean dend og ams based on B ay-Cu is simila i ies among 16S RNA DNA capilla y-
elec opho esis single s and con o ma ion polymo phism p o iles in he p esence o (a) A abian ligh c ude oil and (b) diesel uel incuba ions. The ini ial
communi y (TO); non-amended con ols (squa es); samples amended wi h c ude oil (ci cles) o c ude oil wi h Inipol EAP 22 (in e ed iangles) and
samples amended wi h diesel oil ( hombs) o diesel oil wi h Inipol EAP 22 ( iangles) a e shown o incuba ion empe a u es o 48C (whi e), 108C (g ey)
and 208C (black). Dashed lines indica e cu -o simila i y alue, which we e assigned o 5060% o adequa e clus e de ini ion.
S uc u e o o al e sus ac i e bac e ial communi ies exposed o hyd oca bons A. Rod ı
´guez-Blanco e al.
6
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communi y. These esul s poin o he di e en dynamics
o he o al communi y as opposed o he me abolically
ac i e bac e ial popula ions. The dis ibu ion o he o al
bac e ial communi y was mo e dependen on empe a-
u e a ia ion, e en in he p esence o Inipol EAP 22
when exposed o hyd oca bons. The dis ibu ion o ac i e
bac e ial membe s was mo e ela ed o ac o s playing an
impo an ole in deg ada ion in he Ke guelen expe i-
men , such as a ailabili y o nu ien s and hyd oca bon
composi ion, which is consis en wi h he ac ha
me abolically ac i e membe s a e esponsible o hyd o-
ca bon deg ada ion.
Compa able ends ha e been ound in a simila
expe imen in ol ing seawa e om he Medi e anean
Sea (Rod ı
´guez-Blanco e al. 2010), sugges ing a simila
in luence o empe a u e and nu ien s in he p esence o
hyd oca bons o bac e ial communi ies inhabi ing pola
and empe a e seawa e s. E en hough empe a u e
Fig. 3 Simpson’s index alues ela i e o (a)(d) non-amended con ols o 16S DNA and (e)(h) 16S RNA capilla y-elec opho esis single s and
con o ma ion polymo phism inge p in s ob ained om (a) and (e) c ude-oil-amended cul u es; (b) and ( ) cul u es amended wi h c ude oil and Inipol
EAP 22; (c) and (g) diesel-oil-amended cul u es; and (d) and (h) cul u es amended wi h diesel oil and Inipol EAP 22.
A. Rod ı
´guez-Blanco e al. S uc u e o o al e sus ac i e bac e ial communi ies exposed o hyd oca bons
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(page numbe no o ci a ion pu pose)
usually anges om 28C o88C in coas al seawa e o
he Ke guelen A chipelago (Delille e al. 2009), empe a-
u e can d op below ze o in win e and ise abo e 188C
on wa m summe days. P e ious s udies o Ke guelen
cul i able bac e ia sugges ha a di e se numbe o
op imum empe a u e anges could ha e been ep e-
sen ed a he ime o he expe imen (Delille & Pe e
1989), including ole an psych ophilic bac e ia (op imal
empe a u e be ween 78C and 208C), psych o ophic
bac e ia (be ween 48C and 308C) and ole an psy-
ch o ophic bac e ia (208C o abo e). The e o e, he
simila i y o beha iou be ween Medi e anean and
sub-An a c ic seawa e bac e ial communi ies in he
p esence o hyd oca bons may occu only in speci ic
seasonal pe iods.
Qui e di e en om he Ke guelen expe imen , s uc-
u al changes induced by e iliza ion a he RNA le el in
he Medi e anean expe imen occu ed in he p esence
o bo h c ude oil and diesel uel (Rod ı
´guez-Blanco e al.
2010). Ke guelen ac i e communi y s uc u e was less
in luenced by e iliza ion in diesel incuba ions (Fig. 2).
These quali a i e di e ences ela ed o hyd oca bon
mix u es in Ke guelen seawa e we e also obse ed in
some quan i a i e da a. Biodeg ada ion a es o less
ola ile hyd oca bons (C17/P is ane a io) indica ed
ha e ilize did no inc ease diesel biodeg ada ion
in he same way as i did o c ude oil (Delille e al.
2009). This could be ela ed o he di e en in luence o
di e en hyd oca bon mix u es in he Ke guelen bac e -
ial communi y s uc u e. O he ac o s, such as he abili y
o he Ke guelen bac e ial communi ies o deg ade
ce ain compounds, could explain hese di e ences
be ween he deg ada ion a es o c ude oil and diesel
uel.
Resul s o 16S RNA-based dend og am show an
impo an in luence o ime o incuba ion on he s uc-
u e o ac i e bac e ial communi ies (Fig. 2). These
dynamics we e no obse ed a he DNA le el (Fig. 1b).
Simila esul s ha e been obse ed using a combined
16S DNA/ RNA app oach o s udy he bac e ial com-
muni y o a p is ine mic obial ma exposed o hea y uel
oil (Bo dena e e al. 2007). These au ho s epo ed
ha changes o s uc u e we e obse ed ea lie a he
RNA le el han a he DNA le el. The ela ion among
s uc u e o he ac i e bac e ial communi y and ime o
incuba ion could be ela ed o he modi ica ion in he
hyd oca bons composi ion o o he empo al changes
occu ing in na u al en i onmen s as a esul o biode-
g ada ion (Head e al. 2006). Fo ins ance, alipha ic
hyd oca bons a e deg aded be o e high molecula weigh
a oma ic hyd oca bons (Head e al. 2006). Biodeg ada-
ion o di e en hyd oca bon molecules a di e en imes
is he esul o co-me abolic eac ions in ol ing di e en
bac e ial species (Deppe e al. 2005). Pelz e al. (1999)
showed ha ini ial hyd oca bon deg ading bac e ia we e
eplaced by o he popula ions wi h complemen a y
deg ada ion capaci ies, which could explain he empo al
changes obse ed in he p esen s udy o ac i e bac e ial
s uc u e in he p esence o hyd oca bons. Tempo al
clus e s o 16S RNA-based inge p in s we e also ob-
se ed in a simila s udy made on empe a e es ua ine
wa e s (Coulon e al. 2007). The e y low iscosi y and
pou poin o c ude oil used in his expe ience ensu ed i s
bioa ailabili y o mic oo ganisms om he beginning o
he expe imen . Inipol EAP22 is ela i ely mo e iscous
and has a high pou poin , bu i eadily mixed wi h oil,
did no change he low iscosi y o he mix u e and he
p esence o su ac an s g ea ly enhanced he bioa ail-
abili y o he oil.
These esul s highligh he alue o using a combined
16S DNA/ RNA app oach o s udy he dynamics o he
bac e ial communi y s uc u e in he con ex o hyd o-
ca bon pollu ion. Moni o ing s uc u al changes in he
bac e ial communi y in he p esence o hyd oca bons has
usually been unde aken wi h 16S DNA-based ap-
p oaches (Head e al. 2006). Howe e , limi a ions, such
as a ia ion in he DNA copy numbe among bac e ial
species ha could in oduce biased es ima ion o di e si y
and he lack o in o ma ion ela ed o cellula ac i i y,
make hese app oaches limi ed (Klappenbach e al. 2000;
Acinas e al. 2004). 16S RNA-based app oaches ha e
hei own limi a ions, including g ow h a e a ia ion in
na u al en i onmen s inducing a ia ion in he cellula
RNA con en (Gi sko e al. 1994). Combining 16S
DNA/ RNA app oaches p o ides a mo e comple e and
eliable desc ip ion o he bac e ial communi y s uc u e.
Mo eo e , he use o a high- h oughpu inge p in -
ing echnique, such as he CE-SSCP, allows he com-
pa ison o a high numbe o samples (92), p o iding
accu a e in e -sample analysis (Hong e al. 2007). The
use o a capilla y and s anda dized eagen wi h CE-
SSCP a oids p oblems ela ed o gel- o-gel a ia ion
(Hebenb ock e al. 1995) ha would make o he gel-
based echniques, such as dena u ing g adien gel
elec opho esis, less sui able o he compa a i e analy-
sis o ou samples (Hong e al. 2007). Fu he mo e, no
diges ion wi h es ic ion enzymes is equi ed in he
CE-SSCP echnique, compa ed o o he capilla y-based
inge p in ing me hods, such as e minal es ic ion
agmen leng h polymo phism, a oiding he pu a i e
incomple e diges ions and subsequen o ma ion o mul-
iple peaks pe ope a ional axonomic uni (Mills e al.
2003). The cu en s udy was pe o med unde ield
condi ions ha made he use o eplica es imp ac ical.
S uc u e o o al e sus ac i e bac e ial communi ies exposed o hyd oca bons A. Rod ı
´guez-Blanco e al.
8
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Despi e he lack o eplica es, he esul s om his s udy
showed ha impo an in o ma ion was ob ained bo h
a physicochemical and biological le els.
The Simpson di e si y index showed ha he addi ion
o c ude oil and diesel educed he di e si y in o al
bac e ial communi ies. Howe e , he educ ion was e-
a ded a 48C (Fig. 3). The di e si y o ac i e bac e ial
communi ies expe ienced less ma ked changes a e c ude
o diesel oil addi ion. These esul s a e consis en wi h
hose obse ed in he Medi e anean Sea (Rod ı
´guez-
Blanco e al. 2010). Howe e , e iliza ion inc eased
di e si y o Ke guelen o al communi ies and dec eased
ha o ac i e popula ions, which was di e en om
wha we obse ed in he Medi e anean Sea. This could
indica e ha among me abolically ac i e bac e ia a ew
membe s would bene i om e iliza ion o pe o m
hyd oca bon deg ada ion (Cappello e al. 2007).
The p esen s udy e idenced quali a i e di e ences
in o al and ac i e bac e ial communi y s uc u es in he
p esence o hyd oca bons and he di e en esponses
ela i e o a ia ion in empe a u e and e iliza ion. To
ully unde s and he dynamics o he bac e ial commu-
ni ies subjec ed o an oil spill in cold condi ions, a ia-
ions in hyd oca bon composi ion and e iliza ion ha e
o be aken in accoun .
Acknowledgemen s
ARB was a ilia ed wi h he Pie e and Ma ie Cu ie
Uni e si y and he Na ional Cen e o Scien i ic Re-
sea ch, in Banyuls-su -Me , F ance, a he ime he
esea ch epo ed he e was ca ied ou .
Re e ences
Acinas S.G., Ma celino L.A., Klepac-Ce aj V. & Polz M. 2004.
Di e gence and edundancy o 16S RNA sequences in
genomes wi h mul iple n ope ons. Jou nal o Bac e iology
186, 26292635.
A las R.M. & Ba ha R. 1972. Biodeg ada ion o pe oleum in
seawa e a low empe a u es. Canadian Jou nal o Mic o-
biology 18, 18511855.
Bo dena e S., Gon
˜i-U iza M.S., Caume e P. & Du an R.
2007. E ec s o hea y uel oil on he bac e ial communi y
s uc u e o a p is ine mic obial ma . Applied and En i on-
men al Mic obiology 73, 60896097.
B addock J.F., Ru h M.L., Ca e all P.H., Walwo h J.L. &
McCa hy K.A. 1997. Enhancemen and inhibi ion o
mic obial ac i i y in hyd oca bon-con amina ed A c ic soils:
implica ions o nu ien -amended bio emedia ion. En i on-
men al Science & Technology 31, 20782084.
B agg J.R., P ince R.C., Ha ne E.J. & A las R.M. 1994.
E ec i eness o bio emedia ion o he Exxon Valdez oil spill.
Na u e 368, 413418.
B aks ad O.G. & Lødeng A.G.G. 2004. Mic obial di e si y
du ing biodeg ada ion o c ude oil in seawa e om he
No h Sea. Mic obial Ecology 49,94103.
B aks ad O.G., Nons ad I., Faksness L.-G. & B and ik P.J.
2008. Responses o mic obial communi ies in A c ic sea-ice
a e con amina ion by c ude pe oleum oil. Mic obial
Ecology 55, 540552.
B osius J., Dull T.J., Slee e D.D. & Nolle H.F. 1981. Gene
o ganiza ion and p ima y s uc u e o a ibosomal RNA
ope on om Esche ichia coli.Jou nal o Molecula Biology 148,
107127.
Cappello S., Ca uso G., Zampino D., Mon icelli L.S., Maimone
G., Dena o R., T ipodo B., T oussellie M., Yakimo M. &
Giuliano L. 2007. Mic obial communi y dynamics du ing
assays o ha bou oil spill bio emedia ion: a mic oscale
simula ion s udy. Jou nal o Applied Mic obiology 102,
184194.
Cla ke K.R. & Wa wich R.M. 2001. P ime -E (5) compu e
p og am. Plymou h, UK: Na u al En i onmen al Resea ch
Council.
Coulon F., McKew B.A., Osbo n A.M., McGeni y T.J. &
Timmis K.N. 2007. E ec s o empe a u e and bios imula-
ion on oil-deg ading mic obial communi ies in empe a e
es ua ine wa e s. En i onmen al Mic obiology 9, 177186.
Delbe
`s C., Godon J.J. & Mole a R. 2001. Bac e ial and
a chaeal 16S DNA and RNA dynamics du ing an ace a e
c isis in an anae obic diges o ecosys em. FEMS Mic obiology
Ecology 35,1926.
Deppe U., Richnow H.H. & An anikian W.M.G. 2005.
Deg ada ion o c ude oil by an A c ic mic obial conso ium.
Ex emophiles 9, 461470.
Delille D., Basse
` es A. & Dessommes A. 1997. Seasonal
a ia ion o bac e ia in sea ice con amina ed by diesel uel
and dispe sed c ude oil. Mic obial Ecology 33,97105.
Delille D., Basse
` es A., Dessommes A. & Rosie s C. 1998.
In luence o dayligh on po en ial biodeg ada ion o diesel
and c ude oil in An a c ic seawa e . Ma ine En i onmen al
Resea ch 45, 259268.
Delille D., Pelle ie E., Rod ı
´guez-Blanco A. & Ghiglione J.F.
2009. E ec s o nu ien and empe a u e on deg ada ion o
pe oleum hyd oca bons in sub-An a c ic coas al seawa e .
Pola Biology 32, 15211528.
Delille D. & Pe e E. 1989. In luence o empe a u e on
g ow h po en ial o sou he n pola bac e ia. Mic obial Ecology
18, 117123.
DeLong E.F., Wickham G.S. & Pace N.R. 1989. Phylogene ic
s ains: ibosomal RNA-based p obes o he iden i ica ion o
single cells. Science 243, 13601363.
Ducklow H.W., Mye s K.M.S., E ickson M., Ghiglione J.F. &
Mu ay A.E. 2011. Response o a summe ime An a c ic
ma ine bac e ial communi y o glucose and ammonium
en ichmen . Aqua ic Mic obial Ecology 64, 205220.
E iksson M., Ka J.O. & Mohn W.W. 2001. E ec s o low
empe a u e and eeze haw cycles on hyd oca bon biode-
g ada ion in A c ic und a soil. Applied and En i onmen al
Mic obiology 67, 51075112.
A. Rod ı
´guez-Blanco e al. S uc u e o o al e sus ac i e bac e ial communi ies exposed o hyd oca bons
Ci a ion: Pola Resea ch 2013, 32, 18521, h p://dx.doi.o g/10.3402/pola . 32i0.18521 9
(page numbe no o ci a ion pu pose)