FEMS Mic obiology Ecology,92,2016, iw048
doi: 10.1093/ emsec/ iw048
Ad ance Access Publica ion Da e: 2 Ma ch 2016
Resea ch A icle
RESEARCH ARTICLE
Pho ochemical al e a ion o dissol ed o ganic ma e
and he subsequen e ec s on bac e ial ca bon
cycling and di e si y
Ch is ian Lønbo g1,2,∗,Ma Nie o-Cid
3, Vic o He nando-Mo ales4,
Ma a He n´
andez-Ruiz4, E a Tei a4and Xos´
eAn ´
on ´
Al a ez-Salgado3
1Aus alian Ins i u e o Ma ine Science, PMB 3, Towns ille MC, QLD 4810, Aus alia, 2Cen e o Sus ainable
Aqua ic Resea ch, College o Science, Wallace Building, Swansea Uni e si y, Swansea SA2 8PP, UK, 3CSIC,
Ins i u o de In es igaci´
ons Ma i ˜
nas, Edua do Cabello 6, 36208 Vigo, Spain and 4Depa amen o de Ecolox´
ıa e
Biolox´
ıa Animal, Uni e sidade de Vigo, 36200 Vigo, Spain
∗Co esponding au ho : Aus alian Ins i u e o Ma ine Science, PMB 3, Towns ille MC, QLD 4810, Aus alia. Tel: +61-7-4753-4382; Fax: +61-7-4772-5852;
E-mail: [email p o ec ed]
One sen ence summa y: Sunligh exposu e impac s di e en o ganic ma e sou ces di e en ly, which impac s he physiology and communi y
composi ion o he bac e ia deg ading i .
Edi o : Ga y King
ABSTRACT
The impac o sola adia ion on dissol ed o ganic ma e (DOM) de i ed om 3 di e en sou ces (seawa e , eelg ass lea es
and i e wa e ) and he e ec on he bac e ial ca bon cycling and di e si y we e in es iga ed. Seawa e wi h DOM om he
sou ces was i s ei he kep in he da k o exposed o sunligh (4 days), a e which a bac e ial inoculum was added and
incuba ed o 4 addi ional days. Sunligh exposu e educed he colou ed DOM and ca bon signals, which was ollowed by a
p oduc ion o ino ganic nu ien s. Bac e ial ca bon cycling was highe in he da k compa ed wi h he ligh ea men in
seawa e and i e samples, while highe le els we e ound in he sunligh -exposed eelg ass expe imen . Sunligh
p e-exposu e s imula ed he bac e ial g ow h e iciency in he seawa e expe imen s, while no impac was ound in he
o he expe imen s. We sugges ha hese esponses a e connec ed o di e ences in subs a e composi ion and he
p oduc ion o ee adicals. The bac e ial communi y ha de eloped in he da k and sunligh p e- ea ed samples di e ed
in he seawa e and i e expe imen s. Ou indings sugges ha impac o sunligh exposu e on he bac e ial ca bon
ans e and di e si y depends on he DOM sou ce and on he sunligh -induced p oduc ion o ino ganic nu ien s.
Keywo ds: dissol ed o ganic ma e ; sola adia ion; bac e ial di e si y; bac e ial ca bon demand; bac e ial g ow h e iciency
INTRODUCTION
Mic obial ac i i y in aqua ic sys ems is mainly egula ed by he
ene gy and nu ien s con ained wi hin he dissol ed o ganic
ma e (DOM) pool (Hedges 2002). Coas al wa e s a e he mos
p oduc i e and biogeochemically ac i e ma ine ecosys ems, and
he e o e play key oles in he p oduc ion and deg ada ion o
DOM (Wollas 1998; Lønbo g and ´
Al a ez-Salgado 2012). DOM
in coas al wa e s o igina es om ei he au och honous o al-
loch honous sou ces; au och honous DOM is p oduced wi hin
he sys em, p ima ily by mac ophy es (Sønde gaa d 1981)and
plank onic o ganisms (Kawasaki and Benne 2006; Lønbo g
e al. 2009), whe eas alloch honous DOM is mainly o e es-
ial o igin (Sobczak e al. 2005). The combined e ec s o bo h
Recei ed: 26 Oc obe 2015; Accep ed: 28 Feb ua y 2016
C
FEMS 2016. All igh s ese ed. Fo pe missions, please e-mail: jou nals.pe [email protected]
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2FEMS Mic obiology Ecology, 2016, Vol. 92, No. 5
pho ochemical and mic obial p ocesses ha e been conside ed
he main ac o esponsible o he deg ada ion o na u al DOM
(T an ik and Be ilson 2001). In addi ion, bo h p ocesses a e also
able o p oduce ecalci an DOM wi h li e imes o yea s o mil-
lennia (Mille and Mo an 1997; Benne and Biddanda 1998;Obe -
nos e e , Rei ne and He ndl 1999). The e o e, DOM in coas al
wa e s is a complex mix u e o exuda es, leacha es, and deg a-
da ion and condensa ion p oduc s ha a ies widely in elemen-
al composi ion and molecula s uc u e and, consequen ly, in
eac i i y.
Gi en ha he e o ophic bac e ia a e he majo biological
DOM sink, hey egula e whe he he deg aded compounds a e
used o biomass o ene gy p oduc ion. The a io be ween bac e-
ial p oduc ion (BP) and he sum o BP and bac e ial espi a ion
(BR) has been e med he bac e ial g ow h e iciency (BGE). This
a io a ies widely ( om <1 o 90%) depending on he bac e ial
communi y composi ion (Rein hale , Win e and He ndl 2005),
nu ien and DOM bioa ailabili y (e.g. Ri kin and Ande son 1997;
Apple and del Gio gio 2007; Lønbo g e al. 2010a), wa e empe a-
u e (Ri kin and Legend e 2001) and UV-ligh exposu e (Lønbo g
e al. 2013). The he e o ophic bac e ial communi y consis s o
membe s om a ious phylogene ic lineages, wi h he dis ibu-
ion and abundance o hese species being con olled by bo h bi-
ological and chemical ac o s (Fuh man e al. 2006; Gio annoni
and Ve gin 2012). Howe e , much less is known abou how shi s
in communi y s uc u e may in luence he mic obial ca bon cy-
cling.
Pho ochemical eac ions induced by sola adia ion, espe-
cially in he UV ange o he spec um (UV-B, 280–315 nm; UV-A,
315–400 nm), a e pa icula ly impo an in coas al wa e s wi h
high loads o colou ed alloch honous DOM, whe e hey ha e
been shown o ans o m DOM in o labile ino ganic (mainly
NH4+and HPO42–) and o ganic (e.g. amino acids) compounds
ha can suppo bac e ial espi a ion and biomass p oduc ion
(e.g. Mo an and Zepp 1997; Obe nos e e and Benne 2004). In
addi ion, pho ochemical p ocesses ha e also been shown o
enhance he c oss-linking, humi ica ion and polyme iza ion o
labile biomolecules in o mo e ecalci an compounds (Kiebe
e al. 1997; Benne and Biddanda 1998; Obe nos e e , Rei ne and
He ndl 1999). These sunligh -induced eac ions can be seen as
he abio ic coun e pa o he mic obial ca bon pump, which
sugges s ha he mic obial u iliza ion o o ganic ma e is e-
la ed o he elease o ecalci an compounds ha accumu-
la e in he ocean (Jiao e al. 2010). Sola adia ion has also been
ound o mine alize DOM di ec ly o ino ganic ca bon species
(CO2o CO), ee adicals and eac i e oxygen species (ROS; e.g.
H2O2)(e.g.Coope e al. 1989;Mille andZepp1995). These com-
bined pho ochemical eac ions ha e been demons a ed o ha e
a complex impac on he mic obial communi y ac i i y esul ing
in enhanced, nega i e, mixed o no e ec (see Moppe , Kiebe
and S ubbins 2015 o o e iew). Some s udies hypo hesize ha
hese a iable esponses o sunligh exposu e a e linked wi h
he DOM o igin wi h ecen ly p oduced au och honous DOM ge -
ing less and alloch honous DOM mo e bioa ailable o bac e-
ia upon i adia ion, while o he s ha e ound he con a y (e.g.
Benne and Ziegle 2000; Sulzbe ge and Du isch-Kaise 2009).
While nume ous s udies ha e add essed he link be ween DOM
pho ochemis y and bac e ial g ow h, only a ew (i any) in es i-
ga ed how changes in DOM sou ces and sunligh exposu e in lu-
ence he bac e ial ac i i y and di e si y in coas al wa e s. Since
he impac o pho ochemis y on DOM and bac e ial ac i i y de-
pends on he chemical composi ion, changes in he exposu e o
sunligh and DOM sou ce could impac he mic obial commu-
ni y di e en ly.
In his s udy, we assessed he impac o pho o-al e a ion
on DOM de i ed om speci ic aqua ic sou ces (seawa e , eel-
g ass and i e wa e ) and he subsequen impac o his ex-
posu e on he bac e ial ca bon cycling and communi y s uc-
u e. We hypo hesized ha he bac e ial communi y esponse
will a y depending on he DOM sou ce and sunligh exposu e.
This hypo hesis was es ed using labo a o y incuba ions whe e
sunligh -al e ed DOM was added o su ace seawa e om he
coas al upwelling a ea o he R´
ıa de Vigo (N. W. Ibe ian Penin-
sula) and changes in colou ed dissol ed o ganic ma e (CDOM)
op ical p ope ies and bac e ial abundance, ac i i y (p oduc ion
and espi a ion) and di e si y we e measu ed o e a pe iod o
4 days.
MATERIALS AND METHODS
Si e desc ip ion and sample ea men s
In o de o de e mine he impac o sunligh on speci ic DOM
sou ces and he subsequen e ec s o he addi ion o hese
ma e ials on he bac e ial communi y o he R´
ıa de Vigo, we
collec ed DOM om 3 di e en sou ces: ma ine su ace wa e ,
i e wa e and DOM leached om lea es o he eelg ass Zos e a
ma ina.
Su ace seawa e was collec ed in he coas al upwelling
sys em o he R´
ıa de Vigo. This coas al embaymen is in lu-
enced by wind-d i en upwelling and downwelling episodes,
wi h no he ly winds esul ing in upwelling, which p e ailed
du ing ou sampling pe iod (la e sp ing). The seawa e sample
was collec ed on 30 May 2012 in he middle o he R´
ıa de Vigo,
a sui able si e o e alua ing he in luence on he whole embay-
men (Noguei a, P´
e ez and R´
ıos 1997). The wa e was collec ed
a 5 m dep h using a 12-L acid-cleaned Niskin bo le and com-
bined in o a 50 L acid-washed and aged polye hylene con aine .
A e collec ion, he sample wa e was kep in he da k un il p o-
cessed a he base labo a o y. Wa e empe a u e was measu ed
immedia ely a e collec ion, while aliquo s o he analysis o
salini y we e collec ed and measu ed in he labo a o y using an
Au osal 8400A. Ma e ial o chlo ophyll a(Chl a) de e mina ion
was collec ed by il e ing seawa e (200 mL) h ough a GF/F il e
and analysed a e 90% ace one ex ac ion wi h a Tu ne Designs
10000R luo ome e .
Seawa e il a ions we e s a ed wi hin 1 h a e collec ion.
One pa was il e ed h ough p e-combus ed (450◦C o 4 h)
GF/C il e s o es ablish a mic obial cul u e o be used in all ex-
pe imen s. This was kep in he da k a 15◦C un il use. These
we e he same condi ions used o he incuba ion s udy and
ensu ed ha he added mic obial communi y was adap ed o
hese condi ions. The changes measu ed du ing ou expe i-
men s in he mic obial communi y was he e o e only due o he
changes in he o ganic ma e sou ces and/o sunligh exposu e.
The o he pa o he seawa e was g a i y il e ed h ough a
dual-s age (0.8/0.2 µm) il e ca idge (Pall-Ac opak Supo mem-
b ane), which had been p e-washed wi h Milli-Q (>10 L). The
seawa e was he ea e used bo h as he con ol ea men and
o dilu e he DOM ob ained om he i e wa e and eelg ass
lea es.
In o al 20 L o i e wa e was collec ed in he main ib-
u a y o he R´
ıa de Vigo, Ri e Oi ab´
en-Ve dugo. This i e is
no signi ican ly a ec ed by indus ial o sewage was e, has a
d ainage a ea o 350 km2and ecei es a ain all o 2500 mm
pe yea , which esul s in an a e age low o 15 m3s–1 (Gago
e al. 2005). The wa e samples we e aken in he ups eam
limi o he eshwa e –seawa e in e ace. A e collec ion he
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Lønbo g e al.3
sample wa e was kep in he da k un il il a ion commenced
a he base labo a o y, abou 2 h la e . The i e wa e was
g a i y il e ed h ough a dual-s age (0.8/0.2 µm) il e ca idge
(Pall-Ac opak supo Memb ane) and he ea e concen a ed in
a p opo ion o 1:10 using a me al- ee angen ial low ul a il a-
ion sys em, p o ided wi h a GH2540F30 memb ane (GE Powe &
Wa e – Wa e & P ocess Technologies). Only he DOM ma e ial
wi h a molecula weigh >1 kDa ( ep esen ing 72% o he o al
pool) was used in his s udy.
F esh lea es o he eelg ass Zos e a ma ina we e collec ed du -
ing low ide in he San Simon bay, in he inne mos pa o he
R´
ıa de Vigo. Wi hin an hou o collec ion, he lea es we e b ough
back o he base labo a o y and insed ho oughly wi h he 0.2
µm- il e ed seawa e collec ed in he middle o he R´
ıa de Vigo.
The eelg ass-de i ed DOM was he ea e ex ac ed by adding
app ox. 5 g o we lea es o a glass bo le con aining 1 L o he
0.2 µm- il e ed seawa e . A e ex ac ion (48 h in he da k) he
wa e was il e ed i s h ough a p e-combus ed (450◦C o 4 h)
GF/D il e and hen h ough a dual-s age (0.8/0.2 µm) il e ca -
idge (Pall-Ac opak Supo memb ane).
Expe imen al design
The DOM de i ed om i e wa e and eelg ass lea es we e
added o 10 L polye hylene ca boys, con aining 7 L o seawa e ,
in o de o each a dissol ed o ganic ca bon (DOC) inc ease o
abou 40 µmol L–1, while he con ol seawa e did no ecei e any
addi ion. The samples we e he ea e di ided in o 2 expe imen-
al ea men s: da k ( e med ‘da k’) and ull sunligh ea men
( e med ‘UV’). The da k ea men s we e es ablished by placing
he sample wa e in o UVR- anspa en low densi y polye hy-
lene incuba o s (3.5 L in each) ha we e co e ed wi h aluminium
oil and da k plas ic bags, while he UV samples we e dis ibu ed
in o UVR- anspa en low densi y polye hylene incuba o s ha
we e le unco e ed. No headspace was le in ei he da k o UV
ea men s. The samples we e he ea e placed in a eci cula-
ion wa e ba h (wa e dep h: 25 cm) in he e ace o he labo a-
o y and exposed o 100% na u al sunligh o 4 days encompass-
ing he na u al ligh –da k cycle. Du ing he sunligh exposu e
he incuba o s we e almos comple ely co e ed in wa e (a ound
85%). The empe a u e was no speci ically con olled du ing he
exposu e pe iod bu a cons an low (app ox. 3 L min−1) o cold
ap wa e cooled he samples. This ensu ed ha he empe a-
u e was kep cons an ly low, sugges ing ha changing empe a-
u e did no impac ou UV exposu e esul s. Inciden i adiance
du ing he UV ea men s was aken om he me eo ological ob-
se a o y on he e ace o he base labo a o y, showing ha o e
he 4 days he UV samples we e exposed o 20 MJ m–2 d–1 o o-
al sola adia ion. Be o e and a e sunligh exposu e, subsam-
ples we e collec ed o he analysis o DOC, dissol ed ino ganic
ni ogen (DIN: NH4+,NO
2–,NO
3–), dissol ed ino ganic phospho-
us (DIP: HPO42–) and DOM op ical p ope ies (abso p ion and in-
duced luo escence). A e he 4 days o sunligh exposu e he
wa e samples we e wi hin 15 min combined in o di e en (da k
and UV) ca boys and he seawa e mic obial communi y was
added in a a io o 1 pa o mic obial cul u e, es ablished om
seawa e collec ed in he R´
ıa de Vigo on 30 May 2012, o 9 pa s
o exposed wa e . The wa e was he ea e ans e ed in o i -
een 500 mL glass bo les pe ea men (90 bo les in o al) and
incuba ed in he da k a a cons an empe a u e o 15◦C, wi h
3 eplica e bo les being used o sub-sampling a incuba ion
imes 0, 1, 2, 3 and 4 days. The p ocessing o he samples a ini-
ial ime poin s a ed app oxima ely 1 h a e comple ion o he
sunligh exposu e. Un il e ed wa e om hese bo les was used
o ollow changes in bac e ial abundance (BA), di e si y (using
au oma ed RNA in e genic space analysis (ARISA)), bac e ial
p oduc ion (BP) and bac e ial espi a ion (BR). Samples o he
analysis o DOC, DIN, DIP and CDOM abso p ion and luo es-
cence we e collec ed a e il a ion h ough 0.2 µm il e s (Pall
Supo memb ane Disc), which we e placed in an acid-cleaned
all-glass il a ion sys em unde low N2 low p essu e. All glass-
wa e used was acid washed in 10% HCl and insed wi h Milli-Q
and sample wa e p io o use.
Sample analysis
Samples o BA we e ixed wi h 25% glu a aldehyde (0.5% inal
concen a ion) o 30 min a 4◦C, lash- ozen in liquid ni ogen
and s o ed a –80◦C un il analysed. Thawed samples we e di-
lu ed up o 10- old in au ocla ed 0.2 µm il e ed TE bu e (10:1
T is–EDTA, pH 8.0) and s ained wi h he nucleic acid-speci ic dye
SYBR G een I (In i ogen/Molecula P obes) o 15 min in he
da k and analysed using a FACSCalibu low cy ome e . Bac e ial
biomass (BB) was calcula ed om BA, using a ca bon con e sion
ac o o 12 g C cell–1, which is ep esen a i e o coas al bac e-
ial assemblages (Fukuda e al. 1998).
BP was measu ed by [3H] hymidine inco po a ion (Fuh man
and Azam 1980). Th ee eplica e 9.9-mL samples and 2
ichlo oace ic acid killed samples we e added o an aqueous
s ock solu ion o [3H-me hyl] hymidine (20 nmol inal concen-
a ion). The samples we e incuba ed in he da k a 15◦C o 1
h, 10 mL o ice-cold ichlo oace a e (TCA) was he ea e added
and samples we e il e ed on o 0.2 µmpolyca bona e il e s(p e-
soaked in non-labelled hymidine) and washed wi h 95% e hanol
and au ocla ed Milli-Q wa e . The il e s we e he ea e d ied a
oom empe a u e (24 h) and mixed wi h 10 mL o scin illa ion
luid (Sigma-Fluo ). The adioac i i y inco po a ed in o cells was
coun ed using a Wallac scin illa ion coun e . The disin eg a ions
pe minu e (DPM) o he TCA-killed blank we e sub ac ed om
he DPMs o he samples. Thymidine inco po a ed in o bac e ial
biomass was con e ed o ca bon p oduc ion using he heo e -
ical con e sion ac o s, 2 ×1018 cells mol–1 hymidine (Fuh man
and Azam 1980) and using he same cell- o-ca bon con e sion
ac o as o BA.
The bac e ial espi a ion (BR) was es ima ed using he e-
duc ion o 2-(4-iodophenyl)-3-(4-ni o-phenyl)-5-phenyl e a-
zolium chlo ide (INT) ollowing Ma ´
ınez-Ga c´
ıa e al.(2009). In
b ie , he ac i i y was measu ed using 1 h incuba ions o 3 epli-
ca e samples (10 mL) and 1 o maldehyde-killed con ol. The in-
cuba ions we e e mina ed by adding o maldehyde and il e -
ing on o 0.2 µmpolyca bona e il e s.The il e swe e he ea e
s o ed ozen (–20◦C) un il u he p ocessing. The espi a ion
a es de i ed om INT educ ion (BR, in µmol O2L–1 h–1)we e
ob ained by mul iplying he in i o INT educ ion a e (in µmol
INTF L–1 h–1) by an empi ically de i ed con e sion ac o o 12.8.
The ins an aneous bac e ial ca bon demand (BCD) was cal-
cula ed as he sum o C-con e ed BP and BR:
BCD =BP +BR (1)
The ins an aneous bac e ial g ow h e iciency (BGE) was cal-
cula ed as BP di ided by he sum o BP and BR:
BGE =BP/(BP +BR)(2)
The in eg a ed BCD (BCDin ) o e he incuba ion pe iod was
calcula ed as he bioa ailable DOC (BDOC):
BCDin =BDOC (3)
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The in eg a ed BGE o e he 4 days (BGEin )wascalcula edas
he ne g ow h in bac e ial biomass be ween day 0 and maxi-
mum abundance (BG; BG =BBmax – BBmin) di ided by BDOC:
BGEin =BG/BDOC (4)
The DOC samples we e collec ed in o p e-combus ed (450◦C,
12 h) glass ampoules and p ese ed wi h 50 µL25%H
2PO4
o 10 mL sample. DOC was measu ed using a Shimadzu TOC
analyse (P ca alys ) and 3 o 5 eplica e injec ions o 150 µL
we e pe o med pe sample. Concen a ions we e de e mined
by sub ac ing a Milli-Q blank and di iding by he slope o
a daily s anda d cu e o po assium hyd ogen ph hala e. Us-
ing he deep ocean e e ence p o ided by P o . D. A. Hansell,
Uni e si y o Miami (Sa gasso Sea deep wa e 2600 m) we ob-
ained a concen a ion o 46.0 ±2.0 µmol L–1 (mean ±SD), wi h
he DOC alue p o ided by he e e ence labo a o y being 44.0 ±
1.5 µmol L–1.Thedi e encebe ween heini ial(DOC
0)andmin-
imum DOC (DOCmin) concen a ion o e he 4 days’ incuba ion
was he e de ined as he bioa ailable DOC (BDOC).
DIN and DIP samples we e collec ed in o 50 mL acid-washed
polye hylene bo les and measu ed using s anda d segmen ed
low analysis (SFA) as desc ibed in Hansen and Ko ole (1999).
Abso p ion spec a (250–600 nm) o CDOM we e measu ed on
a Beckman Coul e DU 800 spec opho ome e using Milli-Q wa-
e as a blank. Be o e analysis samples we e wa med o oom
empe a u e and abso bance was measu ed wi h 1 nm inc e-
men s using a 10 cm qua z cu e e. The abso p ion coe icien
a any wa eleng h, aCDOM(λ)(m
–1), was calcula ed as:
aCDOM (λ)=2.303·[Abs (λ)−Abs (600 −750)] /0.1(5)
Whe e Abs(λ) is he abso bance a wa eleng h λ;Abs(600–750)
is he a e age abso bance be ween 600 and 750 nm, which co -
ec s o he esidual sca e ing by ine size pa icle ac ions,
mic o-ai bubbles o colloidal ma e ial p esen in he sample,
o e ac i e index di e ences be ween he sample and he e -
e ence (m–1); he ac o 2.303 con e s om decadic o na u al
loga i hms; and he denomina o (0.1) is he cell pa h-leng h in
me es. The es ima ed de ec ion limi o his spec opho ome-
e is 0.001 abso bance uni s o 0.02 m−1. As he ea men s had
di e en DOC concen a ions, we s anda dize he CDOM abso p-
ion by calcula ing he C-speci ic abso p ion coe icien CDOM
abso p ion spec a (a∗CDOM(λ)). The C-speci ic abso p ion coe i-
cien a 254 nm, known as SUVA (Weishaa e al. 2003), was cal-
cula ed by di iding he decadic aCDOM(254) by he DOC concen-
a ion and exp essed in li es pe millig am pe me e.
The CDOM spec al slope (S) o he C-speci ic abso p ion
spec a was modelled as:
a∗CDOM(λ)=a∗CDOM (375)·e−S·(λ−375) (6)
whe e a∗CDOM(λ)is heca bon-speci icabso p ioncoe icien a
wa eleng h λ,a∗CDOM(375) is he C-speci ic abso p ion coe icien
a he e e ence wa eleng h o 375 nm, and Sis he spec al slope
coe icien o he abso p ion cu e calcula ed o e he ange 250–
600 nm. The CDOM spec al slopes we e also calcula ed o e wo
na ow wa eleng h anges, S(275–295) and S(350–400), using lin-
ea eg essions o he na u al log- ans o med aCDOM(λ)spec a.
These slopes we e used o calcula e he CDOM spec al slope
(S(275–295)/S(350–400)) a io (SR) (Helms e al. 2008).CDOM luo-
escence emission exci a ion ma ices (EEMs) and single-poin
measu emen s we e pe o med on a Pe kin Elme LS 55 lumi-
nescence spec opho ome e equipped wi h a xenon discha ge
lamp, equi alen o 20 kW o 8 µsdu a ion.Measu emen swe e
pe o med a a cons an empe a u e o 20◦Cina1cmqua z
luo escence cell. The EEMs we e gene a ed by combining 22
luo escence emission spec a om 300 o 560 nm a exci a-
ion wa eleng hs anging om 240 o 450 nm a 10 nm in e -
als. Poin exci a ion/emission (Ex/Em) measu emen s we e pe -
o med a wa eleng hs cha ac e is ic o peak-A (gene al humic-
like subs ances, a e age Ex/Em, 250 nm/435 nm), peak-C ( e -
es ial humic-like subs ances, a Ex/Em wa eleng hs o 340
nm/440 nm), peak-M (ma ine humic-like subs ances, a e age
Ex/Em, 320 nm/410 nm) and peak-T (p o ein-like subs ances, a -
e age Ex/Em, 280 nm/320 nm) (Coble 1996; Lønbo g e al. 2010b).
The luo escence measu emen s we e no malized o he Raman
a ea using daily spec a o Milli-Q wa e (Lawae z and S edmon,
2009). As he ea men s had di e en DOC concen a ions, we
s anda dize he FDOM alues by calcula ing he ca bon-speci ic
FDOM as he FDOM signal di ided by he DOC concen a ion.
The limi o de ec ion, calcula ed as 3 × he s anda d de ia ion
o he blank, was 0.03 QSU o peak-A, 0.05 QSU o peak-C and
0.02 QSU o peak-M and peak-T.
Au oma ed RNA in e genic space analysis
Au oma ed RNA in e genic space analysis (ARISA) was
conduc ed wi h DNA om he ini ial communi y and a he
end o each expe imen , o cha ac e izing he ini ial and
inal mic obial communi y o each ea men . Fo he ini-
ial communi y, samples we e p e il e ed h ough a 1.2 µm
po e-size il e (Kleenpak Capsule HDCII), and subsequen ly
2 L was il e ed on a 0.2 µmpo e-sizepolyca bona e il e
(Nuclepo e Wha mann, 47 mm il e diame e ). A he end o
he expe imen s 0.5–1 L om each ea men was il e ed on a
0.2 µm po e-size polyca bona e il e (Nuclepo e Wha mann,
47 mm il e diame e ). Fil e s we e hen s o ed a –80◦C un il
DNA ex ac ion. Mic obial communi y DNA was ex ac ed
using Ul a Clean Soil DNA isola ion ki (MoBio Labo a o ies,
Inc.) and quan i ied in a Nanod op. Bac e ial ARISA was pe -
o med using ITSF/ITSReub p ime se (The mo Scien i ic)
p e iously desc ibed by Ca dinale e al. (2004). The PCR eac-
ion (25 µL) con ained inal concen a ions o 1x PCR bu e
(Genec a ), 2.5 mmol L–1 MgCl2(Genec a ), 250 µmol L–1 o
each dNTP (Genec a ), 250 nmol L–1 o uni e sal p ime ITSF
(5′-GTCGTAACAAGGTAGCCGTA-3′)andeubac e ialITSReub
(5′-GCCAAGGCATCCACC-3′) (Ca dinale e al. 2004), he o me
being labelled a he 5′end wi h he luo escein amidi e dye (6-
FAM), 40 ng µL–1 bo ine se um albumin, 3.5 U o BioThe mD-TM
Taq DNA Polyme ase (GeneC a ) and app ox. 0.13 ng µL–1 o
empla e DNA. The eac ion mix u e was held a 94◦C o 2 min
ollowed by 32 cycles o ampli ica ion a 94◦C o 15s,55
◦C o
30 s and 72◦C o 3 min, wi h a inal ex ension o 72◦C o 10
min. PCR samples we e conduc ed by duplica es o each DNA
ex ac ion ( his compensa es o any anomalously unning ag-
men s bo h in he samples and in he s anda ds). Ampli ica ion
p oduc s we e mig a ed by capilla y elec opho esis on a 50
cm capilla y ABI P ism 3730XL DNA analyse (Applied Biosys-
ems) a Genosc een (www.genosc een. /). The s anda dized
mig a ion cock ail con ained 0.5 µL o ampli ica ion p oduc ,
0.25 µL o in e nal size s anda d LIZ 1200 (20–1200 pb, Applied
Biosys ems) and 8.75 µL o deionized Hi-Di o mamide (Applied
Biosys ems). The mix u e was dena u ed o 5 min a 95◦Cand
kep on ice be o e being u he p ocessed by he sequence .
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Lønbo g e al.5
Capilla y elec opho esis pa ame e s we e as ollows: 10 kV
( un ol age), 1.6 kV (injec ion ol age), 22 s (injec ion ime) and
63◦C (o en empe a u e). Resul ing elec ophe og ams we e
analysed using DAx so wa e (Da a Acquisi ion and Analysis
So wa e, Van Mie lo So wa e). In e nal size s anda ds we e
buil by using a second-o de leas -squa es me hod and local
Sou he n me hod. P o iles we e double checked manually o
pe ec in e nal size s anda d i and s able baselines. Baselines
we e hen ex ac ed, and subsequen ly, peak sizes, heigh s and
absolu e a eas we e de e mined. The same p ocess was done
o he PCR nega i e sample. F om he nega i e sample, he
95 h pe cen ile was calcula ed o he heigh measu emen ,
and used as a h eshold. Sample wi h peak heigh s below he
95 h pe cen ile we e disca ded ( he 95 h pe cen ile o each
duplica ed PCR nega i e p esen ed alues o 9 and 8.7 ela i e
luo escence in ensi y (RFI) espec i ely).
P o ile peaks we e binned and ea anged by ope a ional ax-
onomic uni s (OTUs) by using R au oma ic binning and in e ac-
i e binning sc ip s (Rame e 2009). Binning was ca ied ou in-
dependen ly o he sample (peaks om all samples oge he ).
Only peaks in he ange 200 o 1200 bp and wi h alues abo e
0.09% o o al RFI we e aken in o accoun . Peaks om dupli-
ca es we e manually checked using binned-OTU ables, o a oid
e oneous OTU di isions due o ea angemen o all samples
oge he .
S a is ical analyses
In his pape - es s we e pe o med o es he signi icance o
he di e ences obse ed in bac e ial abundance and ac i i y
be ween da k and UV-i adia ion incuba ions (Sokal and Rohl
1995). The con idence le el was se a 95%, wi h all s a is ical
analyses conduc ed in S a is ica 6.0. Di e ences in ARISA in-
ge pin s be ween ini ial and inal communi y as well as among
ea men s we e analysed by compa ing B ay–Cu is simila i ies
using he package P ime 6 and Pe mano a+.AlogX+1 ans-
o ma ion o ARISA ela i e abundance was used o educe he
dominan con ibu ion by a small numbe o highly abundan
species o he B ay–Cu is analysis (despi e esul s being e y
simila wi h and wi hou ans o ma ions). Simila i y pa e ns
among samples we e hen examined using a hie a chical clus e
analysis. Dendog ams we e gene a ed using he g oup a e age
me hod, and a simila i y p o ile (SIMPROF) es (999 pe mu a-
ions) was applied o es o signi ican ly simila clus e s.
RESULTS
E ec s o sunligh on ino ganic nu ien s and dissol ed
o ganic ma e
The eco ded su ace seawa e salini y (35.4), empe a u e
(17.4◦C), Chl a(4.3 mg m–3) and nu ien le els (HPO42–:0.04±
0.03 µmol L–1;NO
3–+NO2–:0.14±0.02 µmol L–1;NH
4+:1.21±0.17
µmol L–1) we e simila o ypical la e sp ing condi ions in he R´
ıa
de Vigo (Noguei a, P´
e ez and R´
ıos 1997). In all incuba ions, expo-
su e o sunligh esul ed in inc eased NH4+concen a ions ela-
i e o he da k con ol, while inc eased HPO42– concen a ions
we e ound in he seawa e and eelg ass expe imen s (Table 1).
The DOC concen a ions be o e i adia ion we e 99 ±1µmol L–1
in he seawa e , 131 ±4µmol L–1 in he eelg ass and 154 ±2µmol
L–1 in he i e samples (Table 1). The ligh ea men caused a
5±2% dec ease in DOC concen a ions in he seawa e expe -
imen s, while 12 ±8% and 9 ±3% lowe concen a ions we e
ound in he eelg ass and i e samples, espec i ely (Table 1).
The UV exposu e esul ed in a lowe concen a ion o bioa ail-
able DOC (BDOC) o e he 4 days’ incuba ion compa ed wi h he
da k ea men in he seawa e (8 ±1 s17±4µmol L–1)and
i e expe imen s (19 ±1 s26±1µmol L–1), while inc eased
BDOC le els we e ound in he eelg ass expe imen (38 ±2 s20
±5µmol L–1)(Table1).
The ini ial C-no malized CDOM abso p ion spec a measu ed
om 250 o 600 nm showed ha he i e DOM was mo e
colou ed han he seawa e and eelg ass ea men s (Fig. 1a–c).
The maximum pho op oduc ion o CDOM was ound a wa e-
leng hs below 260 nm in bo h he seawa e and eelg ass expe -
imen s, while he la ges loss o ca bon-speci ic abso p ion o
all analysed wa eleng hs and ea men s was ound a ound 310
nm in he i e expe imen s (Fig. 1d). The C-no malized spec al
slope de e mined by i ing he abso p ion spec a o a single
exponen ial decay unc ion (Eq. 6) showed signi ican ly highe
slopes in he sunligh -exposed eelg ass and i e expe imen s
( - es , P<0.05). The C-no malized abso p ion a 254 nm (SUVA)
was he e aken as a measu e o he a oma ici y and abundance
o ca bon double bonds in he DOM (Weishaa e al. 2003) p esen
in he da k and sunligh -exposed samples. Sunligh exposu e
did no signi ican ly impac he SUVA in any o he expe imen s.
The spec al slope a io (SR), which is in e sely co ela ed o
he molecula weigh , inc eased in all UV ligh -exposed samples
(Table 1), sugges ing a pho ochemically induced dec ease in he
a e age molecula weigh (Helms e al. 2008).
The e we e clea di e ences in he C-speci ic FDOM signals
be ween ea men s bo h be o e and a e 4 days’ sunligh expo-
su e especially in he i e expe imen , sugges ing di e ences
in he CDOM chemical composi ion (Fig. 2and Table 1). The ini-
ial seawa e expe imen s had he gene ally lowes FDOM sig-
nals, eelg ass had in e media e and i e samples he highes
alues (Fig. 2and Table 1). Fo he case o he p o ein-like lu-
o escence, he eelg ass showed he highes C-speci ic luo es-
cence, while he i e samples had a gene ally highe humic
con ibu ion (Fig. 2and Table 1). The C-no malized luo escence
signals o he humic- and p o ein-like subs ances dec eased in
all sunligh -exposed samples showing he la ges impac on he
humic subs ances in he i e expe imen (Fig. 2and Table 1).
Bac e ial esponse o DOM o di e en o igin and he
e ec o sola adia ion
Ini ial bac e ial abundances (BA) we e he same (∼2×105cells
mL–1)inall ea men s(Fig.3). Bac e ial abundances inc eased
in all expe imen s (Fig. 3) ollowing he consump ion o DOC
(Table 1), eaching maximum abundances o 23 ×105(da k) and
27 ×105cells mL–1 (UV) in he seawa e , 24 ×105(da k) and 35 ×
105cells mL–1 (UV) in he eelg ass, and 17 ×105(UV) and 19 ×105
cells mL–1 (da k) in he i e incuba ions (Fig. 3). These inc eases
in BA co esponded o an a e age bac e ial biomass g ow h (BG)
o 2.07 ±0.05 (da k) and 2.44 ±0.03 µmol L–1 (UV) in he seawa e ,
2.22 ±0.04 (da k) and 3.27 ±0.04 µmol L–1 (UV) in he eelg ass
and 1.45 ±0.04 (da k) and 1.72 ±0.04 µmol L–l (UV) in he i e
expe imen s (Table 2). The BA was no signi ican ly di e en in
he da k and UV ea men s in he 3 expe imen s. The ini ial BP
a ied be ween 0.14 ±0.09 ( i e –UV) and 1.47 ±0.02 µmol L–1
(seawa e –UV), wi h highe ini ial le els in UV-exposed seawa e
and eelg ass samples, whe eas highe le els we e ound in he
da k samples in he i e incuba ions (Fig. 3and Table 2). The
in eg a ed BP was la ge in he UV seawa e (2.47 ±0.55 (da k)
s 2.89 ±0.31 µmol L–1 (UV)) and eelg ass incuba ions (1.61 ±
0.24 (da k) s 3.25 ±0.55 µmol L–1 (UV)), while lowe le els we e
ound in he i e expe imen s (2.18 ±0.42 (da k) s 1.29 ±0.25
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6FEMS Mic obiology Ecology, 2016, Vol. 92, No. 5
Table 1. Concen a ions o phospha e (HPO42–), ni a e (NO3–), ni i e (NO2–) and ammonium (NH4+), he C-speci ic CDOM abso p ion coe icien a 254 nm (SUVA) and spec al slope in he 250–
600 nm wa eleng h ange (S), a io o CDOM spec al slopes (S(275–295)/S(350–400) a io (SR)), C-speci ic gene al (peak-A∗), e es ial (peak-C∗) and ma ine humic-like subs ances (peak-M∗)and
p o ein-like subs ances (peak-T∗), ini ial concen a ions o dissol ed o ganic ca bon (DOC) be o e and a e 4 days in da kness (da k) o exposed o na u al sunligh (UV). The DOC concen a ion a
incuba ion day 0 (DOC0)and heminimumconcen a ion(DOC
Min)a ealsoshown oge he wi h hebioa ailable ac ion(BDOC)be o eanda e UV-ligh exposu e.Valuesa emeans±s anda d
de ia ion; —, no measu ed.
Seawa e Eelg ass Ri e
Be o e UV Da k UV Be o e UV Da k UV Be o e UV Da k UV
HPO42– (µmol L–1)0.06 ±0.02 0.18 ±0.08 0.37 ±0.11 0.21 ±0.08 0.21 ±0.12 0.30 ±0.08 0.25 ±0.01 0.25 ±0.05 0.24 ±0.20
NO3–(µmol L–1)0.09 ±0.01 0.08 ±0.02 0.30 ±0.01 0.08 ±0.02 0.10 ±0.02 0.09 ±0.02 0.07 ±0.08 0.09 ±0.01 0.13 ±0.01
NO2–(µmol L–1)0.06 ±0.01 0.13 ±0.10 0.22 ±0.02 0.08 ±0.01 0.14 ±0.01 0.11 ±0.01 0.09 ±0.02 0.11 ±0.01 0.14 ±0.01
NH4+(µmol L–1)1.60 ±0.17 1.28 ±0.26 2.17 ±0.33 0.62 ±0.06 0.46 ±0.30 2.61 ±0.76 1.63 ±0.34 1.12 ±0.35 2.31 ±0.31
SUVA (L mg−1m−1)0.95 ±0.04 0.96 ±0.04 1.03 ±0.03 1.16 ±0.03 1.17 ±0.04 1.38 ±0.04 1.99 ±0.06 1.99 ±0.06 1.92 ±0.06
S(nm−1)0.0171 ±0.0001 0.0171 ±0.0001 0.0167 ±0.0001 0.0092 ±0.0001 0.0092 ±0.0001 0.0110 ±0.0001 0.0125 ±0.0001 0.0124 ±0.0001 0.0156 ±0.0001
SR1.59 ±0.04 1.59 ±0.05 1.98 ±0.05 1.38 ±0.04 1.38 ±0.06 1.74 ±0.12 0.84 ±0.01 0.84 ±0.02 1.34 ±0.03
Peak-A∗(×108Lmg
−1m−1)4.02±0.09 4.97 ±0.15 4.13 ±0.09 3.66 ±0.07 3.77 ±0.08 3.81 ±0.06 10.90 ±0.03 12.00 ±0.07 6.38 ±0.09
Peak-C∗(×108Lmg
−1m−1)1.66±0.05 1.81 ±0.01 1.50 ±0.06 2.06 ±0.07 1.92 ±0.03 1.68 ±0.04 5.50 ±0.08 5.75 ±0.03 2.50 ±0.04
Peak-M∗(×108Lmg
−1m−1)1.75±0.11 1.82 ±0.03 1.52 ±0.03 1.93 ±0.01 1.62 ±0.03 1.50 ±0.06 4.33 ±0.08 4.48 ±0.02 2.39 ±0.04
Peak-T∗(×108Lmg
−1m−1)2.36±0.24 3.17 ±0.10 2.66 ±0.08 3.49 ±0.03 3.16 ±0.06 3.44 ±0.18 1.78 ±0.01 2.78 ±0.12 2.19 ±0.05
DOC (µmol L–1)99±198±294±2131 ±4130 ±1115 ±1154 ±2149 ±1140 ±1
DOC0(µmol L–1)—96±193±1 — 117 ±2111 ±1 — 140 ±1139 ±1
DOCMin (µmol L–1)—80±185±1—83±373±2 — 115 ±1121 ±1
BDOC (µmol L–1)—17±48±1—20±538±2—26±119±1
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Lønbo g e al.7
Figu e 1. The ca bon-speci ic abso p ion spec a o colou ed dissol ed o ganic
ma e (CDOM) a e 4 days in he da k (da k) o exposed o na u al sunligh (UV)
in he (a)seawa e ,(b)eelg assand(c) i e expe imen s. The di e ence (da k
minus sunligh samples) in he ca bon-speci ic abso p ion spec a o CDOM in
he seawa e , eelg ass and i e expe imen s a e shown in (d). No e ha in he
case o he eelg ass and i e samples, he blank sub ac ed was he seawa e
used o dilu e he ex ac s.
µmol L–1 (UV)) (Table 2). The BP was signi ican ly highe in he
UV ea men in he eelg ass and in he da k o he i e ex-
pe imen s ( - es , P<0.05, n =4), while no signi ican di e ence
was ound in he seawa e expe imen . The ins an aneous spe-
ci ic g ow h a e (µ), calcula ed as he a io be ween BP and BB
(BP/BB), showed ha he UV p e-exposu e had con as ing e -
ec s on he ini ial µ, wi h an inc ease in he seawa e (3.90 ±
0.01 (da k) s 5.41 ±0.35 d–1 (UV)) and eelg ass expe imen s (1.96
±0.70 (da k) s 3.46 ±1.26 d–1 (UV)) and a dec ease in he i e
incuba ions (3.50 ±1.40 (da k) s 0.65 ±0.47 d–1 (UV)) (Table 2).
The µdec eased o e he incuba ion pe iod in all expe imen s
eaching le els be ween 0.40 ±0.09 (eelg ass–da k) and 0.88 ±
0.16 d–1 ( i e –da k) a day 4 (Table 2).
The ini ial BR was highe in he da k ea men s in all incu-
ba ions (Fig. 3and Table 2). In he seawa e expe imen s, he BR
inc eased a e day 0 eaching maximum le els o 6.52 ±0.41
µmol L–1 d–1 in he da k and 3.49 ±0.15 µmol L–1 d–1 in he UV
expe imen s (Fig. 3and Table 2). In he eelg ass UV incuba ions a
d ama ic inc ease was seen a e he ini ial day, eaching a max-
imum alue o 19.50 ±2.25 µmol L–1 d–1, while mo e s able le -
els we e ound in he da k incuba ions wi h a maximum o 5.39
±0.18 µmol L–1 d–1 (Fig. 3and Table 2). In he i e da k ea -
men he BR emained a a cons an highe le el (max. 8.95 ±
0.50 µmol L–1 d–1) han heUVp e- ea edsamples(max.7.86
±0.61 µmol L–1 d–1) un il incuba ion day 3, whe ea e equal BR
we e ound in bo h expe imen s (Fig. 3and Table 2). The BR and
in eg a ed BR we e signi ican ly highe in he da k ea men in
he seawa e and i e expe imen s, and signi ican ly highe in
he UV ea ed samples in he eelg ass expe imen ( - es , P<
0.05, n=4). In all expe imen s he ini ial cell-speci ic BR was
lowe in he UV p e- ea ed samples (Fig. 3j–l and Table 2). A -
e he ini ial day in he seawa e and i e incuba ions he cell-
speci ic BR was gene ally highe in he da k compa ed wi h he
UV ea men s, while a highe ac i i y was ound in he UV ex-
posed samples in he eelg ass expe imen s (Fig. 3j–l and Table 2).
The ini ial BCD was highe in he da k compa ed wi h he
UV ea men s in all expe imen s (Fig. 4a–c and Table 2). The e-
a e a con inued highe BCD was ound in he da k samples in
he seawa e and i e expe imen s, while in he eelg ass ex-
pe imen highe le els we e ound in he UV p e- ea ed sam-
ples (Fig. 4a–c and Table 2). O e all he a e age BCD was sig-
ni ican ly highe in he da k samples in he seawa e and i e
expe imen s, while a signi ican ly highe BCD was ound in he
UV p e-exposed samples in he eelg ass expe imen s ( - es , P<
0.04, n=4). The UV p e- ea men also dec eased he in eg a ed
BCD in he seawa e (23.7 ±1.7 (da k) s 11.7 ±1.1 µmol L–1 d–1
(UV)) and i e (32.3 ±3.4 (da k) s 23.5 ±1.9 µmol L–1 d–1 (UV))
expe imen s, while an inc ease was ound in he eelg ass (16.4
±1.0 (da k) s 62.8 ±10.7 µmol L–1 d–1 (UV)) expe imen (Table 2).
The ins an aneous BGE a ied ini ially be ween 3 ±1 ( i e –
UV) and 58 ±7% (seawa e –UV) dec easing he ea e o alues
be ween 4 ±1(eelg ass–da k)and14±1% (seawa e –UV) a in-
cuba ion day 4 (Fig. 4d– and Table 2). The ini ial BGE was signi -
ican ly highe ( - es , P<0.01) in he UV p e-exposed seawa e
expe imen s, while equal le els o bo h ea men s we e ound
in he eelg ass and i e incuba ions (Fig. 4d– and Table 2).
The in eg a ed BGE (BGEin )showedhighes aluesin heUV–
seawa e samples (25 ±6%) and lowes in he da k– i e sam-
ples (5 ±1%), wi h no signi ican di e ences be ween he da k
and UV p e- ea men s in any o he expe imen s (Table 2). The
ins an aneous BGE a e aged o e he 4 days’ incuba ion showed
simila le els as he BGEin (Table 2).
The bac e ial communi y composi ion a ime ze o and a
he end o each expe imen was compa ed based on ARISA
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8FEMS Mic obiology Ecology, 2016, Vol. 92, No. 5
Figu e 2. C-speci ic luo escence exci a ion emission ma ix o (a) seawa e da k, (b)seawa e UV,(c) eelg ass da k, (d) eelg ass UV, (e) i e da k, ( ) i e UV, (g)seawa e
da k minus UV, (h) eelg ass da k minus UV and (i) i e da k minus UV samples. Fluo escence uni s a e 106Lmg
−1m−1.
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Lønbo g e al.9
Figu e 3. Time cou se changes in bac e ial abundance (BA; a,b,c), p oduc ion (BP; d,e, ), espi a ion (BR; g,h,i) and cell-speci ic espi a ion (Cell esp; j,k,l)du ing
he 4-day incuba ions in he di e en expe imen s (seawa e , eelg ass and i e ). E o ba s ep esen s anda d de ia ions; whe e no isible e o ba s a e wi hin he
symbol.
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