Con as ing complexing capaci y o dissol ed o ganic ma e
p oduced du ing he onse , de elopmen and decay o a simula ed
bloom o he ma ine dia om Skele onema cos a um
J.I. Lo enzo
a,b,
⁎, M. Nie o-Cid
c
, X.A. Ál a ez-Salgado
c
, P. Pé ez
b
, R. Bei as
b
a
Depa men o Biology, Campus Middelheim, Uni e si y o An we p, G oenenbo ge laan 171/U7, BE-2020, An we p, Belgium
b
Uni e sidad de Vigo, Facul ad de Ciencias del Ma , Dep . de Ecología y Biología Animal, Lagoas-Ma cosende, E-36200 Vigo, Spain
c
CSIC, Ins i u o de In es igacións Ma iñas, Dep . de Oceanog a ía, Edua do Cabello 6, E-36208 Vigo, Spain
Recei ed 2 Feb ua y 2005; ecei ed in e ised o m 28 Feb ua y 2006; accep ed 30 May 2006
A ailable online 25 July 2006
Abs ac
The capaci y o na u al dissol ed o ganic ma e (DOM) p oduced du ing he onse , de elopmen and decay o a simula ed
bloom o he ma ine dia om Skele onema cos a um o complex ee coppe has been ollowed o a 2 week pe iod. Coppe binding
capaci y o he cul u e was measu ed by anodic s ipping ol amme y (ASV) wi h a hanging me cu y d op elec ode (HMDE). The
concen a ion o dissol ed o ganic ca bon (DOC) and wo luo opho es, M (humic-like, Ex/Em: 320 nm/410 nm) and T (p o ein-
like, Ex/Em: 280 nm/350 nm), we e ollowed du ing he cou se o he incuba ion. Models using DOC concen a ions alone could
no accu a ely p edic he complexing capaci y o he cul u e, especially a he end o he bloom, and be e p edic ions we e
ob ained when luo escence measu emen s we e conside ed. They we e help ul in cha ac e ising wo ypes o coppe ligands
p oduced in he cul u e. The i s ype, aced by he luo escence o peak T, was ela ed o labile DOC di ec ly exuded by
phy oplank on. The second ype, aced by he luo escence o peak M, was he e ac o y humic-like ma e ial p esumably
p oduced in si u as a by-p oduc o he bac e ial deg ada ion o phy ogenic ma e ials. Du ing he onse and de elopmen o he
bloom (days 0 o 7), he luo escence o peak T explains 60–80% o he o al complexing capaci y o he cul u e, sugges ing ha
exuded “p o ein-like”compounds among o he exuded complexing agen s e icien ly complexed ee coppe . On he con a y,
du ing he decay (days 8 o 13), hese ligands we e eplaced by humic subs ances as he complexing agen o coppe .
© 2006 Else ie B.V. All igh s ese ed.
Keywo ds: Dissol ed o ganic ma e ; Phy oplank on cul u e; Ligands; Fluo escence; Vol amme y
1. In oduc ion
Ma ine dissol ed o ganic ma e (DOM) is he la ges
pool o educed ca bon on he Ea h's su ace (Hedges,
2002). I is composed o a my iad o di e en compounds,
co e ing a wide ange o molecula weigh s, chemical
s uc u es and unc ions. F om he en i onmen al poin o
iew, ma ine DOM plays se e al ele an oles (Sunda,
1995), some o hem being connec ed o he a e o me als
in he en i onmen : (1) i con ibu es o he ecycling,
accumula ion, and expo o biogenic ma e in ocean
biogeochemical cycles, (2) i is an elec on dono in he
pho o educ ion and solubilisa ion o i on and manganese
and (3) i educes he oxici y o ee me al ions by he
o ma ion o s able me al/o ganic complexes.
Ma ine Chemis y 103 (2007) 61–75
www.else ie .com/loca e/ma chem
⁎Co esponding au ho . Depa men o Biology, Campus Middel-
heim, Uni e si y o An we p, G oenenbo ge laan 171/U7, BE-2020,
An we p, Belgium. Tel.: +32 32653478; ax: +32 32653497.
E-mail add ess: [email p o ec ed] (J.I. Lo enzo).
0304-4203/$ - see on ma e © 2006 Else ie B.V. All igh s ese ed.
doi:10.1016/j.ma chem.2006.05.009
Mos o he ma ine DOM is p oduced in si u om he
accumula ion and subsequen deg ada ion o he p oduc s
o syn hesis o ma ine phy oplank on (Biddanda and
Benne , 1997; Ca lson, 2002), and only 10% o igina es
on he con inen s (D u ele al.,1992). Mechanisms o
DOM p oduc ion include di ec exuda ion by ma ine
phy oplank on, cell au olysis in esponse o nu ien s ess
condi ions o senescence, and zooplank on media ed libe-
a ion in connec ion wi h an ine icien g azing p ocess
(Naga a, 2000). Among he p oduc s libe a ed du ing he
p oduc ion o ma ine phy oplank on a e pho osyn ha es,
essen ially ca bohyd a es and amino acids (Mykles ad,
1995; Biddanda and Benne , 1997; G anum e al., 2002),
mainly he la e ha e high capaci y o complex ee me al
ions. In addi ion, pho ochemical and bac e ial espi a ion
p ocesses gi e ise o a complex se ies o poo ly unde s ood
ans o ma ions expe ienced by ma ine DOM, globally
known as humi ica ion (Hedges, 1988; Kiebe e al., 1997).
Me al complexa ion p ope ies o amino acids ha e
been desc ibed a long ime ago and hey ha e been col-
lec ed and c i ically e iewedin se e al da abases (Ma ell
and Smi h, 1974; IUPAC se ies: Kiss e al., 1991; Pe i
and Powell, 1993; Be hon, 1995; Yamauchi and Odani,
1996). Mos amino acids can ac as biden a e me al bind-
ing ligands h ough hei α-amino and α-ca boxyla e
g oups, and hei s oichiome ic s abili y cons an s a e
hus e y simila o he one om glycine (logK
Cu–L
∼8.1),
because hei side chains a e usually no in ol ed in he
me al binding p ocess. These s abili y cons an s a e low
o e ec i ely complex me al ions in na u al sys ems
because o hei low concen a ions, and specially in sea-
wa e , due o he compe i ion wi h o he ca ions and he
high ionic s eng h. Howe e , his idine and cys eine p e-
sen s ong dono g oups in hei side chains, imidazole
and hiol g oups, espec i ely, being much mo e e icien
complexing amino acids. Mo eo e , p o eins wi h high
me al binding p ope ies (e.g.,me allo hionein, zinc inge
pep ides, me allop o eins) a e also ich in hose amino
acids (Fa kas and Só ágó, 2002).
Humic and ul ic acids, ei he o con inen al o in
si u o igin, a e complex high molecula weigh sub-
s ances, wi h a capaci y o complex me al ions ha de-
pends on he abundance o ca boxylic acid g oups pe uni
weigh (Mido ikawa and Tanoue, 1998). In seawa e , hey
usually cons i u e 5–15% o he o al DOM concen a ion
in ca bon uni s, ca. 8–25 μMC(Benne e al., 1992),
al hough in es ua ies and coas al wa e s hey can each
concen a ions as high as 125 μMC(Ishiwa a i, 1992;
Bu ney, 1994).
Coppe is an essen ial elemen o li ing o ganisms, a
na u al componen o a a ie y o mine al sal s, and also an
elemen used in many indus ial applica ions and in wa e
piping. The e o e, na u al and an h opogenic inpu s o
coppe o he en i onmen a e bo h ele an (Scheinbe g,
1991). Al hough an essen ial elemen , coppe is one o he
mos oxicme als o many o ganismsabo e ce ainle els,
especially o aqua ic in e eb a e la ae (His e al., 1999)
and phy oplank on species (Sunda and Lewis, 1978).
Inc easing a en ion has been paid o he DOM pool in
en i onmen al s udies due o i s ole in bu e ing he
oxici y o me als o aqua ic o ganisms (Campbell, 1995).
The inclusion o DOM in wa e quali y c i e ia has also
been ecommended (No , 1987; Allen and Hansen, 1996).
The e o e, unde s anding how DOM a ec s me al bio-
a ailabili y has been a key issue in ecen yea s. Se e al
models ha e been de eloped o p edic he biological
e ec o me als in he en i onmen . The bio ic ligand
model, BLM (Di To o e al., 2001), combined he ee ion
ac i i y model wi h he gill su ace in e ac ion model, and
hus conside s me al specia ion in aqua ic sys ems oge-
he wi h he e ec o compe ing ca ions a he memb ane
su ace o heo ganisms. Ne e heless,disc epanciesha e
been obse ed be ween he p edic ions made in e ms o
dissol ed o ganic ca bon concen a ions (DOC) and he
obse ed bioa ailabili y da a, sugges ing ha u he DOM
cha ac e isa ion is needed (Playle, 1998; Ryan e al., 2004).
Clea di e ences in me al complexa ion p ope ies ha e
been also ound in na u al o ganic ma e om di e en
sou ces (Benede i e al., 1996; Abb -B aun and F immel,
1999) ha canno be explained in e ms o hei DOC
con en .
The aim o his wo k is o s udy he p oduc ion and
ans o ma ion o he DOM o igina ed du ing he onse ,
de elopmen and decay o a simula ed bloom o he
ma ine dia om Skele onema cos a um and i s implica-
ions o hei coppe binding p ope ies. The luo es-
cence o DOM, in addi ion o adi ional case a iables
such as DOC and chlo ophyll a, is in es iga ed o ge a
be e cha ac e isa ion o DOM in he cul u e.
2. Ma e ial and me hods
2.1. The cul u e
The ma ine dia om S. cos a um was cul u ed in a i icial
seawa e (Lo enzo e al., 2002) in acid-washed 4-
l polyca bona e bo les, wi h he aim o p oducing na u al
DOM. Two eplica es, named A and B, we e pe o med.
Thecul u eswe ekep a 18°Cunde a14hligh :10hda k
pho ope iod (70 μmol quan a/m
2
s) wi h bubbling il e ed
ai (0.45 μm PTFE, poly e a luo oe hylene), and gen le
magne ic s i ing was p o ided when cell densi y inc eased.
On he ini ial day, he a i icial seawa e was en iched wi h
hal o he nu ien s o a s anda d /2 media (Guilla d, 1975)
62 J.I. Lo enzo e al. / Ma ine Chemis y 103 (2007) 61–75
wi hou added EDTA (e hylenediamine e aace ic acid).
The ini ial concen a ions o N and P we e 700 μMand
18 μM, espec i ely. Bo h bo les we e inocula ed wi h a
non-axenic cul u e o S. cos a um in exponen ial g ow h
phase o each a inal densi y o 10
4
cells/ml. The pH o he
medium was kep be ween 8.05 and 8.25 by adding dilu ed
HCl when necessa y. All eagen s used we e analy ical-
g ade quali y, o minimize he me al con en o he cul u ing
media. The cul u es we e moni o ed along he expe imen
ollowing he ime cou se o wo s a e a iables:
(1) Chlo ophyll a(Chl a) concen a ion: 20 ml o he
cul u e we e il e ed h ough Millipo e APFF glass-
ib e p e il e s o o al Chl ade e mina ion. Sam-
ples we e kep a −20 °C un il analyses. Chl awas
ex ac ed in 90% ace one a −20 °C o ca. 24 h.
Samples we e analysed using a Tu ne TD-700
luo ome e calib a ed agains pu e Chlo ophyll a
(Sigma).
(2) Cell densi y was assessed daily using a Neubaue
haemocy ome e .
2.2. Fil a ion p ocedu es
Abou 300 ml o each cul u e we e gen ly il e ed
(∼150 mm Hg acuum) o minimize cell lysis, h ough
acid cleaned 0.22 μm Millipo e PES (polye he sulphone)
il e s. Acid cleaned polyca bona e il e holde s we e used
o a oid me al con amina ion. The il a e was aken e e y
2 days du ing he cul u e and i was used o dissol ed
o ganic ca bon (DOC) and luo escence o dissol ed
o ganic ma e (FDOM) analyses and coppe i a ions.
2.3. Dissol ed o ganic ca bon (DOC)
The il a e was collec ed in o 10 ml p ecombus ed
(450 °C, 12 h) glass ampoules. A e acidi ica ion wi h
H
3
PO
4
o pH <2, he ampoules we e hea -sealed and s o ed
in he da k a 4 °C un il analysis. DOC was measu ed wi h a
Shimadzu TOC-5000 o ganic ca bon analyse . The sys em
was s anda dized wi h solu ions o po assium hyd ogen
ph hala e. The concen a ion o DOC was de e mined by
sub ac ing he a e age peak a ea om he ins umen
blank a ea and di iding by he slope o he s anda d cu e.
The CV was ∼1% and he accu acy o he measu emen s
was success ully es ed wi h he TOC e e ence ma e ials
p o ided by P o . D. Hansell (Uni e si y o Miami).
2.4. Fluo escence o dissol ed o ganic ma e (FDOM)
Aliquo s o he DOC il a e we e collec ed be o e Cu
addi ion in o 20 ml acid cleaned polyca bona e lasks and
ozen a −20 °C un il analysis. FDOM o hese samples
was measu ed wi h a Pe kin Elme LS 55 Luminescence
spec ome e . The ins umen was equipped wi h a xenon
discha ge lamp, equi alen o 20 kW o 8 μs du a ion, and
a 1 cm qua z luo escence cell. Milli-Q wa e was used as
a e e ence o luo escence analysis, and he in ensi y o
he Raman peak was checked egula ly. Disc e e exci a-
ion/emission pai measu emen s we e pe o med a peaks
M (ma ine humic subs ances, Ex/Em: 320 nm/410 nm)
and T (“p o ein-like subs ances”, Ex/Em: 280 nm/350 nm),
speci ically yp ophan (Coble, 1996; Nie o-Cid e al.,
2005; S edmon and Ma kage , 2005). Fou eplica e mea-
su emen s we e pe o med o each Ex/Em wa eleng h
pai s. A ou poin s anda d cu e was p epa ed daily wi h
a mixed s anda d o quinine sulpha e (QS) and yp ophan
(T p) in 0.05 mol/l sulphu ic acid (concen a ions om +0
o +150 ppb and om +0 o +100 ppb o QS and T p,
espec i ely; Nie o-Cid e al., 2005). The equi alen con-
cen a ion o e e y peak was de e mined by sub ac ing
he a e age peak heigh om he blank heigh , and
di iding by he slope o he s anda d cu e. Fluo escence
uni s we e exp essed in ppb equi alen s o QS (ppb eq
QS) o FDOM
M
and ppb equi alen s o T p (ppb eq T p)
o FDOM
T
. No e ha ppb eq QS is iden ical o he
quinine sulpha e uni s (QSU) epo ed p e iously in o he
s udies. The p ecision o he luo escence measu emen s
was ±0.1 ppb eq QS and ±0.6 ppb eq T p, espec i ely.
In addi ion o hese measu emen s, a ial was pe -
o med wi h he il a e om he six h day wi h di e en
addi ions o Cu ( anging om 0 o 5 μM) o check he
e ec o Cu complexa ion in he di e en luo opho es.
Twen y-millili e samples we e spiked wi h inc easing Cu
concen a ions and allowed o equilib a e o 24 h in
he da k. A e ha ime, FDOM
M
and FDOM
T
we e
measu ed.
2.5. Cu measu ing condi ions
Squa e wa e anodic s ipping ol amme y (ASV)
analyses we e ca ied ou wi h a hanging me cu y d op
elec ode, a Ag/AgCl e e ence and a P - od auxilia y
elec ode held in a Me ohm 663 VA pola og aphic s and
coupled o an Eco-Chemie Au oLab PGSTAT10 po en-
ios a . Samples we e placed in a 20 °C he mos a ic
glass cell. A e solu ions had been pu ged o 250 s wi h
N
2
, coppe was accumula ed on a me cu y d op o
0.52 mm
2
a −0.55 V o 15 s a he maximum s i ing
speed (3000 pm) and 10 s o equilib a ion we e allowed
be o e he ol age scan. The condi ions o he squa e-
wa e (SW) scan we e an ini ial po en ial o −0.55 V, an
SWampli ude o 25 mV, an SW equency o 25 Hz and a
scan inc emen o 2 mV. Th ee ol ammog ams we e
63J.I. Lo enzo e al. / Ma ine Chemis y 103 (2007) 61–75
eco ded o each solu ion. To minimize po en ial e ec s
o adso p ion o Cu o cell walls, cells we e only washed
wi h sample solu ion du ing each i a ion. Samples wi h
inc easing Cu addi ions we e measu ed in o de , s a ing
om he cul u e il a e wi h no Cu addi ion.
2.6. Coppe i a ions
Be ween 13 and 16 samples o 16 ml cul u e il a es
we e dosed in 20 ml polyp opylene ials. Solu ions we e
s o ed a −20 °C un il analyses. Be o e analyses,
Fig. 1. Time e olu ion o he Skele onema cos a um cul u e and he dissol ed o ganic ma e concen a ion and composi ion. (a) Numbe o cells
measu ed in cul u es A and B. E o ba s ep esen he 95% con iden ial in e als o he mean o h ee eplica es. (b) DOC concen a ion in cul u es A
and B compa ed wi h cell numbe s. (c) Fluo escence cha ac e isa ion o he DOM pool: FDOM
M
is he luo escence o humic subs ances in quinine
sulpha e uni s (ppb eq QS), and FDOM
T
is he luo escence o he a oma ic amino acids in yp ophan uni s (ppb eq T p).
64 J.I. Lo enzo e al. / Ma ine Chemis y 103 (2007) 61–75
inc easing Cu addi ions ( om 0 o 5 μmol/l) we e made o
each se o samples om a dilu ed spec opho ome ic Cu
s anda d solu ion (1000 ppm, Pan eac). Solu ions we e
kep 24 h in he da k o allow he equilib a ion o he
complexa ion eac ion. All addi ions we e p epa ed and
analyses we e pe o med on a lamina low, class 100
il e ed ai , clean bench. All plas ic lab wa e was kep in
5% HNO
3
a leas 24 h and insed 5 imes wi h deionised
wa e pu i iedbyion exchange( esis i i y≥18.2 MΩ/cm;
Milli-Q®) be o e use.
The peak heigh o each ol ammog am was mea-
su ed, and i was ans o med in o labile Cu concen a-
ions ([Cu′]) di iding by he slope o he linea segmen
o each i a ion (Dona e al., 1994). Labile coppe is he
equi alen concen a ion o coppe ha con ibu es o he
ol amme ic signal o he same ex en as he ee me al
ion (Mulle e al., 2003). The i a ion cu es we e
explained assuming he simples complexa ion model;
ha is, only one ype o ligand and a eac ion s oichi-
ome y o 1:1. Ti a ion plo s we e i ed o Eq. (1),
ob ained om he heo e ical complexa ion model
p e iously explained (Lo enzo e al., 2002):
CuV¼aþffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
a2þ4CuT=KV
p2ð1Þ
whe e a=(−Cu
T
+L+1/K′), and Cu
T
and Cu′a e he o al
and labile coppe concen a ions (mol/l), espec i ely, L is
he o al ligand concen a ion (mol/l) and K′is he
condi ional s abili y cons an o he Cu–L complexes,
alid o he condi ions o he expe imen al medium. This
Fig. 2. E olu ion o he measu ed luo opho es du ing he cul u e. (a) Compa ison o FDOM
T
and DOC; (b) compa ison o ne p oduc ion a es o
FDOM
T
and DOC along he cul u e; (c) compa ison o FDOM
M
and DOC; and (d) FDOM
T
ne p oduc ion a e e sus ime. Reg ession lines we e
i ed o model II in (a) and (b), and o model I in (c) and (d), espec i ely. The eg ession equa ions (±s anda d e o ), he co ela ion coe icien s, and
he signi icance o he ela ionships a e also p esen ed.
65J.I. Lo enzo e al. / Ma ine Chemis y 103 (2007) 61–75

K′is e e ed o labile coppe concen a ions, no o ee
coppe ion concen a ions. I is necessa y o conside
ino ganic coppe specia ion in he medium o con e ing
K′ o K′
Cu 2+
(Dona e al., 1994).
KV
Cu2þ¼KVdaCu2þð2Þ
whe e α
Cu 2+
is he ino ganic eac ion side coe icien ,
which accoun s o he ino ganic coppe specia ion. A
alueo 36wasassumed(Leal e al., 1999) o
calcula ions.
2.7. S a is ical analysis
The bes - i be ween any couple o a iables (X,Y)
was ob ained minimizing he unc ion:
X
i
ðXi−XiÞwXðYi−YiÞwY

2ð3Þ
whe e w
X
and w
Y
a e weigh s o Xand Y espec i ely,
wi h w
X
,w
Y
≥0 and w
X
+w
Y
=1. Any eg ession model
can be exp essed as one o wo ex eme cases: (a) model
I, which should be applied when w
X
=0, w
Y
=1 and (b)
model II when w
X
=w
Y
=0.5 (Sokal and Rohl , 1995).
S a is ical analyseswe epe o medwi hSTATISTICA
e sion 6.0 (S a So ). The pa ame e s o he complexa-
ion model in Cu i a ions we e es ima ed by means o
non-linea i ing p ocedu es wi h Sigma Plo 2001 e -
sion 7.0 (SPSS), which uses i e a i e p ocesses o
minimizing he sum o he squa es o he esiduals by
means o he Ma qua d -Le enbe g algo i hm.
3. Resul s
3.1. Onse , de elopmen and decay o he cul u ed S.
cos a um bloom
Fig. 1 shows he ime e olu ion o he cul u e. Simila
pa e ns we e obse ed in bo h lasks (A and B) o he
di e en measu emen s. Fig. 1a shows he loga i hmic
inc ease in he numbe o cells and chlo ophyll a(Chl a)
concen a ion du ing he ini ial 4 days. No lag ime was
obse ed in he cul u e g ow h. F om days 4 o 6, he
numbe o cells emained s a iona y, and a e day 8 he e
was a con inuous dec ease in cell densi y. The maximum
Chl aconcen a ion (700–1000 μg/l) was achie ed a
day 7, and a e ha , a con inuous dec ease was also
obse ed o Chl ale els.
Fig. 1b shows he high co ela ion be ween DOC
accumula ion in he cul u e and cell densi y du ing days
0 o 7 ( he anabolic phase o he bloom). The e was a
con inuous inc ease in he DOC con en o he cul u e
om he ini ial day, bu his inc ease was much highe
om days 4 o 5. A e day 5, a clea DOC dec ease was
obse ed. A la ge disc epancy was obse ed a e day
8 (named he ca abolic phase o he bloom), when cell
densi y dec eased whe eas he DOC con en inc eased,
especially a he end o he cul u e (no e he change o
scale). High bac e ial densi ies we e obse ed a e day 4
Fig. 3. Fluo escence quenching in he p esence o coppe . Fil e ed samples om he six h day o he cul u e we e spiked wi h inc easing Cu
concen a ions and luo escence o “humic-like”subs ances (FDOM
M
) and o “p o ein-like”subs ances (FDOM
T
) was measu ed a e 24 h. Fi ings
o obse ed da a o exponen ial decay equa ions a e also p esen ed.
66 J.I. Lo enzo e al. / Ma ine Chemis y 103 (2007) 61–75
( e ained in 0.22 μm il e s), and one sample was incu-
ba ed o es bac e ial consump ion o he DOC in he
cul u e. One aliquo o he cul u e in day 5 was il e ed
h ough a 5 μm po e size GF/F il e , which emo es
phy oplank on bu no bac e ia, and incuba ed o 24 h in
he same cul u e condi ions. A DOC dec ease om 0.50
o 0.14 mM C was obse ed.
Fig. 1c shows he e olu ion o he wo luo opho es
measu ed du ing he cul u e. FDOM
T
ep oduced he
same empo al pa e n han he DOC concen a ion
(Fig. 2a). A high co ela ion was obse ed be ween bo h
a iables, bu he co ela ion was e en highe when p o-
duc ion/deg ada ion a es o FDOM
T
and DOC we e
compa ed up o day 11 (Fig. 2b). FDOM
M
showed a
comple ely di e en pa e n. A mono onic inc ease was
obse ed in he cul u e and FDOM
M
was no signi ican ly
co ela ed wi h DOC (Fig. 2c). The same esul was
obse ed when compa ing FDOM
M
and DOC p oduc ion
a es. Howe e , FDOM
M
p oduc ion a es we e highly
co ela ed wi h ime (Fig. 2d).
A signi ican dec ease in luo escence was obse ed
when Cu addi ions we e made o samples con aining
bo h luo opho es (Fig. 3). FDOM
T
was mo e a ec ed
by Cu addi ions han FDOM
M
, and a 32% e sus a 20%
luo escence dec ease we e measu ed, espec i ely.
3.2. E ol ing coppe binding capaci y o DOM
p oduced by he cul u e
Fig. 4a shows he e olu ion o Cu i a ion plo s in
he cul u e o e ime. Some speci ic days we e selec ed
o clea ly show he shi in he plo s o he igh , due o
he inc ease in Cu complexing capaci y. The complex-
a ion model p e iously desc ibed (Eq. (1)) accu a ely
i ed he da a, and he ob ained
2
anged be ween
0.997 and 0.9996, e en when indi idual measu emen s
and no mean alues whe e used o i ing pu poses.
Coppe i a ion i ing pa ame e s (±95% con iden-
ial in e al, CI) a e shown in Table 1. The e was a good
ag eemen be ween he pa ame e s ob ained in bo h
cul u es, as p e iously obse ed wi h DOC measu e-
men s (Fig. 2b). The e was a ma ked inc ease in ligand
concen a ion o e ime (see also Fig. 4b). The ligand
inc ease showed an in e media e end o hose obse ed
o bo h luo opho es: an ini ial and sha p inc ease a he
end o he exponen ial g ow h (days 4–5) coinciding
wi h FDOM
T
and no dec ease be ween days 5 and 8, as
Table 1
Complexing capaci y (L±95% CI, μmol Cu/l) and condi ional s abili y
cons an (logK′
Cu 2+
±95% CI) in cul u es A and B a di e en ime
in e als; n, numbe o coppe addi ions in each i a ion
Time
(days)
L
A
(μmol
Cu/l)
LogK′
Cu 2+
nL
B
(μmol
Cu/l)
LogK′
Cu 2+
n
0 0.04±0.01 9.5±2 12 0.08±0.01 9.4±0.9 14
2 0.12±0.01 8.4±0.2 15 0.13±0.02 8.6±0.5 15
4 0.40±0.03 8.2±0.2 12 0.24±0.02 8.4±0.2 15
5 0.64±0.02 8.60±0.02 15 0.57±0.03 8.2±0.1 13
6 0.64±0.04 8.64±0.04 16 0.58±0.02 8.5±0.1 16
8 0.79±0.02 9.0±0.2 14 0.84±0.02 8.82±0.09 15
11 0.92±0.06 8.3±0.2 14 1.00±0.05 8.3±0.1 14
13 1.12±0.03 8.41±0.09 12
Fig. 4. (a) Time e olu ion o Cu i a ions o he il e ed cul u e. Solid
lines ep esen he bes non-linea squa es i ings o he da a o Eq. (1).
(b) Time e olu ion o me al ligand concen a ion in he cul u e. E o
ba s ep esen he 95% con iden ial in e als ob ained om he
s anda d e o s o he i ings.
67J.I. Lo enzo e al. / Ma ine Chemis y 103 (2007) 61–75
FDOM
M
. Day 8 showed s a is ically highe condi ional
s abili y cons an s, coinciding wi h he ini ia ion o he
ca abolic phase o he cul u e. The ligand capaci y a he
ini ial day o he cul u e was e y low, which was also
e lec ed in he low con idence o he ob ained K′ alue.
The sample om day 5 incuba ed o bac e ial con-
sump ion was also i a ed wi h Cu. The ligand concen-
a ion in heincuba ed sample dec eased om 0.64±0.02
o 0.38±0.04 μM, i.e. a 40% dec ease. This esul con-
as s wi h he 72% dec ease o DOC concen a ion in he
same sample.
3.3. DOM composi ion e sus Cu binding capaci y
Fig. 5a shows he ligand concen a ion measu ed
du ing he cul u e e sus he DOC con en in he il a es.
In he anabolic phase, a signi ican co ela ion was
obse ed be ween he DOC concen a ion and he me al
binding capaci y (ligand concen a ion) o he cul u e. Up
o 70% o he ligand a iabili y could be explained by he
DOC concen a ion. Howe e , when he ca abolic phase
o he cul u e was conside ed, he eg ession did no
p edic he ligand concen a ion. A sha p inc ease in
ligand concen a ion was obse ed be ween days 8 and 11
coinciding wi h a clea dec ease in DOC. The opposi e
end was obse ed on day 13, when a 10- old inc ease in
DOC was p oduced, bu jus a small inc ease in ligand
concen a ion was obse ed.
The wo luo opho es we e used o p edic he ligand
concen a ion in he cul u es (see Fig. 5b and c).
FDOM
T
was he bes p edic i e a iable: 88% o he
a iabili y o he ligand concen a ion in he anabolic
Fig. 5. Rela ionships be ween he concen a ion o ligand in he cul u e and di e en measu emen s o he DOM con en in he medium. (a–c) Ligand
concen a ions exp essed as a unc ion o he DOC, FDOM
T
and FDOM
M
o he cul u e, espec i ely. (d) Obse ed e sus expec ed ligand concen a ions
ob ained om hemul iple model II eg ession. The mul iple eg ession (see ex , Eq. (4)) was calcula ed wi h da a om he anabolic phase.Whi e andg ey
do s ep esen da a om he ca abolic phase i ed o he p e ious eg ession be o e and a e FDOM
T
co ec ions. Black do s and long dashed lines
co espond o da a and i ings ob ained in he anabolic phase o he cul u e. Open do s and do ed lines co espond o da a and i ings om he ca abolic
phase. The eg ession equa ions (±s anda d e o ), he co ela ion coe icien s and he signi icance o he model II ela ionships a e also p esen ed.
68 J.I. Lo enzo e al. / Ma ine Chemis y 103 (2007) 61–75
phase can be explained by FDOM
T
. The eg ession
model wi h FDOM
M
was also signi ican , bu i did no
imp o e he p edic i e powe o he model ob ained
wi h he DOC concen a ion.
None o hese h ee eg ession models es ed in he
anabolic phase can be success ully applied o he
ca abolic phase o he cul u es, and clea disc epancies
wi h he p edic ed alues o he ligand concen a ions
we e obse ed al eady o he measu ed alues on day
8 (open ci cles in Fig. 5a, b and c).
A mul iple eg ession model conside ing he con i-
bu ion o bo h luo opho es o he ligand concen a ion
was also es ed in he anabolic phase o he cul u es. The
ollowing equa ion was ob ained:
½L¼0:05ðF0:03Þþ0:007ðF0:001ÞFDOMT
þ0:008ðF0:002ÞFDOMMð4Þ
whe e L is he ligand concen a ion (μmol Cu/l), and he
s anda d e o o p edic ed pa ame e s a e shown in
b acke s (n=11). The eg ession model explains 96% o
he obse ed ligand a iabili y in he cul u e (see black
do s o Fig. 4d). A signi ican con ibu ion o bo h
FDOM
T
and FDOM
M
o he ligand concen a ion was
obse ed (p<0.001 and p=0.005, espec i ely).
When he same model was applied o he ca abolic
phase o he cul u e, good p edic ions we e ob ained o
day 8. None o he p e ious models conside ing only
DOC, FDOM
T
o FDOM
M
could p edic ligand
concen a ions a his day. Howe e , expec ed ligand
concen a ions (open do s in Fig. 4d) o he las days o
he cul u e (days 11 and 13) depa ed signi ican ly om
measu ed alues. Model p edic ions o e es ima e mea-
su ed ligand concen a ions, coinciding wi h he clea
decay o he cul u e. P obably, FDOM
T
a his pe iod is
no ela ed o labile exuded DOC by algae bu o DOC
libe a ed du ing cell lysis o he dead cul u e. The e o e,
a new pa ame e was in oduced o Eq. (4) o accoun o
a possible dec ease in ligand capaci y o measu ed
FDOM
T
a his pe iod:
½L¼0:05 þ0:007ð1−aÞFDOMT
þ0:008FDOMMð5Þ
whe e αis he % o FDOM
T
wi hou me al binding
p ope ies. Good i ings wi h he obse ed ligand con-
cen a ions we e ob ained conside ing ha 85% and 100%
o FDOM
T
had no me al complexing p ope ies in days 11
and 13, espec i ely (g ey do s in Fig. 4d; Table 2).
The pa ame e s ob ained om Eq. (4) a e no di ec ly
compa able, because bo h luo opho es a e no ex-
p essed in he same uni s. Howe e , i is possible o
calcula e he con ibu ion o bo h ac ions o he ligand
concen a ion by conside ing he measu ed FDOM
T
and
FDOM
M
a any ime (see Table 2). Du ing he ini ial
phase o he cul u e, “p o ein-like subs ances” oge he
wi h o he labile DOC compounds ( aced by FDOM
T
)
di ec ly exuded om phy oplank on li ing cells made up
mos o he me al complexing capaci y o he cul u e
(be ween 60% and 80%). Howe e , a e day 6, humic
subs ance con ibu ion o he ligand concen a ions
s a ed o be dominan .
4. Discussion
4.1. DOC p oduc ion du ing he cul u e
Two phases ha e been dis inguished in he e olu ion o
he cul u e, named anabolic and ca abolic. The anabolic
phase coincided wi h he g owing and s a iona y phases
(days 0 o 7), cha ac e ised by he exuda ion o DOM by
S. cos a um li ing cells. The ca abolic phase coincided
wi h he decay o he bloom (days 8 o 13), p obably
caused by P nu ien limi a ion, cha ac e ised by inc eased
cell lysis.
DOC p oduc ion by S. cos a um has been desc ibed
p e iously in ield (Igna iades, 1973) and in cul u e
(Igna iades and Fogg, 1973) condi ions. The amoun and
composi ion o DOC eleased by ma inedia oms has been
p o en o be dependen on he physiological s a us o he
cul u e (Chen and Wage sky, 1996a; Te zićand Ahel,
1998; G anum e al., 2002). Whe eas low molecula
weigh ma e ials a e eleased in he exuda ion p ocesses
du ing he g owing phases o cul u es, high molecula
weigh ma e ials a e dominan in he senescen phases
(Chen and Wage sky, 1996b).
Table 2
Pe cen age o he measu ed ligand concen a ion explained by he
FDOM
T
and FDOM
M
luo opho es (±s anda d de ia ion) du ing he
cou se o he Skele onema cos a um cul u e acco ding o Eq. (4)
Time(days) %L
FDOM
M
%L
FDOM
T
% Non-complexing FDOM
T
0 28±1 72±1 0
2 22±2 78±5 0
4 37±13 63±23 0
5 28±2 72±6 0
6 44±1 56±1 0
8 58±2 42±2 0
11 92±6 8±1 85
13 100 0 100
The es ima ed pe cen age o FDOM
T
wi hou me al binding p ope ies
acco ding o Eq. (5) in he anabolic phase is also shown in he ou h
column.
69J.I. Lo enzo e al. / Ma ine Chemis y 103 (2007) 61–75