Limne ica, 29 (2): x-xx (2011)
Limne ica, 30 (1): 17-26 (2011)
c
Asociaci´
on Ib´
e ica de Limnolog´
ıa, Mad id. Spain. ISSN: 0213-8409
A sequen ial p ocedu e o he quan i ica ion o biologically p oduced
polyphospha e in sedimen samples
Ma a Reina1,∗, Lau a Se ano1and Han L. Gol e man2
1Depa men o Plan Biology and Ecology, Uni e si y o Se illa, P.O. Box 1095, 41080 Se illa, Spain.
2Rouboslaan 66, 2252 TR Voo scho en, The Ne he lands ([email p o ec ed]).
∗Co esponding au ho : [email p o ec ed]
2
Recei ed: 4/10/10 Accep ed: 25/11/10
ABSTRACT
A sequen ial p ocedu e o he quan i ica ion o biologically p oduced polyphospha e in sedimen samples
Polyphospha e (poly-P) is a phospha e s o age compound widesp ead in p oka yo ic and euka yo ic cells. I s quan i ica ion,
howe e , is no ye well de eloped o sedimen samples because o he di icul ies associa ed wi h using a single ex ac an
o ex ac a speci ic P compound wi hou al e ing o he s. The p esen wo k uses an analy ical p ocedu e al eady de eloped
o measu e poly-P in algal cul u es and la e modi ied he e o sedimen s o s udy poly-P in he sedimen . The p ocedu e
uses he E hylenediamine e aace ic acid (EDTA) me hod o sequen ial P- ac iona ion. This app oach ensu es ha he main
ino ganic phospha e compounds (i.e., Fe- and Ca-bound phospha e) a e ex ac ed p io o poly-P quan i ica ion. We applied
his me hod o suspensions likely o con ain biologically p oduced poly-P, such as labo a o y cul u es o Synechocys is PCC
6803 (Cyanobac e iaceae), Anabaena a iabilis (Cyanobac e iaceae), and Chlo ella sp. (Chlo ophyceae) g owing in a P-
en iched medium, and ac i a ed sludge. Acco ding o his p ocedu e, he concen a ions o poly-P in Synechocys is PCC
6803, Anabaena a iabilis and Chlo ella cul u es we e 15.4, 6.2 and 7.2% o he sum o all P- ac ions, espec i ely, whe eas
ac i a ed sludge showed a lowe pe cen age (3.7%). Known olumes o hese suspensions and a comme cially a ailable
syn he ic poly-P s anda d (T ime aphospha e) we e added o na u al sedimen . The P-composi ion o each sample was hen
compa ed in duplica e o he co esponding con ol samples (sedimen alone). Poly-P in all con ol sedimen samples was
ex emely low (0-2 µgg
−1d.w.). I inc eased in all sedimen suspensions o which biological samples had been added. The
inc ease was pa icula ly no ewo hy o Synechocys is (up o 220 µgg
−1d.w. o poly-P). The eco e y o poly-P in sedimen
samples o which Anabaena cul u es had been added showed some de ia ion om a 1:1 expec ed:obse ed a io, pa icula ly
when 2 ml o he cul u e we e added ( a io o 0.79). Howe e , he eco e y imp o ed o 1.12:1 when a la ge olume was
added (5 ml), p obably owing o he inhomogeneous na u e o he cyanobac e ia cul u e. In con as , he addi ion o 500 mg
o ime aphospha e o he con ol sedimen did no signi ican ly inc ease (p>0.05) he pe cen age ound o he obse ed
poly-P ac ion. Consequen ly, biologically p oduced poly-P can be associa ed wi h he ac ion o poly-P ex ac ed h ough
use o his p ocedu e.
Key wo ds: Polyphospha e, EDTA me hod, P- ac iona ion, sedimen .
RESUMEN
An´
alisis secuencial pa a cuan i ica el poli os a o biol´
ogico en mues as de sedimen o
El poli os a o (poly-P) es un compues o de ese a de os a o ampliamen e dis ibuido en e c´
elulas p oca io as y euca io as.
Sin emba go, a´
un no es ´
abiende inida la me odolog´
ıa pa a su cuan i icaci´
on en mues as de sedimen o, debido a la di icul ad
de encon a un ´
unico ex ac an e pa a compues os espec´
ı icos de P que no al e e a los dem´
as. En el p esen e abajo se sigue
un p ocedimien o anal´
ı ico pa a medi poly-P en cul i os de algas que pos e io men e ue modi icado pa a es udia el poly-P
en los sedimen os median e el m´
e odo EDTA de accionamien o secuencial de P y que pe mi e ex ae los compues os de P
ino g´
anico (P adso bidos a Fe y Ca) an es de cuan i ica el poly-P. Hemos u ilizado es a me odolog´
ıa en suspensiones que
con en´
ıan poly-P p oducido biol´
ogicamen e, como cul i os de labo a o io en iquecidos en P de Synechocys is PCC 6803
(Cianobac e ia), Anabaena a iabilis (Cianobac e ia), y Chlo ella sp. (Clo o i a), as´
ıcomo en angos ac i ados de depu-
ado a. Siguiendo es e p ocedimien o, las concen aciones de poly-P en los cul i os de Synechocys is PCC 6803, Anabaena
18 Reina e al.
a iabilis,yChlo ella sp. supusie on el 15.4, 6.2 y 7.2% de la suma de odas las acciones de P, espec i amen e, mien-
as que los angos ac i ados alcanza on po cen ajes meno es (3.7%). Se en iquecie on suspensiones de sedimen o na u al
con ol´
umenes conocidos de las an e io es suspensiones y con un poly-P es ´
anda come cialmen e disponible (T ime a os-
a o); se compa ´
o, en mues as duplicadas, la composici´
on de P de es as suspensiones con sus co espondien es mues as
de sedimen o con ol. La concen aci´
on de poly-P de e minada en las mues as de sedimen o con ol ue ex emadamen e
baja (0-2 µgg
−1p.s.), pe o ´
es a aumen aba en odas las suspensiones en iquecidas con las mues as biol´
ogicas, en pa ic-
ula con Synechocys is (m´
as de 220 µgg
−1p.s.). La a io en e las concen aciones de poly-P espe adas y obse adas en
las mues as en iquecidas no ue exac amen e 1:1, sino 0.79 al a˜
nadi 2 ml y 1.12 al inc emen a el olumen a˜
nadido de
cul i o de Anabaena (5 ml) debido, p obablemen e, a la al a de homogeneidad del cul i o de cianobac e ias. Sin emba go, la
adici´
on de 500 mg de ime a os a o, un compues o sin ´
e ico, a la mues a con ol de sedimen o no inc emen ´
o signi ica i a-
men e (p>0.05) el po cen aje de la acci´
on de poly-P. Po an o, el poly-P p oducido biol´
ogicamen e se puede asocia a la
acci´
on de poly-P ex a´
ıda siguiendo el p ocedimien o aqu´
ıp esen ado.
Palab as cla e: Poli os a o, m´
e odo EDTA, accionamien o de P, sedimen o.
Abb e ia ions used:
Abb e ia ion Desc ip ion
To -P To al P
i-Pdiss Dissol ed ino ganic phospha e
o g-P O ganic phospha e compounds
FeOOH≈P I on-bound phospha e
CaCO3≈P Calcium-bound phospha e
o g-P→acid O ganic phospha e compounds soluble in acid
o g-P→EDTA Sum o o ganic phospha e compounds soluble in EDTA solu ions
o g-P→NaOH O ganic phospha e compounds soluble in ho NaOH
o g-P es Residual o ganic phospha e compounds
To -P→TCA To al P in he ho TCA ex ac
TCA T ichlo oace ic acid
INTRODUCTION
Polyphospha e (poly-P) is a linea o hophos-
pha e polyme con aining a a iable numbe o
phospha e esidues ha a e linked by ene gy-
ich phosphoanhyd ide bonds. This compound is
widesp ead in li ing o ganisms, pa icula ly mi-
c oo ganisms, and plays a mul i unc ional ole in
phospha e and ene gy s o age, ca ion seques a-
ion, cell en elope o ma ion, memb ane ans-
po , and me abolic egula ion (Ko nbe g 1995).
Low- and high-molecula weigh poly-P occu in
a a ie y o loca ions wi hin mic obial cells, om
cell memb ane o nucleus, as do poly-P-u ilising
enzymes (Kulae & Kulako skaya 2000). The
unc ion o poly-P as a phospha e ese e in
p oka yo ic and euka yo ic cells is well known.
I has long been es ablished ha he in acellula
concen a ion o poly-P depends s ongly on he
phospha e concen a ion o he medium in labo-
a o y cell cul u es (Kulae e al. 2004). The ex-
apola ion o his ela ionship o na u al sys ems
emains p oblema ic. A emp s o use he concen-
a ion o in acellula phospha e ese e com-
pounds in algae as an indica ion o ecosys em
ophic le el and P-a ailabili y o phy oplank-
on ha e p oduced con adic o y esul s (Ken-
ney e al. 2001, Wa e s e al. 2005, Selig e al.
2006). The s o age o poly-P in cells is, how-
e e , caused by he ans e o cells om con-
di ions o s a a ion o a phospha e- ich cul u e
medium and N sho age (Kuesel e al. 1989). The
deg ee o poly-P s o age in algae is no signi -
ican ly a ec ed by a ia ions in phospha e con-
Quan i ica ion o polyphospha e in sedimen samples 19
cen a ion in he medium (Eixle e al. 2005). In-
co po a ion o ex e nal phospha e in o he cell
poly-P pool p oceeds in a ious s eps: anspo
h ough he cell memb ane, phospho yla ion, and
polyme isa ion o elonga ion o he poly-P chain
(Plae ze a e al. 2005). Theo e ically, his up ake
mechanism enables he o ganisms o main ain
con inuous g ow h, bu unde na u al condi ions
i is mo e likely ha P up ake and g ow h a e de-
coupled when g ow h is limi ed by he lack o
ano he nu ien , o en N.
Nuclea magne ic esonance spec oscopy
(31P-NMR), de eloped o he de e mina ion o
poly-P in cell cul u es, has been applied o he
de ec ion o his compound in soil and sedimen .
Howe e , i has long been known ha g ea ca e
mus be aken o in e p e quan i ica ions o o -
ganic phospha e compounds because he ex ac-
an used will g ea ly a ec bo h he concen a-
ion and he o ms o P ob ained in a 31P-NMR
analysis (Cade-Menun & P es on 1996, Gol e -
man 2006). The complexi y o he mix u e o
phospha e compounds in sedimen makes i im-
possible o a single ex ac an o dissol e all
P o ms o o ex ac a speci ic P compound
wi hou al e a ion o o he s. 31P-NMR esul s o
hese subs ances in he sedimen a e only co -
ec i o he in e e ing compounds can be ex-
cluded, e.g., by a p eceding pu i ica ion ex ac-
ion (Gol e man, 2006). Thus, he quan i ica ion
o o ganic phospha e compounds in he sedimen
i s equi es a sequen ial p ocedu e o ensu e
ha main ino ganic phospha e compounds ha e
been p e iously ex ac ed (i.e., Fe- and Ca-bound
phospha e). This ac no wi hs anding, sedimen
o ganic phospha e compounds a e o en quan-
i ied by using 31P-NMR a e a di ec ex ac-
ion wi h a mix u e o NaOH wi h o wi hou
EDTA (Ahlg en e al. 2006, Rei zel e al. 2006,
Hup e e al. 2007). I is di icul , i no im-
possible, o compa e esul s om di e en s ud-
ies wi h NMR in which di e en ex ac an s
we e used when iden i ying and quan i ying o -
ganic P-compounds in he sedimen . The p esen
wo k ollows an analy ical p ocedu e de eloped
o measu e poly-P in algal cul u es (Gol e man
1960) and la e modi ied o sedimen (Gol e -
man 2006). Se e al samples o biologically p o-
duced poly-P we e also analysed o p o ide pos-
i i e con ols o he ex ac ion o poly-P om
he sedimen . This biologically p oduced poly-
P came om di e en sou ces: ac i a ed sludge,
a na u al phy oplank on bloom, and cell cul u es
o Synechocys is PCC 680, Anabaena a iabilis
and Chlo ella sp. Addi ionally, sedimen sam-
ples we e augmen ed wi h cell cul u es, ac i a ed
sludge, and a comme cially a ailable syn he ic
poly-P s anda d compound (T ime aphospha e)
o es o possible in e e ence wi h he de e mi-
na ion o poly-P in he sedimen .
MATERIAL AND METHODS
Two cul u es o cyanobac e ia (Synechocys is
s ain PCC 6803 and Anabaena a iabilis)we e
g own a 30 ◦C wi h BG-11c medium (Rippka
e al. 1979) in 750 ml lasks. The cul u es
we e bubbled wi h a con inuous s eam o 1 %
( / ) CO2in ai unde con inuous illumina ion
(50 µEm
−2s−1). Chlo ella sp. was cul u ed wi h
Sueoka medium in 1000 ml lasks (Sueoka e al.
1967). The e o e, hese cul u e media con ained
a P bu e . Once exponen ial g ow h ended, cells
we e incuba ed o 12-24 h in ligh wi h ex a
phospha e added o hei cul u e medium o a
inal concen a ion o 5 mg l−1o P by adding
he co esponding olume o a K2HPO4solu-
ion (1.6 mM). Then, cul u es we e cen i uged a
12000 pm o 15 min o eco e he cells in pel-
le s ha we e washed wi h ap wa e (<0.1 µgl
−1
o i-Pdiss,0.3mScm
−1) a e each cen i uga ion.
A inal cell suspension o abou 200 ml was ob-
ained o each cul u e. In o de o emo e i-Pdiss
and phospha e associa ed wi h he cell su ace,
25 ml o ichlo oace ic acid (TCA 5 %) was
added o 10 ml o each cell suspension in se e al
eplica es and kep a 3-4◦C o 0.5-2 h. Then,
cell suspensions we e cen i uged a 12000 pm
o 15 min, and supe na an s we e collec ed in a
200 ml lask o de e mina ion o i-Pdiss concen-
a ions. Each pelle was esuspended in 25 ml
o 70 % e hanol a 90◦C o 10-20 min o in-
ac i a e enzyme ac i i y and o emo e lipid-
associa ed P (Gol e man 1960) and la e cen-
i uged (12000 pm, 15 min). Supe na an s we e
20 Reina e al.
collec ed in a 200 ml lask o de e mina ion o
i-Pdiss concen a ions. Pelle s we e esuspended
in dis illed wa e o ob ain he inal cell suspen-
sions. The d y weigh o hese suspensions was
de e mined g a ime ically in 3-6 eplica es o
1 ml-aliquo each. To -P concen a ion was mea-
su ed in 2-6 eplica es o 1 ml-aliquo each as
i-Pdiss ollowing he me hod o Mu phy & Riley
(1962) a e acid diges ion wi h 0.5 M H2SO4and
K2S2O8(0.5-1 g) a 120◦C o 4 h (De G oo &
Gol e man 1990). The op ical densi y a 882 nm
was ead in a HITACHI U-2000 Spec opho-
ome e using ei he a 1 cm o 10 cm cu e e. The
P- ac iona ion o he inal cell suspension was
pe o med acco ding o he EDTA me hod o se-
quen ial ex ac ion o P in he sedimen (Gol e -
man 1996) and la e modi ied o measu e poly-P
(Gol e man 2006). The esul s a e he a e age o
a duplica e o 10 ml aliquo s o Chlo ella sp.,
2 ml aliquo s o Synechocys is PCC 6803 and
5 ml aliquo s o Anabaena.In he i s ex ac-
ion s ep, 25 ml o a solu ion o 0.05 M Ca-EDTA
(pH 7.8) con aining 1 % di hioni e was added o
each aliquo (pelle 0, Fig. 1). This ex ac ion was
pe o med du ing 1-2 h o e con inuous shaking
in a o o . The suspensions we e hen cen i uged
(12000 pm, 15 min) and he concen a ion o
i-Pdiss was measu ed in each supe na an in o -
de o de e mine he Fe(OOH)≈P. Nex , 25 ml o
0.1 m Na2-EDTA (pH 5) was added o he p e i-
ous pelle and shaken con inuously du ing 2-6 h.
The suspensions we e cen i uged again. The
measu ed concen a ion o i-Pdiss in he supe -
na an co esponds o he ac ion o CaCO3≈P.
Figu e 1. The EDTA me hod o sequen ial P- ac iona ion o sedimen (Gol e man 1996) wi h some modi ica ions o he quan-
i ica ion o poly-P as p oposed by Gol e man (2006). M´
e odo EDTA pa a la ex acci´
on secuencial de las acciones P sedimen a io
(Gol e man 1996) con algunas modi icaciones pa a la cuan i icaci´
on de poly-P p opues as po Gol e man (2006).
Quan i ica ion o polyphospha e in sedimen samples 21
The concen a ion o To -P was also measu ed
in he supe na an s ex ac ed wi h EDTA in o -
de o calcula e he o ganic P- ac ion dissol ed
by he eagen i sel (o g-P→EDTA) as he di -
e ence be ween he To -P and i-Pdiss concen a-
ions. Nex , 25 ml o 0.5 m H2SO4was added
o he p e ious pelle (1 h, con inuous shaking).
The suspensions we e cen i uged as usual, and
he concen a ion o i-Pdiss in he supe na an was
measu ed o ob ain he o ganic P- ac ion solu-
ble in H2SO4(o g-P→acid) and o ensu e ha he
ex ac ion o he p e ious P- ac ions was com-
ple ed. Then, 25 ml o TCA (5 %) we e added
o he p e ious pelle , kep a 3-4◦C o 45 min,
cen i uged as usual and he concen a ion o i-
Pdiss measu ed in he supe na an (cold TCA ex-
ac ion). The same suspension was hen hea ed
in a wa e ba h a 90◦C o ano he 45 min.
The concen a ion o i-Pdiss in he supe na an
was measu ed a e cen i uga ion (ho TCA ex-
ac ion). The di e ence be ween he i-Pdiss con-
cen a ion in he ho and cold TCA ex ac ions
equals he poly-P ac ion, acco ding o Gol e -
man (1960), who ollowed he acid-soluble spe-
ci ic me hod o MacFa lane (1936 in Kulae e
al., 2004). The concen a ion o To -P in he su-
pe na an wi h ho TCA was de e mined a e di-
ges ion (To -P→TCA). Nex , 25 ml o 2 M NaOH
was added o he p e ious pelle and kep a 90◦C
o 2 h. The suspensions we e cen i uged, and
each supe na an was b ough o a inal olume
o 50 ml wi h H2O. The concen a ion o To -
P a e diges ion o he supe na an yielded he
o ganic P- ac ion soluble in ho NaOH (o g-
P→NaOH). The nex pelle was diges ed wi h 0.5 m
H2SO4and1gK
2S2O8a 120◦C o he de e -
mina ion o To -P. This ac ion was e med he
esidual o ganic P- ac ion (o g-P es). Owing o
he high concen a ions o i-Pdiss ound in he
Fe(OOH)≈P and in he o g-P→NaOH, bo h s eps
we e epea ed se e al imes o ensu e a comple e
ex ac ion. The di e ence be ween he sum o P-
ac ions and he To -P concen a ion o hese cell
cul u es was lowe han 15 %.
A wa e sample o 1.5 L was collec ed om
he sho e o Dulce pond (Do˜
nana Na ional Pa k,
SW Spain) du ing a phy oplank on bloom in
Sep embe 2007. An aliquo was p ese ed wi h
lugol in si u o axonomic de e mina ion wi h
an op ical mic oscope (1000×o magni ica ion).
Aphanocapsa a . holsa ica (Cyanobac e ia) was
he dominan axon in his bloom. A cell p epa-
a ion was ob ained ollowing he same p oce-
du e as ha applied o he labo a o y cul u es
(cen i uga ion, 5 % TCA and e hanol p e ea -
men s), excep ha no P incuba ion was pe -
o med. Then, d y weigh , P- ac ion and To -P
we e de e mined as men ioned abo e in dupli-
ca e wi h 1 ml aliquo s (Fig. 1). The di e ence
be ween he sum o P- ac ions and he To -P
concen a ion o he phy oplank on suspension
was always lowe han 10 % and o en lowe
han 5 %. D y-weigh and To -P we e also de e -
mined o he o iginal wa e sample be o e ob-
aining he cell suspension.
A sample o ac i a ed sludge was p o ided
by he labo a o y s a o he municipal sewage-
ea men plan (E.D.A.R. Tablada, Se illa) and
analysed a ew hou s a e collec ion. A suspen-
sion o his ac i a ed sludge was ob ained and
p e ea ed p io o P- ac iona ion (in duplica e
wi h 5 ml aliquo s) as men ioned abo e (Fig. 1).
Again, he di e ence be ween he sum o P-
ac ions and he To -P concen a ion o he ac-
i a ed sludge was less han 10 %.
A 200 ml sedimen suspension was made wi h
H2O and d y sedimen collec ed om a em-
po a y pond (Dulce), once he pond had na u-
ally d ied up, o ep esen he con ol sedimen
samples (SED). A olume o 10 ml o he i-
nal cell suspension o Synechocys is PCC 6803
was added o 10 ml o sedimen suspension in
duplica e (SED+SYN). Duplica e sedimen sam-
ples we e also augmen ed wi h 5 ml o he i-
nal cell suspension o Chlo ella (SED+CHL),
1.5 ml o he ac i a ed sludge (SED+ACT), and
Na- ime aphospha e (SED+TMP) in inc easing
quan i ies (100, 250, and 500 mg). The d y
weigh o each inal suspension was e alua ed
g a ime ically (3 eplica es o 1 ml each). A
mo e ca e ul eco e y es was also made wi h
sedimen samples augmen ed using 2 di e en
olumes o Anabaena cell cul u e: 2 and 5 ml
o cell cul u e, wi h an a e age d y weigh o
0.0101 g ml−1, we e added, in duplica e, o a
known amoun o con ol sedimen (2.0135 g on

22 Reina e al.
Table 1. A e age concen a ion o each P- ac ion (µgg
−1d.w.) in labo a o y cell cul u es (Synechocys is PCC 6803, Anabaena
a iabilis,Chlo ella sp.), a na u al phy oplank on bloom, and ac i a ed sludge. Concen aci´
on media de cada acci´
on (µgg
−1p.s.)
en los cul i os celula es de labo a o io (Synechocys is PCC 6803, Anabaena a iabilis,Chlo ella sp.), en un bloom de i oplanc on y
en angos ac i ados.
P- ac ion Synechocys is
PCC 6803
Anabaena
a iabilis
Chlo ella sp. Phy oplank on Ac i a ed
sludge
FeOOH≈P 2539 1677 1750 46 757
CaCO3≈P 683 37 51 63 46
o g-P→EDTA 12836 3249 2211 761 46602
o g-P→acid 135 26 6 2 25
To -P→TCA 4012 9651 6385 2108 9490
o g-P→NaOH 1304 2042 1295 1419 1815
o g-P es 56 222 277 57 69
P- ac ions 21566 16904 11975 4456 58805
a e age). The inal d y weigh o each augmen ed
suspension was he sum o he d y weigh in
each con ol sedimen sample plus he d y weigh
ha co esponded o he added olume o he
cell cul u e. P- ac iona ion was ca ied ou a
he same ime o each amended sample and he
cell cul u e. The concen a ion o To -P was ca -
ied ou as desc ibed abo e using 2 eplica es o
1 ml o each suspension. On a e age, he di e -
ence be ween he sum o P- ac ions and he To -
P concen a ion o he amended sedimen sus-
pensions was less han 11 %.
RESULTS
The concen a ion o To -P was highes in he
sample o ac i a ed sludge, had in e media e al-
ues o he cell cul u es o Synechocys is sp.
PCC 6803, Anabaena a iabilis and Chlo ella
sp. g owing in a P-en iched cul u e medium, and
was lowes in he na u al phy oplank on bloom
(Table 1). As expec ed, he sum o o ganic P-
ac ions cons i u ed he la ges po ion in hese
samples, anging om 78 % in he Chlo ella
cul u e o 99 % o he sum o all P- ac ions
in he ac i a ed sludge, bu each suspension
had a di e en p opo ion o o ganic P- ac ions
(Table 1). The ac i a ed sludge showed he high-
es pe cen age o o g-P→EDTA, whe eas he cul-
u es o Chlo ella and Anabaena and he phy o-
plank on bloom we e iche in To -P→TCA.
Once main ino ganic P- ac ions (FeOOH≈P
and CaCO3≈P) and o ganic phospha e com-
pounds soluble in acid (o g-P→acid)hadbeen
ex ac ed, he concen a ion o poly-P was de-
e mined as he di e ence be ween a cold and
a ho TCA ex ac ion (Table 2). The pe cen -
age o poly-P was he highes o he cul u e
o Synechocys is (15.4 %) ollowed by he cul-
u e o Chlo ella (7.2 %) and Anabaena (6.2 %),
whe eas he phy oplank on bloom and he ac i-
a ed sludge showed lowe p opo ions (3.8 and
3.7 %, espec i ely). This P- ac iona ion p o-
cedu e was pe o med on sedimen alone (as
a con ol) and on sedimen suspensions aug-
men ed wi h biological samples (cell cul u es
and ac i a ed sludge) and wi h a s anda d syn-
Table 2. A e age concen a ion o i-Pdiss (µgg
−1d.w.) ex-
ac ed wi h TCA, a p ocedu e ha yielded measu emen s o he
poly-P pool om se e al biological sou ces: h ee labo a o y
cell cul u es (Synechocys is PCC 6803, Anabaena a iabilis,
Chlo ella sp.), a na u al phy oplank on bloom, and ac i a ed
sludge. Concen aci´
on media de i-Pdiss (µgg
−1p.s.) ex a´
ıda
con TCA y su co espondien e acci´
on de poly-P en dis in-
os ma e iales biol´
ogicos: es cul i os celula es (Synechocys is
PCC 6803, Anabaena a iabilis,Chlo ella sp.), un bloom de i-
oplanc on y en angos ac i ados.
cold TCA ho TCA poly-P
Synechocys is PCC 6803 48 3375 3327
Anabaena a iabilis 26 1067 1041
Chlo ella sp. 76 936 860
Phy oplank on 25 197 172
Ac i a ed sludge 132 2331 2199
Quan i ica ion o polyphospha e in sedimen samples 23
Table 3. A e age concen a ion o P- ac ions (µgg
−1d.w.) in sedimen augmen ed wi h Synechocys is PCC 6803 (SED+SYN),
Anabaena a iabilis (SED + ANA), Chlo ella sp. (SED+CHL), ac i a ed sludge (SED+ACT), and ime aphospha e (SED+TMP)
and hei co esponding con ol wi h sedimen alone (SED). Concen aci´
on media de las acciones de P (µgg
−1p.s) en el sedimen o
en iquecido con Synechocys is PCC 6803 (SED+SYN), Anabaena a iabilis (SED + ANA), Chlo ella sp. (SED+CHL), ango ac i ado
(SED+ACT), y ime a os a o (SED+TMP), as´
ıcomo sus co espondien es sedimen os con ol (SED).
SED SED+SYN SED SED+ANA SED SED+CHL SED SED+ACT SED SED+TMP
FeOOH≈P + CaCO3≈P 89 220 28 71 89 154 28 40 61 409
o g-P→EDTA 10 1164 14 72 10 33 14 161 6 24608
o g-P→acid 9913941445
To -P→TCA 10 648 1 217 10 76 1 44 2 6
o g-P→NaOH +o g-P
es 114 261 37 123 114 104 37 94 95 110
he ic poly-P compound (T ime aphospha e) o
es o in e e ence wi h he de e mina ion o
biologically-p oduced poly-P in he sedimen
(Table 3). The la ges P- ac ion was he sum o
o g-P→NaOH and o g-P es in all con ol sedimen
samples. The augmen a ion o he con ol sed-
imen suspensions changed he P- ac ion com-
posi ion (Table 3), and his change was pa ic-
ula ly la ge o he pe cen ages o o g-P→EDTA
and To -P→TCA (Fig. 2). As expec ed o he d y
sedimen o a empo a y pond, he poly-P con-
cen a ion was ex emely low in all con ol sedi-
SED+SYN SED SED+CHL SED SED+ACT SED SED+TMP SED
%
0
20
40
60
80
100
o g-P_EDTA
poly-P
To -P_TCA
Figu e 2. Pe cen age o a ge P- ac ions in each es o sed-
imen augmen ed wi h Synechocys is PCC 6803 (SED+SYN),
Chlo ella sp. (SED+CHL), ac i a ed sludge (SED+ACT), and
ime aphospha e (SED+TMP) and hei co esponding con ol
wi h sedimen alone (SED). Po cen aje de las acciones de P
en cada cul i o de sedimen o en iquecido con Synechocys is
PCC 6803 (SED+SYN), Chlo ella sp. (SED+CHL), ango ac i-
ado (SED+ACT), y ime a os a o (SED+TMP), as´
ıcomo sus
co espondien es sedimen os con ol (SED).
men samples, almos app oaching he de ec ion
limi (0-2 µgg
−1d.w.), bu i s concen a ion in-
c eased in he sedimen suspensions augmen ed
wi h biological samples. In con as , he sedimen
augmen ed wi h he syn he ic ime aphospha e
showed a highly signi ican inc ease in he o g-
P→EDTA ac ion, whe eas he pe cen age o poly-
PandTo -P
→TCA did no inc ease signi ican ly
(p>0.05) compa ed o he con ol. Fu he ad-
di ions o ime aphospha e (up o 500 mg) o he
con ol sedimen suspension did no yield a sig-
ni ican inc ease (p>0.05) in he p opo ion o
poly-P and To -P→TCA, whe eas he o g-P→EDTA
ac ion accoun ed o nea ly he sum o all P-
ac ions (97.8-98.3 %).
The eco e y o poly-P in sedimen samples
augmen ed wi h he Anabaena cell cul u e did
no show an exac 1:1 a io be ween he expec ed
and obse ed alues. This a io was 0.79 and 1.12
o he addi ion o 2 and 5 ml o he cul u e, e-
spec i ely. The addi ion o 2 ml o he cell cul u e
implied an expec ed poly-P concen a ion o only
10 µgg
−1d.w., p obably oo low a concen a ion
o be de ec ed wi hou e o , whe eas he la ge
olume o 5 ml yielded a mo e p ecise and ac-
cu a e esul (Fig. 3). In con as , he addi ion o
up o 500 mg o Na- ime aphospha e, a syn he ic
s anda d cyclic P-compound, did no p oduce a
signi ican inc ease in he obse ed poly-P con-
cen a ion because he added P appea ed in a di -
e en P- ac ion (o g-P→EDTA) a he beginningo
heP- ac iona ion.Consequen ly,biologicallyp o-
duced poly-P can be associa ed wi h he ac ion
o poly-P ex ac ed h ough use o his p ocedu e.
24 Reina e al.
poly-P ( gg
-1d.w.)
0
5
10
15
20
25
30
2ml 5ml
Figu e 3. Reco e y es o poly-P in sedimen samples
amended wi h 2 and 5 ml o Anabaena cul u es. Mean con-
cen a ion and SE o he de ec ed poly-P a e he augmen a-
ion (black ill) and he calcula ed mean and SE o he expec ed
concen a ion (whi e ill) o each added olume. Recupe aci´
on
de poly-P en mues as de sedimen o en iquecidas con 2 y 5 ml
del cul i o de Anabaena: se mues a la concen aci´
on media y
des iaci´
on es ´
anda (SE) del poly-P de ec ado (en neg o) y la
media y des iaci´
on es ´
anda (SE) de la concen aci´
on espe ada
(en blanco) seg´
un el co espondien e olumen a˜
nadido.
DISCUSSION
This s udy ound he highes pe cen age o s o ed
poly-P in he Synechocys is cul u e (15.4 %).
S o age o poly-P in labo a o y cul u es o au-
o ophic o ganisms a ies widely acco ding o
he li e a u e. The p opo ion o poly-P has
been epo ed o be highe a as e g ow h
a es (Rhee 1973 in Thompson e al. 1994).
In some cyanobac e ial cul u es, poly-P ep e-
sen ed 60 % o he To -P in cells o Oscilla o-
ia edekei,10%inSynecoccus, and be ween 3
and 6 % in Anabaena los-aquae (Thompson e
al. 1994). In Scenedesmus cells, be ween 5 and
32 % o cellula -P accoun ed o poly-P (Gol e -
man 1960). This a iabili y esul s om mul i-
ple ac o s, including he ex en o P limi a ion
in he cell cul u es, g ow h a e, N s a a ion,
and speci ic physiological adap a ions (Kuesel e
al. 1989, Kulae e al. 2004. Compa ison wi h
o he esul s is di icul because poly-P is quan-
i ied by a di e si y o me hods in he li e a u e:
in i o NMR, NMR a e a a ie y o ex ac an s
(NaOH, EDTA, TCA), ho wa e , cold/ho TCA
ea men (Hup e e al. 2008). The elegance o
he me hod p esen ed he e lies in he ac ha
he same eagen was used o he p epu i ica-
ion o he pelle (TCA a 4◦C) and he ex ac-
ion o poly-P (TCA a 90 ◦C) om he same su-
pe na an . Only empe a u e was changed o al-
low o he ex ac ion o his compound, as poly-
P is hyd olysed 105-106 imes as e a 100◦C
han a 0◦C (G eenwood & Ea nshaw 1984). A -
e hyd olysis, he TCA ex ac p obably yielded
a mix u e o compounds (de ined he e as To -
P→TCA) wi h a highe p opo ion o o g-P com-
pounds in he Chlo ella cul u e han he Syne-
chocys is cul u e. Nucleo ide-P and suga -P a e
epo ed o be ex ac ed oge he wi h poly-P in
TCA-soluble ac ions om cell cul u es o pho-
oau o ophs such as Scenedesmus quad icauda
(Gol e man 1960) and he cyanobac e ium An-
abaena los-aquae (Thompson e al. 1994).
In he sequen ial p ocedu e p esen ed he e,
he main ino ganic P- ac ions had been p e-
iously ex ac ed by wo sequen ial ea men s
wi h chela ing agen s (Ca-EDTA and Na2-EDTA)
ollowed by an ex ac ion wi h dilu ed H2SO4
(o g-P→acid) o p e en con amina ion o subse-
quen s eps wi h aces o p e ious ac ions.
As expec ed, his dilu ed-acid washing accoun ed
o a e y small pe cen age o all P- ac ions
(0.1-0.6 %), simila o ha o he inal esidual
pelle (o g-P es). The use o EDTA allowed o he
quan i ica ion o a P- ac ion soluble in EDTA
bu no de ec able un il hyd olysed, and hus de-
ined he e as an o g-P ac ion (o g-P→EDTA).
Howe e , in biological samples, i may co e-
spond o he ino ganic phospha e pool o cells
(Law ence e al., 1998). In i o 31P-NMR spec-
oscopy o Synechocys is sp. s ain PCC 6308
has indica ed ha ino ganic phospha e, bu no
poly-P wi h a molecula weigh >1000 Da, leaks
om he cell as he esul o ea men wi h
EDTA (Law ence e al. 1998). The eason ha
his cellula ino ganic phospha e equi es a hy-
d olysis o i s de ec ion is unclea . The low-
molecula -weigh ime aphospha e also equi ed
hyd olysis o i s de ec ion. T ime aphospha e is
a syn he ic cyclic poly-P compound and appea s
o be mo e s able in aqueous solu ion han lin-
ea polyphospha es. The assignmen o peaks o
poly-P in 31P-NMR spec oscopy o soil and sed-
imen ex ac ions has been adi ionally based
on a s anda d o comme cial ime aphospha e
(Adams & By ne, 1989; Cade-Menum & P e-
s on, 1996). In ou s udy, howe e , we used se -
Quan i ica ion o polyphospha e in sedimen samples 25
e al s anda ds (a comme cial ime aphospha e,
cell cul u es g own in a P- ich medium and ac-
i a ed sludge) o allow an analy ical compa -
ison o biologically s o ed poly-P. These bi-
ological suspensions p o ided a posi i e con-
ol in o de o es he e iciency o his p o-
cedu e o he de ec ion o cell poly-P. The
ime aphospha e p o ided a nega i e con ol, as
i was no p oduced biologically. Al hough his
p ocedu e does no de e mine poly-P molecula
size o sepa a e poly-P om di e en in acel-
lula loca ions, i can be a use ul ool o assess
he ele ance o biologically p oduced poly-P.
This poly-P o biological o igin is e en ually de-
posi ed in he sedimen unde na u al condi ions.
The de ia ion om a 1:1 a io be ween he ex-
pec ed and de ec ed poly-P concen a ions in sed-
imen samples augmen ed wi h Anabaena was
sligh ly highe when 2 ml a he han 5 ml o
cell cul u e we e added. The o me addi ion im-
plied a lowe a ge ed poly-P concen a ion (10
and 26 µgg
−1d.w., espec i ely) and was hus
mo e likely o accumula e e o s. Ne e heless,
only 1 µgg
−1d.w. o di e ence was ound o oc-
cu be ween he eplica es o he poly-P con-
cen a ion de ec ed in he duplica ed suspension
amended wi h 2 ml, whe eas no such e o in p e-
cision occu ed wi h he 5 ml addi ion. The cal-
cula ion o eco e y a ios o augmen ed p epa-
a ions made using o he P sou ces was hampe ed
by se e al ac o s: a) he low biomass o he bio-
logical sou ces implied e y low a ge ed poly-P
concen a ions; b) he cell cul u es p oduced in-
homogeneous suspensions ha caused low p e-
cision among eplica es: c) he numbe o epli-
ca es was se e ely limi ed by he low biomass o
he cell cul u es. The eco e y es was conduc ed
o he augmen a ions in ol ing Anabaena be-
cause hese cul u es p oduced he leas inhomo-
geneous suspensions. Indeed, d y weigh was cal-
cula ed wi h a p ecision 5-10 imes highe han
he p ecision ob ained wi h he o he P sou ces.
CONCLUSIONS
The applica ion o his me hodology allows he
quan i ica ion o he sedimen P- ac ion ha co -
esponds o he P s o ed by aqua ic mic oo gan-
isms. The me hod is easible o his pu pose be-
cause i has been es ed wi h biologically p o-
duced poly-P (posi i e con ol) and a syn he ic
ime aphospha e (nega i e con ol). The ad an-
age o his me hod is ha i ollows he EDTA
me hod o sedimen P- ac iona ion long used in
na u al aqua ic en i onmen s. Mo eo e , i ensu es
ha he main ino ganic P- ac ions (i.e., Fe- and
Ca-bound phospha e) ha e been ex ac ed p io o
he de e mina ion o o g-P ac ions. Addi ionally,
he quan i ica ion o biological poly-P is ca ied
ou sequen ially in he same supe na an a e a
change in empe a u e combined wi h ex ac ion
in cold TCA as a speci ic blank o each
sample. The e o e, his p ocedu e is sui able o
ecological s udies ha aim a he quan i ica ion o
biologically p oduced poly-P in aqua ic sys ems.
ACKNOWLEDGEMENTS
We a e e y g a e ul o he ollowing col-
leagues o p o iding se e al ypes o biologi-
cal ma e ial o ou analyses: Ma ibel Mu o, E a
Rod ´
ıguez, M.aCa men Po illo, Juan Gonz´
alez
and Me cedes Gonz´
alez.
REFERENCES
ADAMS, M. A., & L. T. BYRNE. 1989. 31-NMR
analysis o phospho us compounds in ex ac s o
su ace soils om selec ed Ka i (Eucalyp us di-
e sicolo . Muell.) o es s. Soil Biol. Biochem.,
21: 523–528.
AHLGREN, J., K. REITZEL, L. TRANVIK, A.
GOGOLL & E. RYDIN. 2006. Deg ada ion o
o ganic phospho us compounds in anoxic Bal ic
Sea sedimen s: A 31P nuclea magne ic esonance
s udy. Limnol. Oceanog ., 51: 2341–2348.
CADE-MENUM, B. J. & C. M. PRESTON. 1996. A
compa ison o soil ex ac ion p ocedu es o 31P
NMR Spec oscopy. Soil Sci., 161: 770–785.
DE GROOT, K. J. & H. L. GOLTERMAN. 1990. Se-
quen ial ac iona ion o sedimen phospha e. Hy-
d obiologia, 192: 143–149.
EIXLER, S., U. SELIG & U. KARSTEN. 2005. Ex-
ac ion and de ec ion me hods o polyphospha e