Metabolic and ~ceatlographiccoIIs6QuetUrp_C:of iron deficiency in
hetecotrophic marine protozoa
Zama Chase
Deparcment of Biology. Mffiili University. Montreai
August 1996
A thesis submitted to the Faculty of Graduate S ~ d i c and
s Reseawh in partial fuifiUment of
the quirernents of the d e p of Mas= of Science
Q Zama Chase 1996
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Abtract
Iroa is recognued as a key element regdahg primary production in large mgions of the
ocean, but nothing is known of its d k t effect on higher mphic ieveis. k w t h and
metabolism of two species of heterotrophic protomans fed iron-rich and iron-poor prey
weze Uius examined.
Maximum p w t h rates of Par4physoomo~sUnpeflorm and P.
butcheti were obsemd only when Fe quotas of bacterial prey were greater than 70 p
l
Fe:mol C. At lower Fe:C ratios, but at constant prey bioniass (W),
both species grew
sipnincantly slower. Minimum Fe quotas of the flagellates at these slow p w t h mes
(-
10 p o l Fe:mol C) were simüar to those of b n - h i t e d phytoplankton and bacteria
Growth rate reduction was the result of direct elemental limitation by Fe, judging h m the
protomans'positive response to Fe additions and h m their biochemical characteristics.
Filtrationand carbon ingestion rates inmased under Fe-limitation, but carbon gross
growth efficiency (COGE) d e ~ e a s e dwhen Paraphysumoo~~
Unpefloratoconsumed Kon-
poor bacteria. Ammonium regeneration efficiency was also reduœd. The àecrease in
CGGE was a consequenceof reduced activity of the irondependent electron transport
system, p a t e r DOC excretion, and p a t e r
evolution by Fe-limited flageliates.
Pwaphysomo~~
impe~orutaexcreted Fe, even when limited by this element, and retained
less of the ingestcd ration and thus had a higher Fe regeneration efficiency than when
consuming Fe-rich bacteria. According to ment measuremnts of biogenic Fe:C in the
subarctic Pacific, our results suggest that heteromphic bacterivorous fiagellates may
experience iron-limitation in remote oceanic regions. Such limitation could profoundiy
a f k t C, N and Fe cycling in the sea.
iii
On nxonnait le fer connne un éi6ment réglant la production plimairt dans de nombreuses
régions océaniques. Cependant, on ignore jusqu'à présent les effets directs sur les
broutem. Cette etude a donc comparée la croissance et la physiologie de deux esptces de
protozoaires se nourissant de bactéries contenant des concentrations 6kvées et faibles de
fer. ûn a observt les taux de croissance zrmimaux chez Parqphysommas imperforuta et
P. butcheri seulement quand Ie rapport Fe:C des bactéries dépassait 70 pmo1:mol. Quand
le Fc:C etait plus bas. mais B une concentration de biomass de proies
(ad)
constante, les
taux de croissance ttaient significativement plus lents. A ces taux de croissance lents, les
rapports Fe:C des flageliés (- 10 poVmo1) ressemblaient A ceux du phytoplancton et du
bactérioplancton quand ces derniers sont iixnités en fer. La réduction du taux de
croissance semble être le resultat directe d'une limitation en fer, il en juger par la Ftponse
positive des flageliés aux edditions de fer, et par leurs traits biochimiques. Les
protozoaires limités en fer avaient des taux 616~6sde filtration et d' ingestion de C, mais ils
avaient une moindre efficacite brute de cioissance comparés & ceux qui consommaient
des bactéries riches en fer. L'efficacité de régénération de NHq+ etait aussi réduite chez
ces protozoaires. La diminution en efficacité brute & croissance etait la conséquence
d'une réduction de l'activité du système de transport des tléctrons, une plus grande
production de carbone organique dissout. et une plus grande évolution de CO;? par les
flagell6s limités en fer. Paraphysomo~simperforatu 6xmtait du fer, mêm lorsque
limitk par cet 616ment. Les flagellés retenaient moins du fer consommé quand leurs proies
contenaient peu de fer. et avaient donc une efficacité de rCgen6ration plus éievée que les
fiageliés qui amsommaientdes bactéries riches en fer. Dans le contexte de mesures
dcents du rappon biologique Fe:C dans l 'occanPacinque sub-arctique. nos résultats
suggèrent que les flageU6s bacterivores peuvent êtres limités en fer dans des dgions
Occpniques CloigiiCes. Une tek limitation a d'importantes iniplicatious au niveau des
cycles & C, N et Fe dans ia ma.
Table d Contents
Abstrsct
i
Rtsumt
ii
Table of Contents
iv
List of Figures
v
U t of Tables
M
Acknowledgmtnts
vii
Reface
viii
Background
Backgmund Rehnces
Introduction
Methods
Resdts
Discussion
siimmat~
Acbiowledgments
Refererices
Figure 4. Timcouru of iion dynamies in cultum with birteria and Paraphysomo~s
ini;pe@ormaand conml cultures wirh banaia alone. p. 33
List of tables
Tabk 1. Iron quotas of two proto2108 ancl th& biictaialpny uada high and low irm
cmditions @Fe18 ami pFe21, nspectively). p. 30
Table 3. lron budget f a Piu4physomow hper/ora consuming iron-repletc (pFel8p w n ) or iron-deplete (pFe21-pwn) bacmial prey (main Jd88). p. 38
Table 4. Elecmn transport sysrem aftivity (ETS) per cellular kon in marine
hetaoaophs. p. 43
1have to thanL N d Rice for M g the bcst supMsor eva. His cr~ativity,sense
of scicntific ekgancc, rnd never-cdhg cnthushsm sre an insp'd011.He was always
encouraging. and made me set the positive side of evay expiment. 1sm also grateful to
the mmbers of my supeMsay Committce, Drs. Joe Rasrnusstn and Jaap Kalff, for rhcir
valuabte aiggt~tions.
'Ibe lab was a won-
piace ü, spead many many hours, th&
to Jay, Quis,
Julie and Maite. Our many c01lverSations. whetha about scitncc, politics or fristee, were
always insighaul. Most of aii I t h a d Maite for hcr great patience and sLiu as a teacher,
and f o her
~ benevolent reign over the lab. She always found tim to help me out, and 1
owe much of this thesis to her guidance.
Finally, I thank m y mother, father, brother, gjysha and Bryce, for their love.
niesis specifidons stipuiatc that the fobwing five paragraphs pppcar at the
kginning d aii manuscript-basal theses:
"Candidateshave the option of iocl&g,
as pan of tbe diesis, the tnrt of m e or
mare papas submiuecl or to be submi#ed f a pubïication, a the cclaslyduplicatd text of
one or mae pubîished papers. These tex& must be b o d as an inte@ part of the thesis.
If this option is chosen, ronnechg tethe dimerent papers are ainnâatory.
t h t provide l @ d bridges between
me thesis must bc writtcn in such a way that it is
more than a mcrc collection of manuscxipts; in other words, nsults of a saics of p a p a
must be intcgrated.
The thesis must sdll d o m to ali other requirements of the "Guidelinesfor lhesis
Reparation". The thesis must indude: A Table of Contents, an abstr~ctin English and
Fknch. an introduction which clearly states die mionale and objectives of the shdy, a
comprehensivereview of the literanirt, a final conclusion and sunnnary, and a thorough
bibliopphy or refance list
Additional material must be provided whac appropriak (eg. in appendices) and in
sufficiait detail to allow a clcar and precise judgment of the importance and onginality of
the research reporteci in the thesis.
in the case of manuscripts CO-authoredby the candidate and others, the candidate
is required to make an explicit statement in the thesis as to who contributed to such
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authan of the wauthoreâ papers."
'Ihisthesis is based on a manuscript that wili be submined to thejournal Limnology
and ûceanography with Dr. Neil Rice, my supemisor,as CO-author. 1performed a l l the
experhents and analysis, and wnxe the paper. Dr.Rice contributed valuable insight,
suggestions,and improvements to the text Anton Pitts pvided assismi with the ETS
measurements.
A fraction of the roughly 50 gigatomes of carbon tued annually by oceanic
phytoplankton (Longhurstet al. 1995)malces its way into the deep ocean. This downward
C flux, the biological pump, is a small yet dynamic si& term in the global carbon budget
(McCarthy et al. 1986; Longhurst 1991). Low levels of amiosphenc C@ driring glacial
tixnes have k e n linlred D a strengthening of the pump (Ganeshram et al. 1995; Kumar et
al. 1995), and its present state may constrain the capacity of the ocean to absorb incnasing
levels of atmosphenc C%.
Biological m s f e r of C to the deep sea reflects the balance
between phytoplankton production and heterotrophic remineralization. and thus depends
on determinants of new production, food web structure, and ecotrophic efficiency.
New production represents the maximum exportable carbon h m a system for a
given year (Dugdale and Goering 1967; Eppley and Petersen 1979). It is t h part of the
annual production supported by 'new' nutrient inputs -N0.j- and N2-fixation in the case
of
N-limited systems -, and its magnitude is ultimately set by the iight and temperature
regime of the habitat. Differenœs in the hetemtrophic processing of primary production,
however, can profoundly influence the naiized (as opposed to maximum)aamunt of
carbon exported (Michaels and Siiver 1988; Peiiert et a11989; Legenâre and Le Rvre
1995).
Marine heterotrophy effects a partial transfer of carbon h m phytoplankton cells
into larger, rapidy sinking packages (e.g. fecal pellets), with the concornmitant release of
nspired carbon as Ce. Some ingested carbon is also lost as dissolved organic carbon
(DOC) (Lampert 1978; Caron et al. 1985; Jumars et al. 1989)
which is w n s d by bactcrh anci citba rwntas the
~~chah W the miCrobial
bop (Pomeroy 1974; Azam a aï. 1983) a is rclcased a g a h by virpl lysis (Bratbak et al.
1992; Futirman anâ NoMe 1995). In this way, -hic
BCtivity rrpockages and
. .
mnheralizes the carbon (and nutrîents) aciimililrrl by photasyntbcsis. H c ~ h i c
p w t h efficiency detamines bow much carbon is q a c k a g d as opposcd to respired or
excreted, ansi is thus a criticai paramtter in the ultimate fote of al@
C. Consider two
bounding cases. In the &tg hetcmtmphic effkitncy is high, a d Plmost aîi the new
production is t r a n s f d to large. rapidly sinking packages. In the second case, the same
quantity of new production en-
a network of highly inefficient hetcrotophs, and most of
the algal C is respired. This second system a a y support high leveis of regenerated
production, but the net e f k t of such low heterotmphic efficiency is that l e s new
production gets exported. nius, while phytoplankmn productivity is clearly of great
importancein detennining the s m g t h of the biological pump, the cc010gical details of the
betemttophic community -how many steps, the efficiaicy of cach step, the site, life
history, and physiology of dominant grazes -WU
aiso affect the amount of C rransfemed
ta depth by the marine biota.
Biological ~ceanographershave t y p i d y cunmtrated on determinhg the limits to
phytoplankton carbon fixation, and duected lcss effort towards understanding variation in
heterotmphic efficiency a community structure. Despitc the importance of heterotrophic
growth efficiency as a biogeochemical variable, zooplankton respiration rate, assimilation
efficiency, and growth an often included as constants in ecosystem models (e.g. Harvey et
al. 1935; Conover and Huntley 1980; Frost and -zen
1992; Boyd et al. 1995). Whiie
such simplificationsarc o k n oeccssey,<bey igmre potentially significsnt regionai
chamacrhtics of zooplanhDn production. For example, tempaoture (Pcriçy 1988), food
abundancc (Urobc dWsnntabe 1991). body size (Banse 1982) Md 'food quaiïty' (in a
gentml seose) (ArnoId 1971; Pater 1973; Ahlgrcn a ai. 1990) are known to affect
p w t h cfficiency and rrproductim. Recent work also suggtsts the elemental
composition of aigae (C:NJ) can influena nccpiankton pmductian nad ooanaunity
oomp~sitionmessen 1992; UrPbc and Waiuitshe 1992; Rotbhnupt 1995): The
cansumption of prey items witb a low N:C a RC causes gmwth inhibition of
zooplankton, analogous to nutrient limitation of autooophs. Esscntiaily, hetemtrophic
nutrient limitation leads to reduced yield and grcater rernineralizationin terus of carbon.
as organisns dispose of 'excess C relative to the nuaient in shonest supply. Nutrient
limitation of hetc10troph.i~production thus has important consequenccs for carbon cycling
in a q h systems, yet it has d v e d liak attention bcyond a few laboratory sadies of
aesbwater moplanlaon.
If consideration of metaman gra~rs(zooplanhon) and their regdation was a late
development in the study of planhonic systems, recognition of the importance of
protozoan grazers (2-20 )un. predominantly fiagellates) came even later. We now know
that these small grazers play a signüicant role as cunsumers of both bacteriai and
phytoplankton production in the sa,particularly in oligotmphic waters (see S h m and
Sherr 1994, and references therein). Inâced, in a variety of marine systeau, protomans
consume h m 25-100R, of daily pnduction (Shen and Shen 1994). They an responsible
for the bulL of nutrient r e g e n d o n (Caron and Go1âman 1990), and serve as a link to
higher trophic levels (Giffard 1991). Their abundance and growth rate are ultimately
driwn by the amount of food available, as with metaman grazm. Thus, several reports of
large d e patterns relating the abmdauce of hetemtrophic nanoflageliates to bacmial
abunciana have appeared (Smders et al. 1992; Berninger et ai. 1991), in addition to
numemus laboratory studies demonstrating the dependence of protoz~if~l
yield and growth
rate on prcy density (e.g. Fenchel1982; Shexr et al. 1983; Geider and Mbeater 1988).
Again, relatively liale attention bas k e n paid to the f a c m that rcguïate protozoan
growth efficiency at a given level of fmd abundance. Yet this is a key parameter in
assessing the ecoIogicai and biogeochemical role of these grazers. Aotozoan growth
efficiency has been related to temperature (Choi and Peters 1992)and ceIl volume,
(Fenchel and Fiolay 1983) and some investigators npa chat the biomass-independent
growth rate of protomans is affecteci by prey species type (different algal species, Rubin
and Lee 1976; different bactenal species, Shem et al. 1983; bacteria vs. cyanobacteria,
Caron et ai. 1991). nie potential for these organisms to be nutrient limiteci in the sea has
not even been considered. Indeed, the elemental requirements of phagotrophic protomans
are virtuaily unknown. Goldman et al. (1987) and Eccleston-Parry and Leaàbeater (1995)
saidied nutrient r e g e n d o n by flagellates consuming algai and bacterial pley of different
nutritional States (C:N:P). Though they were not explicitly testing the effect of prey
chernical composition on flagellate growth, and thus did not contml for ciifferences in
initial pity biomass, their data, if not their interpretation of the &ta, provide prelMinary
evidence of slower growth when the protozoans consumd nutrient-deficientprey.
The abundana of the major nutrients, particukly nitmgen, bas traditionaily been
considereâ a limiting factar fbr primary production. Recent work, however, shows that
iron limits new production in duee large areas of the ocean.aamly the subarctic Pacinc
(Martin et aL 1989), the equatorial Pacific (Ricc et al. 1991; Martin et ai. 1994) and the
Southern Ocean (deBaar et ai 1995). It has been suggested that iron fertilizaton, induccd
through changing wind patiems, for example, wuià enhaace bio10gical productivity and
change these areas h m net sources to si& of <%.Such fertilization, particularly of the
Southem Ocean, could fonr interglacial-glacial shifb in global amrospheric
levels
(Martin 1990, Kumar et al 1995). The magnitude of any effect of iron fertiiization on
CO2 levels will clearly &pend on the ecosystem response to iron addition: How do
grazas respond to an increase in biomass and iron content of their pny? In the ironlimiteâ ntgions, small phymplaaktDn (C2 p)
ami bacteria dominate the autotrophic
biomass (e.g. Riia et al. 1990, Boyd a al. 1995). Rotomans, which efficiently
bacteria and small algal ceils (Fenchel1982; GolQian and Caron 1985; Caron et al. 1991;
Shem and Shen 1994), are thus the most important consumers in these regions, in terms of
b o t . abundance and gxazing impact (equatorial Pacific, Qiavez et al. 1991; subarctic
PaQfif,Smm and Welchmyer 1991). Aithough the relative geochemical importance of
the biological pump and the 'chernical pump' are stiU àebated (Peng and Brœker 1991).a
snidy of the iron requiremnts and the potential for iron limitation of protozoan grazers
has clear implications for undeatanding the ecology of large areas of the sea. and thus
fomis the basis of my thesis.
ïkttalizationtbatiiwplnysakcyroleinaranicpsim;iypmiucti~~~~
crimnlalrA much rrstarch into the iroa qiiircmtnts and gcnaal Ilon physidogy of
phytoplanhnn_ Irwcaitainiag r c k airymsa essential in tbe rroctioasof
photosyn~
a d nitra&a s s h î h i r n Bioiogicaî
tbt haaooophic m
t
bave iargdy ignacd
y in oiis comcxk rssumk>g (Wells a al
1995) riniil noently
flortciï a aï. 19% ) tbu iwmmphs, lacking a phawyottm. w u l d have reiarivtly low
iroo caatair MicmbI*
however, bave h g ken mtcresa in tbc ifon physiobgy
of heurouophic bctcrb, and to a lessaextent protozoans, because of the link bctween
iron aquisition and pathology in maay nrifiobts (e.g. Buücn a ai 1974). Iron ans as an
elecDonCamainthcrrspir<uoryelectroniransportchain.aodisthusCnticallyarsociated
with aaobic hetff~mphicmttaboIism IndeeQ a calculadon of the minimirm hcm
~oquu#oentsof heterotropbic bactaia (J. Granga, in
m.),
found die bulk of
iD~11ularkisaFsociatedarithtbcelaniarranspatchain,Pndrhat~gcellular
rcquirrments f a maximum groMh (60pmoiFc :d C )arc highcr thm tbosc predicted
for pbotoautotrophs ( 23 p.molmol; Raven 1988). Muistxemtnts of the iron wntcnt of a
number of bacterd isolates and of the in-situ comniunity Fe:C (0.2-lm)in the ironlimited subarctic Pacitic cortell et ai. 1996) have codkmed the p r e d i h that bsncria
require large axtmmts of iroa
kcniming rhat aii ktmtmphs have Pmilar rrspinuay pattiways. eulraryotic
hcu~r~trophs
should have tbc sam (high) Fe xcq-ts
as bectaia k a u s e bocraia
and phytoplankm in m t e oceaaic regions arc v a y Fe-poor (Suuda et ai. 1991;T m i l
a al 1996),thm promzloan gra+as might have difnnilty acquiring su&citnt Fe ffor
By d o g y with th& rde in major nutrmt rtgaiefatiDn (Caron a d Goldman
1990). protomans should also k important in k m cycling in surface aratas. Barbeau et
ai. (1996) dernomratcd the ability of fiagellates to effect a transfa of iron h m the
coiloicial (unavailable to phytoplanhnn) to the dissolved (availablc) phase through grating.
In their mode1 of tbe surface iroa cycle. howeva, Bruland a al. (1991) proposeû that
cuiloidal irw fOrmaeion and dissolution arc in9gnifïcant pnxrsses in iron-poor regiw.
where the in&
rc-supply of dissolved Fe arurs primarily througb the release of
biologicaily-boundFe. Hutchins et aL (1993) tested ttiis mode1in the field by adding
5%c4abeled cyanobactcria and diatoms to scawatef samples. 'Ibey foui evidence f a the
oansfer of iron from snall cells (cymbacteria) to indige~~ous
h g e alls in both high and
low-Feenvironmenrî. and f h n large ceils (diataus) to indigenous srnail ceiis in the low-
Fe environment only. Lara laboratory work. focushg on copepods p z b g b t h diatoms
and protou>ans, demonsaatcd grazer-nrAiatcd regenaation of essential rnce metals,
including Fe (Hutchins anci Bnilaad 1994). Rccent work by Hutcbins et PL (1995)
e x a m i d iron rcgeaer;rtionby copepods m m cbsely in remis of wimilatioa efkiency
and prey œiiular irw parütioaing.
AUofdicse~genaatiaisadieshpw~pcrfamedotFc~tratiOnSwcu
rbon those found in tbe bon-poor waters. Since rcgencration of maja numcnts &pends
on tbe supply ratio in the food relative to the r~~uircments
of die graztt (e-g- Olsea et al.
1986; CMa and Ooldmnn 1990).regencd011studies with inm-rich p y tnay yicld
srtincinlly bigh estimates Fiinbamac,ttuie hs been no stuc& of pzcr-mnliatrA
mgeneratiow of Fe bmm hctci~,tr~phic
bctaia kcawc of tb&
rtlatively bgh iroa
content and large biomas. betcro~rophicbacerinreprrsent the largcst pooi of biogenic
iron in the subamic Pacifie ûccan (TocteU a al. 1996). and pPcsumably in the otha imnlimiteci rcgions. Rcgeneration of this psvticuiatc pool to the dissolved phase through
gxazing activity by bacterivorous flagellates might t h ~ t f ~ rbc
i tan impowrt vcctor f
a the
supply of dissolved Fe. A second compontnt of this thesis thus assesses the potential of
£ i a g e Wpmtotoans to micase bactffial-bound Fe. and explicitly tests the effect of bon-
poor prey on such release.
In this study of the iron requircments and iron regeneration of protozoans, 1useû
as a modtl system the non-pigmented chrysomoaad fïageilatc Parqphysomoll~~
h p e @ k u u (VSI). isolated ftom Vineyard Sound, MA, and a marine banerium (soain
Jul88) isolated h m the Sargasse Sea nie flageliatc, which is rapidly becoming the E.coii
of aquatic p r o t ~ ~ ~ ~ lisoubiquitous
gy,
in coastalregions (Lucas 1%7). and has been found
in the oligotmphic Gulf of Elat (Goldman and Caron 1985). It is capable of both
bacterivory and herbivory (Goidmm and Caron 1985). Md if thus a good mode1 organi~m
In an attempt to gen&
my w&
I perfomed a subset of cxperiments using a different
bacterial strain, and an oceanic species ofPcucrphysomo~s.
Ahlgren, Ge,
L.Lundstcàt, M.B m t and C Faskrg. 1990. Lipid compo~itionand food
quaîity of socne kshwattr pbytoplanhnn for cladocffaa tooplanhefi. J.
Planlrton &S.
12: 809-818.
Anroici, DE. 1971. Ingestion, assimilation, sunival, a d rrpmduction by Dqiuiiapulex
fed scven q&cs of bluc-grcm algac. Limaol. ûccanogr. 16: 906920.
Azam. F., T.Fcnchel, J.O. Field, J.S. Gray,LA. Meyer-Rd and F. Thingstad. 1983. The
ecological rolc of wateralumn microbes in the sui. Mar. Ecol. h g . Ser. 10:
257-263.
Banse, K. 1982b. Mass scaled rates of respiration and inirinsic p w t h in very srnail
invertetmates. Mar.Ecol. h g . Sa.9: 28 1-297.
Barbeau, K., J.W. Moffett, D.A. Caron, P.L.Croot and DL.Erdner. 1996. Role of
protousan graziag in nlieving iron limitation of phytoplanhon. Name 380:
6 1-64.
Berninger, U.-G., B.J. Finlay and P.Kuuppo-LcinürLi. 1991. Rotozam control of
bacterial abundances in h s h wata. Limnol. Oceanogr. 36: 139-147.
Boyd, P.W., S. Strcm. F.A. Whimey. S. Doherty. ME.Wen, P.J. Harrison, C.S. Wong
and DE.Varela. 1995. nie NE subamtic PPcific in wintec L Biological
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Bratbak, G., M. Heldal, T.F. Thingstad, B. Riemann and O.H.Haslund. 1992.
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appmach. Mar. Ecol. Rog. Ser. 83: 273-280.
Bruland, K.W.,J.R. Donat and DA.Hutcbins. 1991. Interactive influences of bioactive
trace mctals on bioIogicai production in oceanic waters Timno1. ûccanogr.
36: 1555-1577.
Builen, M.,W.
Rogas MdE.Griffiths. 1974. Bactedl Poa mttabOîh in infection and
immuniry, p. 518-551. Iii J.B. N e W [d]
Miciobial
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iron mttab0iis.m:A
comp~~=htnsivc
aatise.Acadtmic.
Buma, A.G.J., H.J.W. de Baar, R.F. Nolting ad A.J. van Bemekom. 1991. Metal
enrichment expcrimcnts in the Wedâelll-Scotiaseas: Effecü of Fe end Mn on
various planlrton mtnmunities. Limnol. 0cea~)gr.36: 1865- 1878.
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Roceatly. T e i l a ai. (1996)
mwlilnA ôy decrrased
activity of the iron-dependtnt lltspiratory (EîS). Because phymplankton and
bactniaplanhnnin iilon-poor, occanic waters contain vay l i e iroa (Sunda a al. 1991;
Maîdorisrdn and Ricc 1996; TomU a aL 19%), graurs in tbcsc regions may have mwible
acquiiing sufficent Fe for growth. E i ~ ~ ~ l t ~limitation
ltal
of hemotioptiic eukuyotes
(mainly Marater zooplanhnn) has kena m i d a d in the cases of N anci P (Hessen
1992. Umbe and Waaatlbe 1992; MüIler-Navarra 1995; Rothhaupt 1995). but the effect
of iroa content of prey on consuma mctabîism has not becn Pddrrssed
Pbssibly the xmst important conauac~to evaluatc in the mtext of iron-limitation
are the protomans. Tbcse small gnuns are membas of411 planhonic systems, where
rhey consume bacteria and phytopiankton (Goldman and Caron 1985; Caron et al. 1991;
Sherr and Sherr 1994).regencratc nutrients (Caron and Go1Anr?in 1990). and sewe as a
iinlc bctween the microbiai loop and the miauran fmd chah ( G i n d 1991). Protozoans
are paniculariy s
w
i c
a
n
t in t
k hm-limitai occanic rrgions ( S m and Weischwyer
1991) because production is domiriated by picophytopiankton ( P e a et al. 1990; Miller et
al. 1991; Rice eE ai. 1994). Measurements of the iron content of marine heterotrophic
baciaia demonstrate that this community consritutes a substantial pool of particulate Fe in
the subarctic Pacific ûcean (up to 5û%,Torteii et al. 19%). and presumably in other ironlimitai waters. As the most important consumers of bacteriai biomass, the protozoans
thus rrprrscnt a potcntjaily signifiant pathway f h m parriailate to dissolved Fe via
exnetion. Whik bottie incubation experimcnts (Buma a al. 1991; Chavez et ai. 1991)
and a mesoscale fatilization experiment (Manin et ai. 1994) repn an iacreasc in the
abiidaace of micn>heterotrophsfollowing Fe-addition, tbcy canna distinguish direct
stimiilation of ptozoans by iron from the indirect e E k t of an inc~tasein prey ûensity.
H a t we prcsent the rcsults of a sefies of labonuay urpcrimnts that demonstrate
Fe-Limitationof bactaivorws flagellates and document its mctabolic caisequences. A
S n d y orgMhanr
-Taro chxysomonadmicmflageihcsof th genus P(~~aphysonwas
were examinad: Pwaphysomotuw iinpeM014to (clone VSI), a ooasral species isolatcd
nOm Vieyard Saiid (MA). and P. butckri (clone SS), an ocepaic spccics from the
Sargasso S u Par4physomoll~~~
bpe#orrorcr (66pdiameter) is omnivan,us, and
ubiquitous in marine waters (Goldmsn and Caron 1985).
sîightly d
Par4physointo114~butekri is
a (3-5 p).
but otherwisc morpho10gically v a y similar to P. imperforoto.
Bamrial soains used as prey, clones Tef2 and Juî88, w a e bah isolatcd h m the Sargasso
Sea on nument-enriched agar plates. They were maintaincd in batch cultures that were
paiodicaiiy rcnewed M m stocks fiozen in îiquid nitmgen. Protoman cultures were
renderd monobacterial by repeated transfer (> 10 rimes) into stationary-phase cultures of
the prey swins.
Gmwth conditions ami mediundïhe artifîcial seawater medium A q d (Rice et al.
1988/89), containhg the full complement of inorganic autrienu (N,P,Si), was used for al1
culairing and cxperiments. 1opM EDTA was used to b a e r asce mttals. with Cu. Mn,
Zn and Co additions adjusted to achieve fke-ion concentrationsof 10-13.79~.1 0 - 8 - 2 7 ~ ,
10-10-88M,
and 10- 1 0 - 8 8 ~respcctively.
.
A total of 8.4pM or 12.5 nM iron was added
separately as a prc-mued Fe-EDTA(1: 1) cornplex, resulting in a pFe (= -logpe3+])of
18.18 and 21, respectively. Fne metal ion concentrations w a e calculated using the
chcmical equiiihium modcl MINEQL (Wcstall et al. 1976). Stale mdium was enricbed
with filter-stcdhi (acid-washed, 0.2pm Acmdisc) glucose which was punfied of trace
=ta1 contaminants using Chelex 100 ion exchange resin. Because carbon growth
efficitncy of bacteria is k-dependent (Tortell et al. 1996). K)m@ and lOOmg/L glucose
w a e added in the pFel8 and pFe21 trcatments, rcspectively. Such unequal concentrations
yielded similar bacterial biomass in b t h Fe treatments.
I m quota measwements
- W e useddoubly bbeled barnrialptey to meastue iron
quotas @molFdmol C) of the flagellates. Bacteria w a t grown in the medium described
above wifh the addition of d o d y iabeled 14~-giucosc(Amcrsham, 3mCi/mmil) to a
fimi activity of 2.SpCï LOI, and eitha 1% (pFe18)a 10& (pFe21) total Fe as S S F ~ C ~ ~
(specific activity 25-40 m-g,
phase (- 107cells/ml). die cul-
DuPont Canaàa). Whcn the bactuia rcached stationary
was split into four bottles. nine of these were
inoculattd with protozoans (-103 aiidmi) and one savcd as a conml. At s e v d
intavals during mid to late-exponentialphase, 1@2ûxniof culture were filtered onto 2pm
pdycarbonate Ntm,Msed with Ti 0 EDTA-citrate solution to remove Fe hydroxides
and extracciidar1y-bwndFe (Hudson and Mon1 1989). and then rinsed wice with Sm1
SOW. To avoki damage to the flageiiates, vacuum pressure was maintained below U) mm
Hg.and the filters w a c not aüowed to nui dry (Nagata and Kirchman 1990). &cause
we
used small inocula and ailowed at least 8 ceîî divisions in the labcling medium, the barnna
and flageliatcs had rcachcd isotopic quilibrium at the time of saxnpling. The quotas were
messurrd during that part of the protoman growth ccwe whae protoman abundance was
rriaximal and barnrialabuadaate minimal. At this tirne.bactcria ritprtsented l e s than 0.5
96 of the total biomass on the 2pm filtcs, and piotozoan retention w u > 95%. Rotoman
quotas w a e thus calculated by converthg the ratio of S S ~ e - d ~ /r n14c-dpm on the 2.0
pm füters to pmolWmolC using the specifc activity of C and Fe in the medium, after
CW&R a d iron metrrbolism
-'Ihe spmc doubly labled beEmidprey w m used to
examine C snd Fe mttabolisrn in P C V ~ ~ ~ ~ ~ ~S rOf ~o W
r oMt cSIn
~ these
.
expaiments,
stationary-phase bacuria wwe harvtsted by centrifugaticm (1WOO x g .4û m h ) and then
MsCd and resuspended in a small v o l ~ m eof Aquil cwtainiag no trace &S.
no glucose,
and 1OqiM EDTA as a 'dissolvedtrap' (Hutchins and Bruiand 1994). This resuspension
medium was r e f e d to as 'Aquil-TMS' (Aquil minus îrace mtals). An aliquot of the
bacterid concentrate was counted, and appropriate volumes wcre m s f e m d to four
25(hnl pdycarbonate W
e
s containing U)(kil of Aquil-TMS, to achieve an initial C
concentration of about 2E-7mUd. Acciimated ptozoans were inoculatedinio two of
the bottles and the remairhg two bonles saved as controls. Bacteriai and protozoan
densiries, and particdate and dissolveû I4cand 5 5 ~ activities
c
werc m o n i t d at 12h
intervals over a 1 10h period.
Samples for bacterial enurnemion were premed in 3.6% fornialin in fdtered,
synthetic ocean water (SOW) and then countcd by epifiuonscenct micn>scopy (PoMand
Fcig 1980). Rotozoans w a c p ~ e s c ~ ewidth Lugol's solution, and their density m e a s d
by W t mic~oscopyin a Palmer-Maloney cbamkr. For particdate activity. 1 0 - 2 M of
culnue WCIC Nteredont0 a O.Zpm polycarbonatc filter, and then ~ s c twrc
d
with Sm1
SOW.
or dissolved activity (DOW
and 5 5 ~ ~1m.l
) . of0.2pfilt1ate (~crodiscwith
syringe), was msferred to a g h s scintillation via1 wntaining 1opi of 1N HU,and
CO2 was ailowed to purge for 24h. During fdtration. some culture was dways left in the
CO2 ewolwion
- An additional expairnent examined C mctaboiism m o ~ thocoughly.
e
Bacteria acn p w n in the 1'k-glucost d u m describeci above (witbout 55~~).
harvtstcd and nsuspcndui in Aquii-TMS. and uKn heu-Hied at 70°C f
a 30mlli. The
efficacy of the heat -nt
was vcrificd by inocufating aîiquots of the bacmial
Juspension imo organic-e~ched
seawater medium. No p w t h was obsavcd Bacterial
cultures were allowed to sit for 16h bcforc thcy w a c cenaihiged, nsuspendcd in Aquil-
TMS. and inocuiatcd with P w q p I r y s o m o ~Mpe~orata
~
as desCnbtd abovc. Only one
bacterial control was useci. In addition to measuremenu of DOC and PûC. C e evolution
was monitortddirectly over the tirne course by trapping the C e fram acidified -les
desnibed by Rice and Hanison (1987). Recovay of 1-
Electron Tramport System (ETS) activity
as
was 100%.
-Par4physomu~Unpe~orarawas ftd pFe18
and pFe21-grown. heat-Ued bactmia in dupiicate cultures. Durir~gthe exponential
growth phase of the flageiiatcs, duplicate samples of 351x11were gently filtaed onto
combusted (42S°C for 4h) Whamuui GF/C fiîters. ETS activity was measund aooording
u> Packard and Williams (1981). except samples wem homogenizcd with a Mini Bead
Beater (Xymotech). Tetrazolium (INT) reducaon nues were convated to rates of 02
consumption using the extinction coeflkient for INT-fomiazan repaicd by Kemer and
Ahmcd (1975). We normalized ETS activity to ocii âensity and to protein concentration in
each sample, demmined using the fluOQeScamincmethod of Udeaaitnd et al. (1972).
NHq ucretion
-Ammonium exaction was monitored in a m
g experhent where
prey bacteria were kiiied by UV-irradiation: Bacterial cuiwes were pdnsfemd u> plastic
beakers covend with cellophane and imdiated with a 30 Watt GE germicidal bulb four
rimes foi five minutes, with gentle mixing betanen expoancs. This W-nRdiation
inhibitcd bectainl~tplication.kit not respiration. NH4+ conanmtions in 0.2)im-filtrate
wac mznired by die mcthod of Solomno, as dcscribed by PPMas a al. (1984).
In
coatrd cultures, which containcd W-irradiated baneria without flageiiatcs,
ooucencration did not change ovcr timc. Expaimntal barles w u e camcted f a this
oonstaat background concentration.
Cdcrclorion of metabolic parruneters
-Rotozoaa p w t h rate was calcdated during the
expnential piedby linear mgrtssion of ln (d
#) VS. W. Gros giowîh efficiencies for
carbon and iron w a c calcuiated for a thne inaval t, for cach replicate. following Geider
and Lcadbeater (1988):
whac GGE, is the gross gmwth efficiency for element x, Fxt is the activity 8SSOCiated with
the flagellates at tixne t, and I,, is the activity ingested by the flagellates during the interval
t*
More expiicitly, Fxtwas obtained after wrrection for bacterial biomass maining
in the boales and caught on the 0 . 2 N
~t-
whcre
is the total particdate activity ( 0 . 2 fîiter)
~
and Bxt = (bacteridml) x (14cor
55Fc activity/bacterium). Activity/bacterïum was determincd h m the conml(s),
Ingestion was calcuiatcd as
whaeB,andB,
ud ad a the in-
~nt~bscminlZIiCtiYityUstheflagc~bonkatcbebegiamng
nspsctivtly.
Time t was Pkm nail the bcgiamngofcqonatial grpwth ofdie fiagellates until
k i r psL in biomass, as detcimind b m h p c d o n of the p w t h curve. Since the
~gt~hade~sentiallyaot~uiy~~bKbeginningofthisiaotnr~sll
thcactivity atdiis time wassssociaaedwith thebiwt&a,andBmwas taken to be the total
particdate activity. F a comparative piirposes. ,
F and IN w a t convmcd to mol/pior/d
using die q e d i c mivity (SA) of the bacceria (dp-1)
and the logarithmic mean number
of protomans d u h g the hiaval. Elemntai content of the flagellates (moUprot) was
obtained by dividing (Fxt) (SA) by the nmber offlagehtes/ml a& thne t
Excretion rates (DûC, Fe and NHq) f
a a given tùne intaval wcie calculateci by
dividing the slow of cxmtion c w e s (moI/ml/h) by the logarithmic man numkr of
proto~3ans.Rates were corrtctedfor any appeanuice of dissolved elements in the
controls, and convertcd to rcgencration efficiencies by nosmaüring to the puantity of the
clemnt ingested during the same perid
Unless othenvisc noted, ali the data reported represcnt mean f standard deviation.
Growth Md quotas- Pwîzpkysonior~~~
Unpe~orazagrmu significafltly sbwa (by 44%)
and achieved l o w a lcvels of biomass when comming Fc-poor as apposcd to Fe-rich
bactcria @ < 0.005) (Fg.1).
Indeed.the growth rate of the flagellatcs dsacPsed
C0agstmt.y with dencaSag iroa content (Fe:C)of theh bectaialprey
and initiai
parricuiatc I,in the medium mg. 2). SmaU diff~tncesin initial becmial C bicnnass in
these expaimcnts could not account f
atbe growth ~spoiisc(Pcamm product moment
COR..
p = 0.241). nor was bacterial Fc:C rclated to total particulatc C (mol Ud)
(Pearson p d u c t moment corr.. p = 0.226). Maximum p w t h rates of the prot0~)5~1s
w a t obsaved only at bacterial Fe:C ratios p a t e r than 70 p U m o I . and growth rates at
low and high Fe w a e independent of bacteriaiprey @CS
(Fig. 2). Similar rates of slow
growth were o b m e d wben protozoans were inoculaicd into cultures of bacteria that
were in stationary phase or were Msed and rrsuspended in Fe-frrc medium. As the
stationary bacterial cdains contained about 8 nM of Fe wmplexed to EDTA,the
flagellates w m appazentiy unable to use such low conantrationsof dissolved Fe diraaly
for growth.
Iron quotas @mol Fe:mol C)of the oceanic isolate, Pwqphysomo~~
buchri,
w e n higher than those of Porqphysomoltc~shpe~or4t4under both high and low imn
cunditions (Table 1). ?bey decrrased in both species whcn consuming iron-poor baftena
to about a tenth of their maximum value. Thc low bon quotas, which rrprrsent the first
measunment of their kînd far hcterotmphic pmtists. arc at the high end of those r
e
m
for a number of iron-Mted phpplankton (0.7- 14; Maidonado and Rice 1996) and
marine heteromphic bacteria (2.3 - 14; Torteîi a aL 1996). Unàer the low Kon conditions
of our experimnts, iron quotas of both kgellates w a c significantly higher than thosc of
their prey (p < 0.005).
F i 1 Onwth of Pwqp@sonrow inqp#oratu 0snd dcnrase in bancrial prey
M t y (O) in rcplicatc batch culmes. A Iran-rcp1eot bacteria; hgc-
p = 232 f 0.08
d-1. B. Inw-dcplettbpacrll; flageIlatr_p= M f 0.1 del. '2bepnwomiins wcre
~~lodwPpcyfaovafivetrarisC~sdinociilotedint0satiaipY-p~
bactaialcllbcs-
Tirne (days)
Fig. 2 Protozoan p w t h rates (d-1) in batch culaae as a function of (A) prey iron quota
@molFtm01 C) and (B)the initial oonoentration of particulatt iron. Pïu4physomotua.s
butcheri growing on saaia JuIy88 0.
and P a r 4 p h y s o ~ c iimpe~otam
i
gtowing on
swia Id88 (@). and strah Tef2 (O). initial baaaiaï biomoss rangcd h m 1E-7to 4E-7
umlC/ml, and was indeptndtnt of the Fe:C ratio. M c ~ i c n r ein
s bafterial Fe:C were
obtauied by a i t c ~ medium
g
chemistry an&
allowing the bacteria to starve. The curves
w a t fincd to the Michaelis-Menton cquation by non-lincar regression.
A. p-
= 2 . 4 ~ - 1and Km = 4.60 p
l Fe:md C.
O
50
100 150 200 250
Bacterid prey Fe:C (pmol:mol)
Figure 2
Table 1. Im quotas of t
~prot020a
,
aad thcir baaerial p y uDder high and low
coaditioas @Fe18 ami pFe21. rrspcctively). Vaitws rcprrsent mean f SE.
To test whetha the obsaved p w t h inhibition was a âircct c o n ~ ~ ~ u cof
nee
minerai im limitation a an indhxt cffM of stcondary (biockmicai or moephological)
diffkrences ktwten iron-rich and iron-poof brtaiilprey, WC performcd the foîiowing
expaimnt: A c d t m of hi88 wss p w n to stationary phrrse in pFc21 medium, hcatkiiied, and ~ s e oncc
d in Aquii-TMS. Ibe cuiaaic was then divided e q d y hm two
centrifugebotiles. and one boale W ~ tmiched
S
with 8.4jiM of Fe-EDTA. Afta 2h. b t h
culturc t2it8trnents wcrc harvested ft~~spcndcd
in Aquii-TMS, split in two, and imrulated
Mth acclinrated (low-Fe) Pw4physomoo~cimpe?foram. Prtvious elpairnenu with 5 5 ~ e
demonstrated bat the added Fe was rapidly suwcngcd onto tbe dead bacteria, yielding
'hn-coatad', biochemidy irondeficient bac*
Thcsc ϔis had about 20 amd Fe
bound cxaa~ciîuia~ly.
mghly tm t i m s rmrc than the intraceîlular Fe Foatent of hmplete bacteria P a r q p h y s o m ~ Unper/otma
s
grew sigaificantly ( ~ 4 . 0 1faster
)
when
consuming these iroaaated bacteria (3.21 f 0.04 h l ) than when consuming the standard
bon-poor prey (2.59 f 0.08 d- l ). Inexplicably. the growth of P. impe~orratowas
unusualiy fast in both treatments of dris cxperimcnt (c$ Fig. 2).
A series of grazing experiwnts w a c pafomied with Pcvophysomonar
Mperform to d e t e d e the metabok wnsequences of iron liantarion. For these trials
we used 'standard'pFe 18 and pFe21-gmwn bacterial prey (Ju188; sec Methods) as the
hn-rich and iron-poor treatments (mean quotas reported in Table 1). While the
protomans grazed the duai-labeled bacmial pny and increased in number (Fig. 1).
particdate C dccrcascd by 57% and 83% in the pFel8 anci pFe21 matmentS. nspactively
(Fig. 3). and particdate Fe dccrcased by 70% Md 85 % in the same barles (Fig 4). A
concomitant appeaxance of DOC
3) and dissolved Fe (Fig.) occumd in the bttles
inoculated with fhgtliatcs. Control bonlcs containing aaly bscteria resuspendcd in
carbon-free madirun shcweù littic or no change in the particdate or dissolved fiaction of
either C or Fe (Fig. 3.4). Bacterial density in the controls did not change over the course
of the experiment (data not shown).
Fig. 3 Tuncoourse of carbon dynamks in cuîairits with ôactaia and
Pcu4physomon~~
hpet$oram (.;repiicatcs show) and control cultures with baftaia
aiwe (O; mcan ISD of rcplicatc boles). Particdate (A) a d dissolved (B)organic
0
cafbOn in the hi@ irwi (iron-rkh bactaia) matmcnr
PartiCulate (C) and dissolvcd
(D) mganic carbon in the low-irw (bon-poa ktcria) treatmcnt.
O
1
2
3
Time (days)
4
5
O
1
2
3
Time (days)
4
5
F i s 4 Tir.œ+muxseofiron dynamics in niltiaeswith biictaiaanû Par4physomom
&e#ioru&z (.replimes sbown) and conml cultures with bacteria abne (0;man f SD
of rrplicpte boaks). Mculatt (A)and dissdvcd (B)iron in the hi@ iron mtment.
Partidate (C) and dissoIvaî (D) bon in the low-iron trratment.
Particulaie Fe (prnovml)
Dissolved Fe (pmoVml)
Dissolved Fe (pmoVml)
Corbon metobolism
-Slower p w t h of protozoans fed the Von-poor diet (Fig. 1) was
not a CO~~SCQU~~~CC
of lower ingestion
as these priotozoans consumd mare bacterial
C thaa thosc fed iron-rich prey (Table 2). 'Lhe ingestion rates pondc cd m filtration
rates of 26.41 f 0.09 and 60.0 f 0.6 nUprot/d f
apFel8 Pad pFe 21, respectivcly, similar
to maximum rates observed for ocha bactcrivorous flagellates (Caron et al. 1985). The
differenœ in filtration rate &es na reflcct differcnccs in the carbon ccmtent of the
bactcrial pmy, which was not afftcted by iron staais ( 4 2 f 0.3 and 4.4 f 0.4
fmolç/bacterium; mean f SE of pFel8 and pFe21 cultures). nie carbon content of
P~c~,hysomonas
imper/orata was also not affected by iron status (0.243 f 0.005 and
0.245 I0.005 pmolUccU; mean f SE of high and low-iron culnuts). Dissolved organic
cart>on exmetion @molC/pmt/d) was m
e
r in the iron-limitecl protomans. cven when
normalized to their greater ingestion rate, resulting in a lowa carbon assimilation
efficiency (CAE) @ < 0.025) (Table 2). Carbon gros gmwth efficiency (CGGE) of the
flagellates was also lower in the low-iron treatment. This decrease in CGGE,however,
could mt be accwntod f a solely by the differenice in CAE. Mass balance calculations for
the expriment with live pny suggestcd the decrease in CGGE was also due to greater
Co2 evolution by the iron-limited prot~mamuable 2). This result was confinned in
experiments using heat-kiiied bacterial prey where 14c02evolution was measured
directly. Thus, in the low Fe marnent P. hiperforutureluised significantly more ingested
bacterial C as
(C@/ingestion; Table 2) @ < 0.025).
Table 2. Carbon budget for Paraphysonwllcls impetforrora consuming livc and heat-killed im-npletc (pFe18-grown) or
imn-&pletc (pFe21-pwn) bacterial prey (strain Ju188). The carbon gross growth cfficiency (COGE)is cakulated as
Gmwth/lngcstion, and the assimilationcfficiency (CAE)as (Inges(ion-Exmtion)/Ingestion. Values rcpcnted are means f SD
of duplicate bottlcs.
pFc2l
pFc 18
live prey
Initial bactcrial C (mM)
Ingestion (pmol C@rot/d)
Growth (pmol Uprotld)
mz evolution @mol C/pmt/d)
Excreaon (pmol C/prot/d)
CGGE (Q)
CAE (96)
heat-icillcd
live p y
hcat-killed
Although imn-limiteci protozo~llsrcleased marc C
w they did not have
greater respiration rates. Indeed, ttdvity of the Pondependent electron transport
system (ETS) decrrased icn-foid when Par4physomonas tnpe@orm was fed
inon-poar prey (fiam 4 f 1 to 0.36 f 0.06 pmolo2/prot/d in pFel8 and pFe21
cultures, rtspectively). This rcsult was n a due to diff~ritntialretention of
flagellates on GF/C Nters.as the same trcad was obscrved whcn ETS activity per
filter was nomialized to the total protein ofeach sampIc (182f 51 and 39 f 10
mm01 02/g proteMd in p F d 8 and pFe2l cuirures. rrspectivdy). ETS activity
masures the respiratory capacity ofan organism, and should nonndy exceed the
respiration rate. Xn iron-limited protozoaas. howeva, w h m the ETS has
presumably beui duced to a minimum, we expcct the ratio of ETS to respiration
approaches unity.
N h g e n rnetclboiism
-Absolute rates of NH4+ excretion by exponential-phase
Paraphysomonas impe@ioratawere not affected by iron nunitional statu ( 0.24 f
0.01 and 0.233 f 0.005 pmol NHq+MageIlaWday f
a h - s u n i c e n t and irondeficient c e h , respectively). These rates w a c expresseû as regeneration
efficiencies (NRE =100xNHq+ excreteü/ N ingested) by converthg C ingested to
N ingested using the previously published C:N ratio f a strain Ju188. This ratio
was the same in iron-sufficient and irondefitient bacteria (3.4 f 0.7 and 3.9 f 0.3,
~espectively;Tortell et al. 1996). NRE of imn-deficicntprotozoans (1 5.3 f 0.8)
was signiticantly lower than that of iron-sacient c c b (24.4 f 0.8) (t-test, p <
0.05).
Iron mefrrbolLrm -We calcuiated an iron budget uable 3) for Porqphysomu~s
Unperforatu ushg the Fe data h m the duai-label cxpcriment (Fig. 4).
Surprisingly, the iron-limitai protomans excreted more of the ingested Fe than
protozoans coasuming ~II-richprcy, and thuJ had a Iowa Fe gmss growth
tfficiency F E )Oabk 3). W e notc that in the bigh-iron expaimnt @k18),
Fc Excraion + &wth < Ingestion, so thu miss b a h c e was not achicved. The
discrrpancymayarisebrcaust the pariicularr!sampla. uredm&tQmine Griowth,
WCIC
rinsed twiœ with SOW, while th-
for dissalved Fe (Exctetion) wett not.
The missiag Fe may thus k tbe Fe rcmoved by the SOW rime and hence not
mght cm the po1ycafbD~Nta (srcTwiss and Campbell 1995). This SOWlabilc Fe was Iikcly vcy loosely-sssociatedwith ceils and within dttrital matrices.
In the Fe meobolism expairnent parricul- samples wcrc rinsed only with
SOW, and not with ïï, as b e y were for the quota wasurcments. However,
washing a subsample with both SOW and the Tkitrate rcagent mrealed that very
littk iroa (C 1%) was bound to extracellular ligands. The pmcess of rinsing and
resuspending the prey d s in A q d containhg 1WpM EDTA was appaxently
suffiCient to remove the more tightly bound Slltface Fe.Th nmaining exhacellular
Fk was p~e~umably
the loosely-bound, SOW-labile Fe described above. The
EDTA prcsent in the medium also served as a dissoIvcd 'eap'(sensu Hutchins and
Bruland 1994) for regenerated Fe, p r c v e n ~ gadsorption to ceil surfaces and the
sides of incubation botties. When the same expairnent was pedorrned without the
addition of
1WpM EDTA,FeRE for both treatnitna was about 8Wb less than that
observecl in the presenct of EDTA, suggesting that a large amount of the
~tgeneratedFe was chemicaily maive.
Tabk 3. IIMl ôudget f
a P W ~ ~ ~ S O ~Ypuforoto
O M S comdng iron
rrpletc (pFcl8-pwn) a irondcpIetc @F&Lgrown) banaialprcy (saain
Ju188). T h e F e G G E w a s c a l c u l a t e d i n t b e s a m t ~ r r a s ~
Rt&taciatimefficiency (FeRE) is loOX(Excrcti~gcstion).Values
rrpated are mean f SD of dupliate bonlcs
Initial bacterial Fk (nM)
Ingestion (am01 Fe/Pmt/d)
Growth (am01 Fdprot/d)
ExCretion (am01 Fe/Pmt/d)
FeGGE (%)
FeRE (46)
17.7 f 0.2
139 f 10
39i4
55f 5
28 î 5
59î4
1.8 î 0.2
14f 2
2.00 f 0.01
*
9.5 0.2
14k2
84f 2
D ' i o n
Growth rorr -Pwaphysomon~~
inpe@oratu a d P. buchen w a t unable to
achkve maximum growth rates when consrrmingFe-par bactub. Growth
inhibition of bah species was obscrved reg*
of whrh soPia was off& as
p y . with a composite haif s a ~ a t i o nconstant of 4.6 )un01 Fe: molC mg. 2).
Whïît these data fors a ratber piccetneal functional rrsponr, they show clearly
that protozuans grow subr m c h a î l y at prey k : C ratios l e s than 15 pml:mol.
Such s b w p w t h rates w a e obsained solcly by variation in prey iroa conteah
b u s e prey biomass (C) was constant o v a the range of imn mtments Growth
limitation by Fe can thus k induced in phagotmphic protomans. An imponant
issue. however, is whether this limitation was the direct rcsuit of k m deficiency in
the protozoans or an indirect effect of low iron on their bacterial prey.
In the pFe21 growth medium used to cultivate iron-poa bacteria, b t h
strains were growth rate limited; iron-suffinentbacteria grew at maximum rates at
pFel8. n i u s if iron-Ümitation durad the bacteria in otha ways, iron content
might not be the only difference between the two types of p y . Morpbological
and structurai changes to algae. for example. are known a> be induced by nutrient
deficiency. and to reduce theu digestibility (van Donk and Hessen 1993).
Although they might influence consuma growth. such indinct effects of Fe are not
Uely to have been important in our expairnenu. Elongation of the bacterial $train
Jd88 was occasionaiiy o h e d in low hm medium, bit no gros ~~y)rpholopical
changes were observed in Tef2. which eliciteâ the samc g r o d reductim in the
protomans (Fig.2). Biochcmid changes diat accompany nutrient limitation may
a h innuence prey nutritional quality. Indaed what scems to bc P limitation of
Daphnia is appmntly mm a consequeme of the low levels of eicosapentaenoic
acià in P-limited algal prey (Müller-Navarza 1995). Although we know the buik
chernid content of the prcy bacteria (C:N)was nos affccted by ÿon, a large and
unspdied lut dpoanripl diff~itncesexists betwecn iroa-Icufncicnt and ~RIU&fiCient bacmia. Wc thus ad&csd the issue of niincralversus biockmkai
limitation by comppriag
of f h g d k s on iron-poar prq with and witbout
addcd surface-bunâ Fe. niat the growth ofP. impe$iorata was sipifïcantly
faster in dre fomvr case is ~hn,ngmidena for dirrct elemcntal limitation by h n .
Thc mctabolic signature of the siow growing ptozoans provideci fiirther
support f a direct irm limitation. Rotozoan ingestion rates would be expected a
decruise, if iron-poar bacteria wcre indigestible a difncult to capm. However,
ingestion rates inmasecl, and slowa growth was a resdt of lowa growth
efficiency (Table 2). Thc roughly 5096 decrease in CGGE was reflccted in a
dramatic decline in the activity of the elecmn trax~sponsystem (ETS). Becaw it
contains the buik of the iron in a heterotrophic ceIl, reduccd ETS activity under
low-Fe conditions was anticipated ETS reduction appears to bc a specfic
c01lquence of iron deficiency ,b u s e the mtabolic rcactions that maintain
filtration rates,for example, w a c aot similarly dom-reguiatd
In total, the thrce ünes of evidencc presented above - the relationship
between protoman growth rate and bacteriai Fe:C, the stimulation of growth by
ironaateù bacteria, and the biochemistry of growth limitation - demonstrate
elemental iron limitation in marine p m t o m ~The question aanvally anses: Is this
Ymply an intcrtsting laboxatcxy rcsuit, or couid ktaotcopic flageiiates be ironlimited in the ocean? Sincc protozoans arc apparently not able to use dissolved
iron for growth, their potential fœ iron limitation shouïd dcpend entinly on the
irw content of their food. A low Fe:C of 9 )~moi/moIin the bacterial site fraction
(0.2-1 pm) of the open s u M c Pacinc (Torteli et al 1996) suggests their food
could be Fe-poor. Indeed, as it falls within the range of prcy Fe:C found here to
inhibit growth, hetetotrophic flageiiates and other protoroans may well expenence
iroa deficiency in parts of the sea Although this specuiation clearly awaits fieldconfirmation, and may depend on in sim prey abundance (Sumer and Robinson
1994; Rothhaupt 1995). or on the types of prey items naturaiiy consumed by
protozoans. it provides an explanation for the observation that rnicroheterotroph
biomass inacases following iron andition in bottle incubations (Buma et al. 1991;
Chavez et. al. 1991)and mesoscale f-tion
experlments (Martin et ai.1994).
Herbivoiy is not iikely to provide an additional source of Fe for promzoans, as
oceanic eukaryotic phytoplankton have even lower Fc:C ratios than bacteria
(Sunda et al. 1991; Maldonado and Riœ 19%). However, protou>ans may fced
selectively on iron-rich prey. such as cyanobacteria (Brand Ml), or use nonbiogenic particdate Fc sources such as colloids or Fe bound in &trita1 matrices.
Indeed, our results with 'iron-coated' bacteria suggest protozoans are able to use
such Fe bound indiscriminately to ceU sinfaces. If protomans in Fe-poor waters
aie able to supplement their diet in this manner, they may avoid Fe deficiency.
Iron quoius -The Ùon quotas (Fe:C) reporteci here for heterotrophic flagellates
provide the f h t measure of a parameter of ecological and biogeochemical
importance in ngions of the ocean where iron is the c m n c y of production.
While acknowledging that a few laboratory strains cannot be iepresentative of the
diversity of naturai protozoan assemblages, we feel the inclusion of species isolateci
h m an oceanic (Pataphysomon~~
burcheri)and a coastal environment (P.
imperforam)adds some generality to our conclusions.
Iton quotas of the protozoans measured under low Fe conditions were
higher than those of phytoplankton (Maldonado and Rice 1996) and a number of
marine hetemtrophic bactezia (Torteil et al. 1996). This nsult is perhaps
surprising, because a heterotrophic moâe of existence la& the highiy Fe-
dependent photosynthetic nactions, and might thus incu less of a demand for Fe.
Yet the high concentrations of ETS &x
pnottias rquiFed to maintain d v e
heiaotiophic metabolism can i m p a a suôstantial Fe reqPirrmcat.
Indeed.in Fe-
limitai ba*clia.whac thc majonty ofceiiuiar Fe is associated with the ETS
(Righclato 1969; Light and Qcgg 1974; Grangcr a d Rice in prrp.). Fe quotas are
high. Assumiag that aU hetemtlophs have similar rcspiratœy patbways, WC
expcctcd protomansto have minimumFe quotas simüer to a less than those of
bacterh Indeed, tkir quotas wouid most ükely bc Iowa. kcausc the spccitic
qiration rate (nspiration/bianass), and hence ETS/biomass, of hehetaotrophs
demeases with inmashg size (Ptters 1983). h n shouid b u s bc '&-magnitied' as
it passes q the food chain. ûur rcsuits, howevcr, scem to nui counter to this
sllometric argument, because die fîagellates in the low Fe expcrimnts had higher
Fe quotas than their prey. This m y be a question of sample si= - since only two
species of a single genus werc examinai -, or a question of scale - since the
allometric relationship holds best when considering aganisms that dina in size by
many orciers of magnitude. An alternative explanation for the h .quotas is that
eukaryotic metaboiism is nlatively mm imn-stiy
than prokaryotic metabolism.
or uses large amounts of Fe not associated with the ETS. Indeed, when we
consider bacteria, pmtozloans and 200p1anhon. wc find a dnunatic dccrease in the
ratio of ETS to Fe (Table 4). sugges~ga p a t e r i m p o m c e of non-ETSiron in
larger organisms. From this analysis thae docs not appear to bc any obvious a n d
in Fe:C with sizc or trophic level. Whik the Fe nutritional status of the
meso~~)p1ankton
in Table 4 is unknown. we note that with such high Fe:C ratios,
the herbivoms representativts couid also have âifiicuity acquiring sufficient Fe
fiam the very Fe-poor oceanic prey.
Tabk 4. Electron pdnspoit systcxxt activity (ETS) pa celiular iron in marine
hetcrotmphs. A range in s k is shown whcn data for multiple spccies wcrc avcraged.
Bactaial &ta arr h m T m i i et al (1996). Zooplanhon ETS activity and dry
weight wn taken h m King and Packard (1975). and Fe quotas w a e calculated
h m Martin and Knauer (Montaey Bay) (1973) assuming C = 0.4 x dry weight
Values rrportcd npresent mean f SD.
iron deficient
iron suffiCient
P~u4physomo~s
impe@oratîz
irondeficient
iron-suffiCient
2.43E-7
2.4s-7
8.2 f 0.1
98 f 3
271 f 45
176 f 44
Corbon ami nimgen nierobolisn,
-hn-limitation profou~ldlyaffectcd carbon
mtnboiism in Potaphysomonp~Ypeifotct14. Thcsc protomans converteci less
pxcy C into biomass, iad rcleased more ingestcd C as DOC and C%, wnipafed to
those consuming Fe-rich pny. The validity of such oanpPrisoarests on the qua1
fulfillmnt in both Fe mamcnts of assumptions made in caiculating CGGE. Most
impartant among rhcse. WC assumcd that at the end of the flagellate growth
intuval POC consîsted entirely of protoman aeiîs and uneattn bacteria GGEs
determincd fbm measuirernents of pmtozoan volume cxceed by about 12%those
measincd by the meuiod we employed (Fenchel1982; Caron et ai. 1985b;Geider
and Leadbeater 1988). Presumably the discrrpancy exists becausc some ingested
POC is egested as uninco~poratedPOC (see Stoecker 1984). If it is retained by
the fdters,egested PûC wouid increase the measureâ G and demase 1, so that
COL evolution calculateû by d i f f a n a (C@ = 1- G - E) would be lower than
CO2 measured diroctly. Since the ratio of C@ measured to C% cdculaud in
our experiments was 1-08and 1.27 for the bon-richand bn-poor treatments,
overestirnation of CGGE was. if anything. more severe in the low-Fe treatment
Thus we feel that the obsewed differences in C metabolkm between Fe treannents
are real*
The ammonium excretion results showed that N metabolism was also
affected by Fe status. Lower NHq+ regeneration efficiencies of hn-limited
protozoans impiied they exmted more of their ingested N as DON or urea, or
accumulatcd more N in biomass. An iacnased N conrent of the protozoans seems
unlilrely, however, because Uprotozoan was the samc in both Fe mtments and
C:N ratios of other planhon are hown to be independent of Fe (Maldonado and
Rice 1996; Toneii et al. 1996). Nitmgen could not have accumdated in the
bacterial prey because they were W-kiUed and thus did not assimilate NH&
Our
mass balance approach suggests that protomans excnted mon DON when they
consumed iron-poor pny.
AU nutrient limitations of phagotrophic heteroqhs lead to decreaseà
carbon growth efficiency (Hessen 1992). but diere may be important differenœs
between limitation by catalytic (Fe) and structurai (Pmnutrients. Nutrient limited
grazers dispose of excess C (relative to the limiting resource) eiuier through
reduced C assimilation or increased respiration (CO2 evolution). in the case of
iron limitation, we obscrved reduced C assiaiilatiw. Indecd, this is the only
option. ôecause respiration via the electron transport chah is suppressed under low
Fe conditions. There are cuirently no estimates of die relative importance of these
two pathways during N and P limitation. but diese issues deserve f i e r study,
given the emerging importance of DOC flux in oceanic carbon budgets (Carlson et
ai. 1994; Toggweiler 1989).
A mDdestly speculative interpretation of the &ta provides the foliowing
descxiption of hn-limiteci metabohm of protozoans, and its potentiaï
oceanographic consequences. Iron deficiency inaases flagellate filtration rates to
m a x h h ingestion. Unda low Fe conditions, insuf'fïcient Fe is assimilateù to
support rapid rates of elcctron transport by the ETS so that energy extraction
(ATPproduction) from the C ration is inhibited. More carbon dioxide is evolved
by these cells. suggesting they have a greater diance on substrate-level pathways
to generate ATP,or that they process more C through the TCA cycle. This latter
scenario is diffscuit to envisage. as it would presumably resuit in excess production
and accumulation of miuctant. Cellular feedback mchanisms mediated by
reduced ETS activity deaease the amount of C and N t r a n s p d across the food
vacuolar membrane. A greater portion of the ingested ration is thus excreted as
Dot (and DON),although s o m may be coiioidal rather than truiy dissolved (e.g.
Tmvik 1994). Imn-iimited pmmans have lower ammonium regeneration
efficieIIcieSismonoftbeNrationiPegestCdaaddydicarnnnetriifraction
t
.
undagoes deamination.
If protozoam expaicnœ Fe deficicncy in Fe-poorregions of the awui,
then the supply rate of DON and WC should be enhanccd. Stcady-statc
concentrations of these substrates, which depend on tbc balance betwœn supply
and consumption, d bt hi*
than in Fe-su&cient wotcrs if bacteria prr also
Fe-limitcd ('ïcstcll et al. 19%). Tbe concept of the mimbial lmp as a sbort
circuit may thus k particularly applicable in this sctnario, b s e piiotoman
production wodd be highly inefficient.
Iron ossunilcuion and regenerution -nie Kon GGEs reponed b a e for
Pmaphysomo~simpeiforata are very similar to those of metazoan grazers (7%25%. Hutchins and Bruland 1994; Hutchins et al. 1995). Onc of the most
surprishg resulu in this regard was the importance of the Fe content of the prey:
Fiagellates assimilateci considerably l e s Fe fnwn the Fe-poor bacteria (a).
despite
being limiteà by the element (Table 3). We note that the assumptions made in the
calculation of CGGE apply equally to FeGGE. We fiuther assumtd that if the Iive
bacterial prey reassimilated somc of the regenerated Fe,they did so to the same
extent in both Fe treatments. The accordance between carbon GGE measured with
live and heat-lrilled bacteria suggests that reassimiiation of C did not oocur.
.
Reassimilation of Fe would lead to an undecseimation of 1and an overestimation
of G (as remaining bacteria accumulate Fe relative to the contriols). with the net
effect that FeGGE would k overestimated -ter
FeGGE obûmed in the high-
Fe treaemcnt could thus ôe explaineci if bacterial assimilation of excnted Fe was
-ter
under these conditions. Such scoondary uptake. however, is more likely to
be greater in the low-Fe treatment, where the bacteria have presumably inaximized
their iron uptake systcms.
An alternative way to evaluate the validity of the iron rcsuits is to look at
their intanai consisttncy. It shouid bc that
whae Fe and CGGE rrfa to the flagehtc. W e nad tbat the ight haad side of this
relationship exceeds the left hand 4dc by about 14%. f
a both low and high iron
tmtments. This suggcsts that whüc th-
arc some mcthdologicai
inconsistencies, they act equally in both trcatments. W e arr thus confident that the
problems of msimiiation and particdate egestion do not confound our
intqretation of Fe mtaboIism.
A biological explanation for the paradoxical nsuk of lower FeGGE h m
Fe--
prey may Lie in differences in the assimilibility of iron from iron-suffiCient
and deficient bactaia If iron-poor bacteria contain relatively large pools of nonassimiiabIe iron, the low assimiktion of iron h m such prey could k cxplained.
Rice (1968) proposed that the oràer in which cellular iron p i s wen lost as iron
limitation p r o c d reflects the sangth of cellular 'iron chelates', so that severely
iron-Wted plant cells main only that which is most tightly bound. Such chemical
fonns should be l e s reactive and hena less availabie for bidogical assimilation.
Although this mode1 has yet to k confirmecl, bacteria grown with excess Fe an
known to prcxiuce soluble iron s t ~ a g proteins
e
such as bactcnofdtin (Harrison
et al. 1980, Peny a al. 1993) and contain rdatively m m non-berne iron
(Righeiato 1969; Light and Clegg 1974; Archibald and DeVœ 1978) than Fe-
limited cells. During preliminary investigations i n t iron
~ partitionhg in strain Jd88
we found the Ilon of replete cells was more rractive towards glass beads than that
of Fe-deplete celis (data not shown). nie relative availability of any of these fonns
to protoman assimilation is not known. Cellular partitioning of Fe within prey
ctlls might also afEéct pro-
..
rmmrian'0%ûS it h With ~ t s t ~ i l l l
(Rcinfclda anci Fisha 1991; Hutchins et ai. 1995). Clcarly. the f
m that
regulate metal a s s b i h i r n by p r o t o in~ gcned, d 1&ir possible interaction
Mth prey nutritional statu in pmiCU18t. descM fiinha attention.
For example,
even Fe-poar phytoplanhon might providc a good SOUTCC of dietary Fe if their
cellular pools an morr &y
. . than those of bac&
sssmilaied
RemjIlcralization of allular irai is believcd to be a kcy pÿocess in t
k
surface-watcr biogachtniisay of imn (Bniland a al. 1991). Hutchins et aï.
(1993) demonseated iroD cycling Mthin coastal and oceanic plankmn
communities. but the mechankm of transfais stiU n a entirely cl-,
if it t inindeed
pax-mediated (Hutchins and Bruiand 1994). which arc thc most important
grazcrs? We observed very hi@ iron regeneration efficiencies for
Porq~hysomo
imperforata
~~
consuming bactexial prey (59%and 84%.for ironreplete and highdeficient prey. rrspectively). Couplai with the large amount of Fe
in the bacterid wrnmunity (Tortell et ai. 1996). this rcsult suggests prototoan
bacterivory is a potentially important source of regenaarcd Fc.
To evaluate the contribution of protoman bacterivory and me*izoan
herbivory to Fe regeneration, we calculami community xegeneration rates in the
subarctic Pacific. R.otozoans in this region consume 5.8E-7mola'ld-1 of
bacteria (Putland a al.. in p p ) . which conesponds to 5.3E-12 mol Fe^-Id- 1
(taking an Fc:C of bacteria of9 p 1 : m o l fiam TurtcU et aL 1996). Assuming an
Fc mgenaation eff~ciencyof 72% (ave. of high and low iron trcatmtnts reponed
h a ) . ptozoan bactcxivory thus nleescs 3.8E-12 moîFe~-ld-l to the dissolvd
phase. This value agrees ~tmarkablywcii with the calculatecl steaûy statc Fe
uptake rate for the entire community in the subarctic Pacific (3.12E-12moiFeLId-1: Tom11 et ai. 19%). By cornparison, copepods in the same region consume
8E-8moKX ldo1of phytoplaxikton (Dagg 1993). or 3.2E-13mol Fe^-Id-1 (Fe:C
of phytoplankton = 4 pmol-mok Tortcil a al 1996),snd diey rclcasz obwt 2û%
of ingesrcd diîtom iroD 00 tbc disso1ved phase (Hutchins and Bnilaad 1994,
Hutchins a d.1995). Copepod h a b i v q thus rcgaeratcs an esrUnated 8-1E14
11y)FkL-Id-1.U K ~ lcss
< than pmtozc~~cn
bactaivory. This compPisoa bttwœn
piotOu>pn and mttazwin regmeration is a d a l i w y noc Cumple~t.kcaust
coppods in the subarictic Pacifie obtain a substantiiai partion of meir C ratios 6rom
protoloan prey
(Ginard 1993). However, baruse v i a & a ~rclesse even
iess Fe fkm such p ~ c ythan from diatoars (Huochins and Bnilaad 1994). th& tMal
contribution to Fe r e g e n d o n is still expcctd m bc nUless than that of
protozoans. The relative imponaace of protoman ~genciationwiii lilccly depcnd
on their conwrunity composition and graziag behaviour. For example, relatively
ünlc Fe was rcgenerated by a Porciphysomor~~~
sp. grazing Synechococcrcs sp. (a
-
maximum of 6%of initial p y Fe;Hutchins and Bniland 1994). 'bis may reflect
species Merences ammg £lagellatesordi£ferences in the lability of cellular iron in
hettromphic and phototrophic bacrcna
Ihefmgoing dculation demonswtts protoman bacterivory uui supply
high levels of dissolved Fe to support regen-
production, but the chexnical
nature and reactivity of this Fe remain unlaw,wn. Such mtals may not k
immcdiately avaiiable for phytophkmn use. For example, Gd,Zn and Cd
rrgeaaated by a fnsbwater nanoflageïiategrazing Synechococw wese less
available far rrsorption by picoplanlrton than if -nt
as inœganic complexes
n
M
s
sand Campbell 1995). h n regcnaatcd from bon-Rch p y may k
qualitaovcly diff-t
h m that regencrateci nOm iron-pax prcy, just as iron-
suffkient and & f i e n t pmmmam may 1t1tase chcmically distiaa Fe. Iroa
reluiscd to the dissdvcd phase drrring grazing W ~ S ~ Sboth
D of iagcstcd Fe which
.
*
was never assubllated and a fraction which was hrst a s h i h & and then W e n
down and regenerauâ (c$ f.Laadry
1992). These two forms arc d o g o u s to the
rchsc of DON and NHq? Thus, aït&ugh Fe-iimW P a r 4 p k y s o m o ~ ~
ûqq$oratcr have a rrlativcly high Pon regaaeTatimtfnc*acy, mwt of th
dissokad Fe tbcy rele~sehPs wt fint b m .mrriilntrA and may thus bc less
avaiiablt to phytophkrm t h truly rcgeaaated fania Mœd, the tiansitl~;eof
the biologid (phytoplankton) rtspaise to WC-scak
k addition (Mama a aï.
1994, &ale
a PL 1996 OccBn S c i a r e s Meetirig. Absenrt OS41G-9) spcak~
against r@d and cfficitnt -Ling
of œiluiar Fe.
Tbe hsuits of this stdy daoonsmct bon-limitation of ktemmphic
Cagcatts in culture, d show how such limitation prOroundly affkts C and N
~ t a b Maximum
o ~
gmartti
of P w 4 p h y s o m o ~inrpe?formand P.
buahen werr obsavcd d y when Fe quotas of benaialpley w a t grca!cr tban 70
p d Fe-i
C. At l o w a Fe:C ratios but pi constant pny biomass (Ud).
both
..
specics grew sipnincantly slower. Minimum Fc quotas of the flagellates a< these
slow p w t h rates (- 1O pmol Fe:mol C) wcrc sinrilar to thosc of iron-limited
phytoplankmn and bacteria Growth nite reduction was the rcsult of direct,
elementai limitation by Fe,judging h m thc pmtomam' positive nspaise to Fe
additions and theù biochemical characteristics. Unda Fe limitation, P. impevorato
had a l o w a CGGE, excreted more DOC and DON, and regenerated less NHq+.
Rotozoans in Fe-poor waters may thus bc less efficient at convcrring bacterial
biomass into protoman biomas, which wiIi in ~m affect the rolc of the rnimbial
loop as a remincralization pathway vasus a link to highcr împhic lcvels. Although
our nsults show protomans released a large quantity of ingested bactcrial Fe to
the dissolved phase. even when Fe-Limite& the contribution of this dissolved Fe to
phytoplanhon nutrition is not known. Togethex, these resulu suggest that the
concept of iron-limitation be extended to hetemtrophic organisms, and fhat the
entire b l o g y of oceanic regions could bc moduiated by the supply and availability
of iron.
WCthank P.Torteii for providing the baaaial C quotas and Fe:C ratios
used in Table 4. M.MaldOI18d0 for usthil comu~11tsthroughait the siudy, and A.
Pins for assistance with the ETS assay. We are gnucfui to A. Chan and C. Suttic
(University of Texas, -sas)
for pmviding lisctaialstrains, anà to D . k n
(WHOI) for the flagellates.
This w a k was fimded by the Naîuxai Sciences and E n g i n d g Research
C o d of Canada, and the McGül University Facuity of Graduate Studies and
Research. Z Chase was suppoited by a feliowship h m thc Fonds pour la
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