Microb Ecol (1994) 27:1-17
MICROBIAL
ECOLOGY
?D 1994 Sprnger-VerlagNew York Inc.
Does Microbial Biomass Affect Pelagic Ecosystem Efficiency?
An Experimental Study
J.D. Wehr,J. Le, L. Campbell
Louis Calder Center, Fordham University, PO Box K, Armonk, NY 10504, USA
Received:9 May 1993; Revised:12 October1993
in the pelagiczone participate
Abstract. Bacteriaandothermicroorganisms
in the recyclingof organicmatterandnutrientswithinthe watercolumn.The
microbialloop is thoughtto enhanceecosystemefficiencythroughrapidrecycling andreducedsinkingrates,thusreducingthe loss of nutrientscontainedin
organismsremainingwithinthe photiczone. We conductedexperimentswith
lakecommunitiesin 5400-litermesocosms,andmeasuredthe flux of materials
and nutrientsout of the water column. A factorialdesign manipulated8
4 phosphoruslevels x 2 nitrogenlevels. Totalsedimentanutrienttreatments:
tion ratesweregreatestin high-Nmesocosms;withinN-surpluscommunities,
3 1,UM P resultedin 50%increasein totalparticulatelosses. P additionswithout
addedN had small effects on nutrientlosses fromthe photiczone; +2 jLM P
tanksreceived 334 mg P per tank, yet after 14 days lost only 69 mg more
particulate-Pthan did control communities.Nutrienttreatmentsresultedin
markeddifferencesin phytoplanktonbiomass (twofold N effect, fivefold P
effect in +N mesocosmsonly), bacterioplankton
densities(twofoldN-effect,
twofoldP effects in -N and +N mesocosms),andthe relativeimportanceof
autotrophic
picoplankton(maximumin highN:Pmesocosms).Multipleregression analysisfoundthatof 8 planktonandwaterchemistryvariables,the ratio
of autotrophic
picoplanktonto totalphytoplankton
(measuredas chlorophylla)
explainedthe largestportionof the total variationin sedimentationloss rates
(65%of P-flux, 57%of N-flux, 26%of totalflux). In each case, systemswith
greaterrelativeimportanceof autotrophicpicoplanktonhad significantlyreduced loss rates. In contrast,greaternumbersof planktonicbacteriawere
associatedwithincreasedsedimentation
ratesandlowersystemefficiency.We
suggestthatdifferentmicrobialcomponentsmay have contrastingeffects on
the presumedenhancedefficiencyprovidedby the microbialloop.
to: J.D. Wehr.
Correspondence
2
J.D. Wehr et al.
Introduction
The loss of planktonicmaterialsto the sedimentsof lakesis one of the fundamental
processesin freshwaterecosystems.Theselosses leadtowardthe immobilizationof
organicmatterandnutrientsto the sediments,andtheeventualrecyclingof material
followingdecompositionandturnover.Despitethe factthatmostof the sedimented
organic matterproducedby phytoplanktonis decomposed[45], autochthonous
sinkingparticles(deadphytoplankton,fecal pellets, organicdetritus)representa
net loss of nutrientsfrom the photic zone in most stratifiedlakes, until after
turnover.Traditionally,nutrientcycling in lakes is thus thoughtof in termsof a
phytoplankton-zooplankton-fish
model, in which the returnof nutrientsdepends
on settledparticlesbeing decomposedand remixedinto the watercolumnin the
spring or autumn.Factorsaffecting the flux rate of planktonicmaterialsmay
includesystemproductivity,planktonparticlesize, andbasinmorphometry.
However, within a given productivityrange, otherbiotic processesmay enhancethe
regenerationof nutrientswithinthe watercolumnandreducesystemlosses to the
sediments.Bacteriaandotherpelagic microorganisms
participatein the recycling
of organicmatter[3, 5, 6].
It has been proposedthat a microbialfood web consistingof bacteria,picophytoplankton,and herbivorousflagellatesand ciliates can act to shuntprimary
productionandnutrientsawayfroma "linear"metazoanfood chainto a morerapid
andefficientrecyclingmechanism[3, 33]. The importanceof this microbialloop
for pelagic ecosystem efficiency and the retentionof inorganicnutrientsin the
photic zone needs to be quantified.Argumentshave followed that predictthat
microorganisms,
dueto theirhighsurfaceareato volumeratios,rapidgrowthrates,
and slower sinking rates, will enhancethe likelihood of nutrientscontainedin
organismsremainingwithin the photic zone [37]. Thus, a yardstickfor pelagic
ecosystemefficiency may be measuredaccordingto reducedrates of downward
nutrientflux (="losses"), andmay be comparedwith recentlyproposedmeasures
of bacterialandothermicrobialbiomassandgrowth.
Basedon the ideasrepresentedin microbialloop theory,rapidgrowthratesand
efficient remineralizationof nutrientscan be maintainedonly if there is tight
couplingbetweenprotistgrazersand theirbacterialprey [18, 37]. However,it is
not clear whetherthis predictionconsidersboth heterotrophicbacteriaand autotrophicpicoplanktonin this size range.Thesetwo components,whilephysiologically quitedistinct,competefor some of the sameresources.Recentexperiments
indicate that the growth of heterotrophicbacteriamay at times be limited by
inorganicnutrients[15, 40], suggestingthat these microorganismsmay at times
become a sink for nutrientswithin the microbialloop. Indeed, Caron[14] has
shownthatphytoplankton
typicallyhave C:N andC:P ratioshigherthanthe Redfield ratioof 106C:16N:iP,while planktonicbacteriatend to have C:N and C:P
ratiosbelo-v this ratio.
Studieshaveshownthattherelativeabundanceof autotrophic
picoplanktontends
to be greatestin oligotrophic(typicallyP-limitedin freshwater)ecosystems[13, 35,
43], but apparentlyis not limitedby the supplyof dissolvedinorganicphosphorus
(DIP)in eithereutrophicor oligotrophiclakes [41, 44]. Surprisingly,the growthof
heterotrophic
bacterioplankton
was shownto be directlylimitedby DIPavailability
in the samesystem[40]. Theseexperimentsraisequestionsconcerningthe apparent
MicrobialLoop Efficiency
3
universalimportanceof P limitationin freshwaters[32]. The ecosystemimpactof
andprotozoamaythusdepend
moreefficientprocessingby certainmicroorganisms
not only on the totalproductionof microbialorganisms,but also on the differing
withinthe microbialloop.
nutrientconstraintsthataffectthe compartments
In this study we proposethat differencesin total microbialbiomass and the
relativeimportanceof heterotrophic
andautotrophicpicoplanktonwithinthe photic
zone will affectsystemefficiency.Basedon earlierresultsindicatingdifferencesin
nutrientlimitation,we predictthatautotrophicpicoplanktonin particularwill have
a positive effect on the retentionof nutrientsin the watercolumn (i.e., reduced
nutrientsedimentation).We testedthis idea by manipulatingpelagiclake communitiesin largemesocosms,andmeasuringthe flux of materialsandnutrientsout of
the watercolumn.
Materials and Methods
Site Description and Design
Experimentswere conductedat the ExperimentalLake Facility(ELF) at the Louis CalderCenter,
FordhamUniversity(Armonk,NY, USA). The facility contains24 fiberglass,outdoormesocosms
(1.9 m diam;2.1 m high;filled to 5400 litersper tank)thatare directlypump-fedwith CalderLake
water[see 41, 42 for detailson the lake] throughprewashed,opaquepolyethylenepipe. Mesocosms
were filled in darknessduringan 8h periodon 1 June 1992, whenCalderLakewas weaklystratified
(AT 2?C/m).Night collectionensuredthatverticallymigratingzooplankton(principallyDaphnia
pulicaria)occurredin all ELFtanksat similarlevels. Nylonnetting(1 cm mesh)was placedovereach
to minimizeinputsof leavesandwoodydebris.
A factorialdesignconsistedof 8 nutrienttreatments:
4 P levels (0, 0.5, 1.0, or 2.0 ,umolK2HPO4/1
added)x 2 N levels (0 or 10 ,umolNH4NO3/l),with eachblockreplicatedthreetimes. The intentof
this design was to manipulatenutrientlevels andN:P ratiossuch thatphytoplankton
productionand
communitysize structurevariedover the full rangeobservedin earlierstudies[41]. Nutrientswere
addedon day0 duringfilling to allow sufficientmixing,anda secondidenticalset was appliedon day
14 followingthe thirdweeklysampling(see below);the experimentranfor 28 days. Theprimarygoal
was to examinewhetherlosses of planktonicmaterialsfromthe watercolumnincreaseor decreaseas a
functionof nutrientconditionsand microbialprocesses. The basic models tested include (1) an
ANOVAmodel, in whichmanipulated
variables(N, P, N x P) areclassifiedas treatments,and(2) a
regressionmodel, in which observedplanktonvariables(levels of planktonicbacteriaand "algal"
andmicroplankton)
areclassifiedas independentvariables(see also later
picoplankton,nanoplankton,
in DataAnalysis).
Field Sampling
A series of samples were collected weekly startingon day 0 (4h after filling) throughday 28.
Temperatureand pH were measuredat the ELF tanks in situ. Waterand planktonsampleswere
collectedby peristalticpumpfrom0.5 m depth.Pairsof waterchemistrysampleswerefilteredin line
(WhatmanGF/F);one set was acidifiedto pH 2 (withH2SO4), andthe otherset was frozen(- 15?C).
Planktonchlorophyllwas collectedin 1-literbottleswhile prefilteringmicroplankton
through20-p.m
meshsize Nitex. The remainingfiltratewas keptrefrigerated
(-4?C) untilreturnedto the lab.
To measureratesof planktonand otherparticulatelosses, a free-standing,verticalsedimenttrap
(withweightedbottom)was placedin the center-bottomof each ELF tank(retrievedby two nylon
stringsattachedto the rimof the tanks).The cylindricaldesignfollowedgeneralrecommendations
of
Hargraveand Burns[20]and Bloesch and Burns[8], with a length/widthratioof about5.5:1 (total
4
J.D. Wehret al.
volume550 ml) to minimizeturbulence.Trapswere constructedfrom55 mm ID whitePVC, with a
polypropylenefunnelfittedinsideat the bottomto focus the settledsediment.A clampedplastictube
was attachedto eachfunnelso thatweeklysamplesof trappedsedimentcouldbe siphonedintosample
bottles(storedat 4?C)andthe trapsreturnedto eachELFtankimmediatelyaftersampling.Following
Kirchner[24], 90 ml of 5% NaCl was first placedinto the bottomof each trapto createa density
gradientand preventmaterialloss while traps were raised to the surface. Tests with saline plus
methyleneblue indicatedlittleor no loss duringtransfer.
Laboratory Methods
Waterchemistrysampleswereanalyzedforconcentrations
of soluble-reactive
phosphorus(SRP)using
theantimony-ascorbate-molybdate
method[1, 91 andfor totaldissolvedP as above, followingpersulfate digestion[29]. NH4+-Nconcentrationswere measuredby the phenol-hypochlorite
method,and
NO3-(afterreductionto NO2-in a Cd-Cucolumn)via reactionwithsulfanilamide-NNED
[1, 10, 11].
Soluble-reactiveSi (as Si02) was measuredby the molybdosilicatemethod[1, 12]. Methodswere
modifiedfor automatedanalysisandrunon a Traacs800 automatedanalyzer(Bran+LuebbeTechnologies, Inc., BuffaloGrove,IL).
Sediment-trap
samples(in water)were mixed thoroughly,split into four parts,and filteredonto
either25 mm (for particulateN, P) or 47 mm (totaldry weight, pigments)diameterWhatmanGF/F
glass fiberfilters(volumesrecorded).Portionsof filteredresidueused for N, P, andpigmentanalysis
wereimmediatelyfrozenuntildigestion;totaldrymasssampleswereplacedin a dryingoven (105?C)
for48h, thencooledin a desiccator.Particulate
N andP weredigested(30 min)underpressure(120?C,
15 psi) in Pyrextubesusingacid (H2SO4) perchlorate[29]. Dry mass accumulationwas measuredto
the nearest0.1 mg. Sedimentedpigments(chlorophylla and pheophytina) concentrationswere
measuredfollowingextractionin glass tissue grinderswith -4 ml neutral90%acetone(+MgCO3).
Extractswerepouredintograduatedcentrifugetubes,madeup to knownvolume,andfurtherextracted
in the dark (<4?C) for 18-24 h. Samples were centrifugedand measuredspectrophotometrically
(ShimadzumodelUV-160)[1, 27]. Themajorityof measuredpigmentin thesedimenttraps(;80% on
average)consistedof pheophytin.
Bacterioplankton
in watersamples(=5 ml) werepreservedwithglutaraldehyde
(to 2%),stainedfor
fluorescentmicroscopyusing DAPI, filteredonto Irgalanblack stained25-mmpolycarbonate
filters
(0.2 p.mporesize;PoreticsCorp.),andstoredat - 15?Cuntillaterobservation[22, 31]. Countswere
made at x 1250 magnificationusing a Nikon Labophotepifluorescencemicroscope,using 365 nm
excitationand 520 nm barriersets. A minimumof 10 grids and 300 cells were countedper sample
replicate.Phytoplankton
biomass(measuredas chlorophylla) was size fractionated
(usingpolycarbonate filters)accordingto categoriesdefinedpreviously[34, 41]. Briefly, microplankton
are cells or
colonies20-200 ,im, nanoplankton
are2-20 jim, andpicoplanktonare0.2-2 ,um.Filteringmethods
havebeendescribedpreviously[41, 42]; chlorophylla was measuredby spectrophotometer
(corrected
forpheophytin),as describedabove.
Data Analysis
Datawerecompiledandanalyzedusingthe SYSTAT4.2 program[46]. Two typesof hypothesistests
wereset up to evaluatethe models(nutrientandplanktoneffects)outlinedin the experimentaldesign
section. For both, the dependentvariableswere particulatelosses out of the watercolumn to the
sediment,measuredas (1) totaldry weight, (2) particulateP, (3) particulateN, and (3) pheophytin
deposition.The first hypothesistests were conductedusing two-wayANOVA of manipulatedvariables.Treatmentswere(a) nitrogen(2 levels) and(b) phosphorus(4 levels) treatments,and(a x b) an
interactionterm(N x P),-as categoricalvariables.Effectswerejudgedto be significantif theprobability (oa)of an eventoccurringdue to chance(P) was less than0.05.
A secondset of testswasusedto derivepredictionsabouthow measuredkey water-column
variables
affectsedimentationrates,using stepwisemultiplelinearregression(MLR).In these, 8 independent
5
MicrobialLoop Efficiency
mesocosmsat thestartof the
Table 1. Physicalandchemicalconditionsmeasuredin 24 experimental
experiment,classified accordingto fertilizationtreatment.Temperaturewas measuredin ?C, and
chemistryvaluesareexpressedin ,umol/l 1 SD (n = 3; SRP, soluble-reactive
phosphorus)
Phosphorustreatment(>M)
0.5 P
0P
NO N ADDED
Temp
pH
SRP
NH4
NO3
SiO2
+10 FM NH4NO3
Temp
pH
SRP
NH4
NO3
SiO2
18.5 ?
7.56 ?
0.04 ?
6.7 ?
0.34 ?
11.7 ?
0.1
0.12
0.01
1.9
0.01
0.3
18.8 ? 0.5
7.54 ? 0.27
0.03 ? 0.01
10.6 ? 0.5
11.5 ? 0.3
10.9?0.1
18.8 ?
7.56 ?
0.38 ?
6.7 ?
0.31 ?
11.1
0.9
0.14
0.02
0.4
0.02
0.1
18.6 ? 0.1
7.54 ? 0.12
0.34 ? 0.02
14.8 ? 0.7
11.4 0.2
11.4?0.1
1.0P
18.4 ?
7.54 ?
0.81 ?
6.1 ?
0.30 ?
11.1
2.0P
0.2
0.29
0.06
0.2
0.04
0.1
18.6 ? 0.1
7.49 ? 0.08
1.71 ? 0.05
6.0 +0.1
0.29 ? 0.04
11.4 0.03
18.3 ? 0.5
7.53 ? 0.22
0.80 ? 0.02
9.7 ? 0.5
11.6 0.3
11.3?0.4
18.4 ? 0.6
7.58 ? 0.22
1.76 ? 0.04
14.6 ? 0.3
11.6 ? 0.3
11.3?0.1
variableswere testedfor theireffects on sedimentationlosses: bacterioplankton
density,autotrophic
picoplankton,autotrophicnanoplankton,autotrophicmicroplankton,ratio of pico: SChla, ratio of
bacteria:lChla,[SRP],anddissolvedinorganicN ([DIN]). Ratiosof pico:lChla andbacteria:lChla
were includedto test whethera predominance(ratherthansimple density)of microbialcells, both
altersystemslosses andecosystemefficiency.The maximumcriterion
autotrophicandheterotrophic,
for inclusionof independentvariablesinto the modelwas (x = 0.05, andthe minimumfor exclusion
was a = 0.10. All datawerecheckedforassumptionsof normalityandhomogeneityof variancesprior
to analysis.
Insummary,thetwo modelsmakedifferentbiologicalassumptionsabouttheproximateandultimate
factorsleadingto particulatelosses to the benthos.The ANOVAmodelsexaminesolely the effect of
manipulations(i.e., N andP loadings),which may have directeffects on sedimentation,or indirect
effects throughchangesin planktonbiomassor shifts in communityand size structure.The MLR
modelsidentifywhichof severalmeasuredvariablesin thewatercolumn,singlyorcollectivelyexplain
the differencesin sedimentationlosses over time. These factorsmay be thoughtof as havingmore
immediateand directeffects on sedimentationrates, despitehavingnot been directlymanipulated.
Each approachhas its limitations,but both are useful startingpoints for makingpredictionsabout
ecosystemefficiencyunderdifferentnutrientandplanktoncommunityconditions.
Results
Physical and Chemical Conditions
The measuredconcentrations
of SRP at the startof the experimentwere somewhat
below nominaltreatmentlevels for P added(about80%), but N treatmentswere
close to targetvalues (Table 1). Nutrientswere nonethelesssignificantlydifferent
acrosstreatments(ANOVA;P < 0.01 in all cases) andnot significantlydifferent
withintreatments(ANOVA;P > 0.5 in all cases). ActualP treatmentsthusrepresenteda gradientof about1x, 0x, 25 x, and50 x of ambientconditionsin Calder
6
J.D. Wehret al.
+10 FtMNH4NO3
No Nitrogen Added
8 -
*
Z
0
?ojNg6
-
WE4
R -
*No P Added
+0.5 [M P
+ 1.0 1MP
-8
7
?6
4
UWc6m3
3
2
2
1
1
0
0
-J3
0
0
7
14
21
28 0
7
14
21
28
20-
-20
0
TIME
(Dy1s5
a c 10
10
0
0
0
7
14
21
28
0
7
14
21
28
TIME (Days)
Fig. 1. Cumulative,total particleflux from the watercolumn to the sedimentsas a functionof
nitrogenand phosphorusmanipulations,measuredweekly in CalderLake mesocosms. Rates are
measured in terms of total dry weight (upperpanel), and pheophytin a accumulation (lower panet). All
dataareaverageratesfromthreereplicatemesocosmsfor eachnutrienttreatment.
Lake.All non-manipulated
variablesmeasured,suchas temperature,
pH, andother
chemicalconditions,remainedsimilaramongall 24 tanksthroughoutthe study.
SedimentationLosses
The firstgoal was to determinewhethermanipulations
of nitrogenandphosphorus
levels affect total and specific losses of materialsout of the euphoticzone. A
particularinterestwas whetherlosses observeddiffer from those predictedby
simple increasesin planktonbiomass. Over 28 days, sedimentationproceededat
markedlydifferentratesaccordingto treatmentconditions(Fig. 1). Each pair of
treatmentgroups(no N addedvs. 10 ,umolNH4NO3addedperliter)is presentedin
two plots representingthe effect of P additionson particulatelosses to the sedi-
MicrobialLoop Efficiency
7
ments.Sedimentationof planktonicmaterials(dryweight)out of the watercolumn
was notdifferentacrossa wideP-treatment
gradientin mesocosmswithoutaddedN
(Fig. 1, upper panel). However, losses overall were nearly twice as great in
systemswith 10 ,umolNH4NO3addedperliter, whichcorrespondwith a doubling
in surfaceplanktonbiomass (see below). Withinthese N-fertilizedmesocosms,
phosphorustreatments 1 ,Iumol/lresultedin about 50% greatertotal plankton
losses thancontrol-Ptanks.Thiseffect was apparentby day 14 of the study.
Phytoplankton
deathandsinkingrates,as judgedby the weeklyaccumulationof
pheophytina, weredramaticallyaffectedby bothaqueousN andP conditions(Fig.
1, lower panel). Nitrogen-fertilized mesocosms received up to three times more
deadalgalmaterialthanthe sedimentsof N-limitedsystems,althoughthis was not
observedin communitieslackinga P addition.The P treatmentsalso elicitedearlier
(by day 14) and strongerresponsesthan were observedfor total or particulate
nutrientsedimentation.The rangeof 0 to 2.0 ,umolP addedper liter (z0.4 to 1.8
resultedin an averageincreasein the cumulativeloss ratefrom3.9
FIMP measured)
to 18.8 [ig pheophytinper cm2 of sedimentsurface,nearlyfivefold (i.e., 500%)
greater.This greatlyacceleratedpigmentaccumulationcorrespondswith only a
50%increasein the totalmassof sedimentedplanktonicmaterials.
Particulatenutrientlosses were not necessarilyrelatedto increasedloadingsof
those nutrients.In CalderLakewater,P additionsalonehadno apparenteffect on
losses of thisnutrientto sediments(Fig. 2, upperpanel). Particulate-P
accumulated
in the sedimentsof N-limitedsystemsat similarratesacrossthe wide rangeof P
treatments.This meansthata 50-fold increasein DIP loadinghadlittle effect on P
losses in thesemesocosms.Becausethe totalvolumeof each mesocosmis known,
differencesin the retentionof P withinthe euphoticzone can be estimatedaccurately.Systemssuppliedwith2.0 ,umolP/Ireceivedon day 0 an additional334 mg
of P (as K2HPO4).After 14 daysthesecommunitieslost only 69 mg moreparticulate-P (24.9% greater)than did controlcommunities.However, the additionof
inorganicN greatlyacceleratedP sedimentation,withmaximumlosses observedat
P (0.5-1.0 ,umolP addedperliter)treatments.By the end
highN andintermediate
of theexperiment,sedimenttrapsin phosphorus-limited
mesocosmsreceivedabout
28%less particulate-P(- 16.8 [Lg/cm2)thanthose receiving2.0 ptmolP/1(=23.3
pLg/cm2).Nitrogenlosses exhibiteda similarpattern(Fig. 2, lower panel), although
P treatmentshadapparentlylittleeffect on N retentionor N loss in most systems.
The significanceof direct nutrientmanipulationson sedimentationrates was
consideredfor each dateby two-wayanalysisof variance(Table2). The totalloss
of materials(as dryweight)was not significantlyaffectedby eitherN or P untilday
14, andby P treatmentsonly by day 28. Nitrogenclearlywas the most important
treatmentvariablecontrollingtotal sedimentationrates. ANOVA was not able to
detect any significantN x P interactioneffect (such as N:P ratio in the water
column)on these rates. Losses of P, however, were affected by both N and P
treatments(andN x P interactions)in the watercolumnby the end of the experiment.Systemlosses of N weresignificantlyaffectedby N additionsthroughoutthe
study,butthe analysisindicatedthatP additionshadno significanteffects. Unlike
the othervariablesmeasured,pheophytinsedimentationwas significantlyaffected
by both N and P treatments,and exhibitedsignificantlytreatmentinteractions.
Fromthis perspective,P treatmentsresultedin significantlygreaterloss of algal
materialwhen N was also added, even on day 7. The importanceof N x P
J.D. Wehret al.
8
+10 FiMNH4NO3
No Nitrogen Added
*
*
30 -
A
4
225
z
ZE
7
No P Added
+ 0.5 FM P
+ 1.0 RMP
P7
280
- 30
2- 25
20 -20
z
w ~- 15
15
-
7510 -10
077
5-
O.0
0
0
0
7
14
21
28 0
7
14
21
28
70 -
-70
zZE 60 --60
-
04
20
20
0
z1-1
-0
07-4
1
280
TIME
74421
2
(Days)
nutrientflux fromthe watercolumnto the sedimentsas a functionof
Fig. 2. Cumulative,particulate
nitrogenand phosphorusmanipulations,measuredweekly in CalderLake mesocosms. Rates are
measuredin termsof particulateP (upperpanel), andparticulateN accumulation(lower panel). All
dataareaverageratesfromthreereplicatemesocosmsfor eachnutrienttreatment.
interactionon algal losses contrastswith the repeatednonsignificanceof such an
interactioneffect on totalsedimentationrates.
Microbial Biomass and Sedimentation
A secondobjectiveof this studywas to examinethe relationshipbetweenmicrobial
planktonbiomass(algalandbacterial)andsystemefficiency.Directmanipulations
of N and P supplyto the euphoticzone alteredtotal biomasslevels as well as the
relativeimportanceof varioussize fractionswithinthe plankton(Fig. 3). Control
(no P or N added)densitiesof phytoplanktonand bacteriawere not significantly
andbacterioplankton
producdifferentfromlevels in CalderLake. Phytoplankton
systems,butonly bacteriaincreasedin
tionlevels weregreatestin N-supplemented
responseto P additionswithoutadded N. Biomass responsesto P additionsin
MicrobialLoop Efficiency
9
in experimentalmesocosms,as characterTable 2. Effectsof fertilizationon planktonsedimentation
ized by total(drymass),phosphorus,nitrogen,andpheophytinloss rates(see Fig. 1 for details).Data
arefroma two-wayanalysisof varianceof theeffectsof N addition(noneor 10 FAMNH4NO3)and/orP
addition(none,0.5, 1, or 2 FM K2HPO4)on ratesof totaldrymass (g drywt/cm2 perday), P, N (p.g
nutrient/cm2
per day), and pheophytin(p.g pheo a/cm2 per day) accumulationmeasuredat weekly
intervals(a nominalP level of "0.000"is some unknownprobabilityless than0.001)
Day 7
Treatment F-ratio
P-value
Total sedimentation
N
0.327
0.576
P
0.488
0.696
Nx P
0.830
0.497
Phosphorussedimentation
N
0.013
7.798
P
0.047
3.299
Nx P
1.102
0.377
Nitrogensedimentation
N
6.394
0.022
P
0.676
0.580
Nx P
1.434
0.270
Pheophytinsedimentation
N
47.069
0.000
P
6.089
0.006
Nx P
6.136
0.006
Day 14
Day 21
Day 28
F-ratio
P-value
F-ratio
P-value
F-ratio
P-value
15.346
0.914
1.820
0.001
0.456
0.184
6.867
2.335
0.866
0.019
0.115
0.086
38.607
4.112
2.795
0.000
0.024
0.074
12.763
3.931
1.338
0.003
0.028
0.294
15.706
0.424
1.112
0.001
0.739
0.373
64.374
8.553
5.002
0.000
0.001
0.012
9.343
1.272
2.356
0.008
0.318
0.110
9.101
0.319
0.466
0.008
0.812
0.710
46.280
2.630
0.014
0.000
0.086
0.014
89.321
16.628
17.955
0.000
0.000
0.000
12.549
5.841
3.123
0.003
0.008
0.057
14.106
4.273
3.936
0.002
0.021
0.028
N-fertilized mesocosms also differed temporally among the 4 plankton size classes.
Autotrophic picoplankton levels peaked on day 7, while bacterioplanktonnumbers
tended to increase later in the experiment and remain at enhanced levels over longer
periods. Differences in these biomass maxima and minima in turn altered the size
spectrum of the community over time. The proportion of total chlorophyll a
measured in picoplankton cells ranged between 70% in low-P mesocosms (high
N:P ratios), to only about 30% in high-P, high-N mesocosms. Over this spectrum,
total algal biomass increased twofold, mainly within the nano- and microplankton
size classes. The ratio of bacterial numbers to total phytoplankton chlorophyll a
(-bacterial importance) also varied with nutrient treatment. The relative importance of planktonic bacteria was greatest in high-N (50-100% increase) and high-P
(80-130% increase) systems, yet the reverse trend was observed for autotrophic
picoplankton.
These biomass and size shifts had apparenteffects on the quantity and quality of
water column materials lost to the sediments (Table 3). Simple bivariate correlations revealed greater overall sedimentation rates in all systems with greater total
phytoplankton and bacterial biomass. The most important variables leading to
greater total sedimentation and sedimentation of particulate P and N were autotrophic nanoplankton and microplankton, and heterotrophic bacteria. Larger algae and cyanobacteria consisted primarily of Coelosphaerium naeglianum, Ceratium hirundinella, and Anabaena cylindricum. Two marked exceptions to this
positive feedback patternwere (1) a nonsignificant relationship with the biomass of
autotrophic picoplankton, and (2) a significant negative relationship with pico-
J.D. Wehret al.
10
-0-Microplankton
0- Nanoplankton
--10 imol NH4NO3
Picoplankton
0 Bacterioplankton
added / L
No N Added
1-6
No P Added
6-
42-
4.A'
2 -
... ... . ............. 2t
[0.5
60-
4
Imol
P added /
6
-
C
4~~~~~~~~~~~0
(U
oL
4X
0.
2 -
22
0
~ ~
0 -
4
8
4 NII~~20
2 olPade
0
~
~
~ ~
7
o ~~ ~ ~
phosphous (tretments
0
7
~~Tm
noe
14
21
.,
2
28
m~~~~/~
(days
.,ad umolP0 . addeprlir)mnuatosn
/
28 0
.....4
7
14
21
28
Time (days)
Fig. 3. Effects of nitrogen(treatments= no N addedvs. 10 p.molNH4NO3addedper liter) and
phosphorus(treatments= none, 0.5, 1.0, and 2.0 RiMOlPG4 added per liter) manipulationson
phytoplanktonbiomass (measuredas chlorophylla) in three size fractions,and bacterioplankton
densitiesin CalderLakemesocosms(n = 3; errorbars= ?1 SE;errorbarssmallerthansymbolsize
not shown).
planktonas a percentageof totalchlorophylla. As thesebiological(andseveralkey
chemical) variablesmay collectively influence ecosystem processes, and may
themselvesbe autocorrelated,a multipleregressionanalysiswas used to identify
rates,andto producea
the mostimportantfactors(+ or -) affectingsedimentation
morecompletemodelthatcoulddescribethe controlson systemefficiency.
A stepwise multipleregressionmodel evaluatedthe effect of 8 water-column
variables:(1) bacterialdensity, (2) autotrophicmicroplankton(>20 ,um), (3)
11
MicrobialLoop Efficiency
Table 3. Relationshipsbetweenmicrobialplanktonbiomassin mesocosmsandparticulatelosses out
of thewatercolumn.DataarePearsonproduct-moment
correlationsbetweensedimentation
rates(total
dry mass, particulateP, particulateN, and pheopigments)biomass levels of the autotrophicand
bacterialplanktoncommunityin CalderLake;relativemicrobialdominanceestimatedby the ratio
andtotalchlorophyll
betweenbacteriaandtotalchlorophylla, andtheratioof autotrophic
picoplankton
a (n = 24)
Sedimentationvariables
Totaldry mass
ParticulateP
ParticulateN
Pheophytin
per day) (jig/cm2per day)
(mg/cm2per day) (pug/cm2per day) (pLg/cm2
0.721***
0.499*
0.744***
Bacteria
0.499*
-0.096
-0.253
0.219
0.051
AutotrophicPico
0.734***
0.626**
0.845***
0.449*
AutotrophicNano
0.612**
0.593**
0.653**
0.514*
AutotrophicMicro
-0.804***
-0.755***
-0.764***
Auto-Pico:IChla
-0.515*
-0.101
-0.300
-0.039
Bact:IChla
-0.099
Planktonfactors
*P < 0.05; **P < 0.01; ***P < 0.001
autotrophicnanoplankton(>2-20 ,um), (4) autotrophicpicoplankton(>0.2-2
,um), (5) the ratio of bacteria:YChla,(6) the ratio of autotrophicpicoplankton:lChla(labeledas "%Pico"), (7) measuredSRP concentration,and (8)
measureddissolved inorganicN (DIN) concentration.The variablesbacteria:
communitieswithgreateror lesser
IChla andpico:lChla wereusedto characterize
importanceof microbialbiomass.Total sedimentationrateswere consideredfirst
(Table4).
With a greaterpercentageof the phytoplanktonbiomass in small cells, total
ratesweresignificantlyless. The variable"%Pico"was identifiedas
sedimentation
the primaryvariableexplainingtotal system loss, althoughthe completemodel,
with the inclusionof aqueousDIN concentration,could explainless thanhalf of
thisresponse.In contrast,over 80%of the variationin particulateP losses couldbe
predictedby a modelincluding%Pico (-), bacterialdensity(+), andautotrophic
microplankton
(-); nearly60%of theN-loss rateswereexplainedby %Pico alone.
Inbothinstances,a greaterimportanceof picoplanktonresultedin reducedlosses of
nutrientelements. Planktonicbacteriaare predictedto have no such effect on
enhancedecosystemefficiency.In fact, greaterbacterialbiomasswas identifiedas
a factorthat may lead to increasedP losses. Algal pigmentsedimentation(measuredas pheophytin)rateswere stronglyinfluencedby greaterautotrophicnanoplanktonlevels, with the completemodel predictingmore than 75% of this reratesrespondedsignificantlyto N andP manipulations
sponse.All 4 sedimentation
(Table2), yet theirrelationshipto bioticfactorsfromwithinthe planktonwerenot
alike. Pigmentlosses, unlikethe othersedimentationvariables,werenot relatedto
indexesof microbialprocesses(e.g., %Pico, or bact:lChla).
Differencesin these relationshipsare clearlyrevealedin bivariateplots of the 4
measuresof sedimentationloss against the primaryindependentwater-column
variableidentifiedby each multiple regressionmodel (Fig. 4). In 3 out of 4
measures,the most importantpredictor(% Pico) exerteda negativeinfluenceon
systemlosses. Thesemodelspredictthata greaterretentionof materialswithinthe
euphoticzone resultsfrom a picoplankton-dominated
phytoplanktoncommunity.
12
J.D. Wehret al.
Table 4. Resultsof multipleregressionanalysisof the combinedeffects of biologicalandchemical
in surfacewaterson ratesof sedimentationlosses. Eightindependentvariablestested
characteristics
were bacteriadensity, autotrophicpicoplankton,autotrophicnanoplankton,autotrophicmicroplankton, ratio of pico:lChla, ratio of bacteria:lChla, [SRP], and [DIN]. The minimumcriterionfor
inclusionof independentvariablesinto the modelwas a = 0.05, and0.10 to removethem. Independentvariablesarelistedin orderof inclusion,withthefirstin eachset explainingthe largestproportion
of the total variance(n = 24; coef, coefficientof slope; SE, standarderror;t, Student'st-score;P,
probability;
F, F-ratiofromANOVA;r2, finalcoefficientof determination
Step:indepvar
Dependentvariable=
1: % Pico
2: [DIN]
Y-intercept
completemodel
Dependentvariable=
1: Nano
2: Bacteria
Y-intercept
completemodel
Dependentvariable=
1: % Pico
2: Bacteria
3: Auto-Micro
Y-intercept
completemodel
Dependentvariable=
1: % Pico
Y-intercept
completemodel
coef
SE
total sedimentation
-0.002
0.0005
0.001
0.0002
0.232
0.032
F = 10.045
pheo sedimentation
0.412
0.085
0.176
0.065
-0.329
0.157
F = 39.041
P sedimentation
-0.010
0.002
0.066
0.016
-0.194
0.066
0.969
0.138
F = 34.663
N sedimentation
-0.014
0.003
2.421
0.149
F = 29.117
t
P
-3.189
3.031
7.291
0.004
0.006
<0.001
P = 0.001
r
=
0.489
<0.001
0.013
0.048
<0.001
r
=
0.788
<0.001
<0.001
0.008
<0.001
<0.001
r
=
0.839
-5.396
<0.001
16.202
<0.001
P = <0.001
r
=
0.570
4.827
2.698
-2.099
P =
-5.463
4.235
-2.953
7.038
P =
Planktonicbacteriadidnot, however,exhibitthisrelationship.Totalsedimentation
rateswere less effectivelymodeled,but morethan50%of the variationin bothN
and P retentionor loss could be predictedby % Pico alone. In contrast,reduced
ecosystemretentionof phytoplankton
pigmentswas predictedto be the resultof a
greaterbiomassof autotrophicnanoplankton.
Discussion
Nutrientmanipulations
in lake mesocosms,consistingof two pulses (on day 0 and
day 14) of nitrogenand/orphosphorus,affectedthe biomassand size structureof
the planktoncommunity,and also the flux of sedimentexportedout of the water
column.Maximumloss rateswere observedin high N (10 ,umolNH4NO3added
perliterandP (2 ,umolK2HPO4addedperliter)systems,withN exertingthe larger
effect. Because fertilizationwill often result in greaterplanktonbiomass, it is
expectedto somedegreethatnutrient-surplus
conditionscan leadto greaterratesof
sedimentation[e.g., 7]. Otherstudieshaveshownthatphosphorussedimentation
in
lakescan be partiallypredictedby levels of epilimneticP, but also by the amounts
of particulateP containedwithinplanktonicmaterials>20 ,m in size [28]. The
MicrobialLoop Efficiency
13
TotalSedimentation
P Sedimentation
0.4
1.0
2
2
r.=0.265l0
SedimentPi<o0.05
0.30.
e~~~~~~~~~~~~~~~~~~~~i
~ ~ ~ ~ ~ ~ ~ ~
E
~
C.)
~
*
~0.6%
Phephy0i8
E0
.
S
r=0.646
i<me.naio
~a)
T
0
E
0.2
sO
0.0
0
20 40 60
Autotrophic-Pico:
0.0
80 100
E Chia
N Sedimentation
0
20 40 60
AutotrophicPiao
(
80 100
Chia
Pheophytin Sedimentation
2.5
2.5
*
E 2.0E
Z
r= 0.570
< 0.001
~~P
N2.001.5-
co1.5
Co.1.0
C.
0
~~1.0
*
0.5 *r=0.715
o..~~~~~~E
E
0.5
0
20 40 60 80 100
Autotrophic-Pica: y- Chia
P < 0.001
0.0
2
0
1
3
4
Autotrophic-Nano
(jug Chia/I L)
variables)versus4
Fig. 4. Bivariateregressionplotsof thekey watercolumnpredictors(independent
measuresof particleflux intosedimenttrapsin CalderLakemesocosms.The mostimportantindependent variablein each case was extractedby stepwisemultipleregression(n = 24; lines fit by least
For detailsof each independent
squaresregression;P, probability;r2, coefficientof determination).
variable,see text.
mechanismbehindthese dynamicsmay not simplybe a matterof greaterplankton
biomass,butalso enhancedecosystemefficiency.
Ourexperiments,whichdirectlymanipulatedshort-termnutrientconcentrations
(two discretepulses), were designedto cause a shift in the biologicalcommunity
andin longer-termchemicalconditionswithinthe euphoticzone. Thesecharacteristics, particularlyphytoplanktonsize structureand the relativeimportanceof a
pelagicmicrobialloop, shouldbe given particularattention,as they arebelievedto
play a centralrole in regulatingecosystemefficiency[3, 33]. The theorystatesthat
materialsthatpass througha microbialloop aremoreefficientlyremineralized
and
fed back into solublepools. Experimentalstudiessupportthis theory.The uptake
and regenerationof both N and P within surfacewatershas been shown to be
regulatedmainlyby planktonicorganisms<3 pumin size [4, 16, 17], and less by
herbivorouszooplankton,such as cladocerans.Other studies have shown that
smaller protist grazers, such as heterotrophicnanoflagellatesand ciliates, can
acceleratemicrobialregenerationof nutrients,particularlyP, within the water
14
J.D. Wehr et al.
column[5, 6]. Sedimentationstudiesin a tropicalmarinesystemhave shownthat
picoparticlesdo not tend to settle out over 24h, while significantpercentagesof
autotrophicnanoplankton
(- 15%)andmicroplankton
(-30%) areexportedto the
benthos daily [23]. For these reasons, it has been specifically suggested that
nutrientscycled within the microbialloop should have a greaterprobabilityof
remainingwithinthe photiczone [37]. Hence, whole-systemmanipulations
which
lead towardgreatermicrobialprocessingmay be used to test whetherecosystem
efficiencyis trulyenhanced.
This study demonstratesthat changes in the size structureof a phytoplankton
community,thatis, a shiftfrompredominantly
large-celledto small-celledassemblages, correspondwith a greaterretentionof materialswithin the photic zone,
includingboth P and N. Sedimentationrates of particulateP regressnegatively
against a simple index of microbial dominance, the ratio of autotrophic
picoplankton:lChla.The slopepredictsthatfor every 10%increasein thepredominanceof picoplankton,the system(on an arealbasis)will retain7.4 pugmoreP and
13.6 ,ugmoreN/M2 perday. The ecosystemsignificanceof this effect in lakeswill
furtherdependin parton protozoangrazingactivity,butalso the depthof thephotic
zone that will supportautotrophicpicoplankton.Thereare datathat suggestthat
phycoerythrin-rich
picoplankters,such as some species of Synechococcus,may
utilize reducedlight field more efficiently than other autotrophs[e.g., 19, 26],
which may favor their greaterrelativeabundancewith depth [44]. Thus, low-P
(high N:P ratios), deep lakes may be those ecosystems with greatestpelagic,
microbialrecyclingandnutrientretention.
OurdatasupportearlierstudiesindicatingthatgreaterN:P ratiosin freshwaters
not only alter phytoplanktonproductionlevels, but may result in communities
dominatedby bacterial-sizedcyanobacteria,such as Synechococcus[36, 41, 44].
SuttleandHarrison[38] have shownthata high affinityfor DIP in Synechococcus
spp. may be criticalin favoringsmallerautotrophsin P-limitedsystems. Ourdata
revealthattheecosystemimplicationsof thishigheraffinityforP arereducedlosses
of nutrientsout of the watercolumn,as well as significantlylowertotalsedimentationrates.The lackof a similareffect by planktonic,heterotrophic
bacteria(Tables
3, 4), whichalso fall intothis size range(or even smaller),is not easily explained.
Recentstudieshave shown, however,thatproductionratesof planktonicbacteria
may be directlylimitedby inorganicP in freshwaters[15, 40], andthis limitation
may occurindependentlyof a phytoplankton
response[25]. It has been suggested
thatDIP uptakeby planktonicbacteriamay exceed demandsandresultin P04-P
becomingimmobilized,unless there is considerableprotozoanactivity [2, 39].
Similardatahave led some researchersto statethatplanktonicbacteriamay be a
phosphorussink [6]. It is still difficultto explainhow in the same communities
from CalderLake, that greaternumbersof autotrophicpicoplanktonwill lead to
increasedP retention,while presumablymorerapidly-growing
(andgrazed)bacteria may have the opposite effect (Table 4). Effectivenessand mechanismsof
nutrientturnoverby heterotrophic
(andmixotrophic)flagellatesmayperhapsdiffer
accordingto food item, withmorerapidmineralization
occurringwithherbivoryon
autotrophiccells suchas Synechococcus.
Further,the ratioof recycledC:N:Presultingfromzooplanktonherbivoryis the
resultnotonly of the grazingrateandthe nutrientcompositionof food items,butis
also affectedby differentnutrientdemandsamong variouszooplanktonspecies
MicrobialLoop Efficiency
15
[21]. For example, low P:C ratiosin P-starvedbacteriamay lead to still lower P
releaseratesby grazingflagellatesthathavehighP demands.Phosphorusremineralizationmayeven approachzeroduringactivegrazingif P:Cratiosof bacteriaand
phytoplanktonfall below critical levels [30]. Net mineralizationmeasurements
[e.g., 6] and sedimentationefflux measurements(the presentstudy) reflect the
combinedeffects of nutrientturnover(leakage, excretion, grazingeffects) and
utilization(uptake,storage,growth)by each microbialcomponent.Ourdatasuggest thatheterotrophicbacteriaand autotrophicpicoplanktonmust differ in some
criticalbutnotfullyknownwaysthatleadto theirrolesas eithersinksor sourcesfor
P andN withinthe photiczone.
Acknowledgments.
Thisstudywas fundedin partby theNSF, grant# DIR-9002145,andby theLouis
CalderCenter,FordhamUniversity.We thankKenCavassifor helpfulassistancein the field.
References
1. AmericanPublic Health Association(1985) Standardmethodsfor the analysis of water and
wastewater,16thed. APHA,WashingtonDC
2. AndersenOK, GoldmanJC, CaronDA, DennettMR (1986) Nutrientcyclingin a microflagellate
food chain.III. Phosphorusdynamics.MarEcol ProgrSer 31:47-55
3. Azam F, Fenchel T, Field JG, Meyer-ReilLA, ThingstadF (1983) The ecological role of
water-column
microbesin the sea. MarEcol ProgrSer 10:257-263
4. BermanT (1985) Uptakeof [32P]orthophosphate
by algae and bacteriain Lake Kinneret.J
PlanktonRes 7:71-84
5. BermanT, NawrockiM, TaylorGT, KarlDM (1987) Nutrientflux betweenbacteria,bacterivorousnanoplanktonic
protists,andalgae. MarMicrobFoodWebs2:69-82
6. Bloem J, AlbertC, Bar-GillisenM-JB, BermanT, CappenbergTE (1989) Nutrientcycling
throughphytoplankton,bacteria,and protozoain selectively filteredLake Vechten water. J
PlanktonRes 11:119-131
7. BloeschJ, UehlingerU (1986) Horizontalsedimentationdifferencesin a eutrophicSwiss lake.
LimnolOceanogr31:1094-1109
8. Bloesch J, BurnsNM (1980) A criticalreview of sedimenttraptechnique.SchweizZ Hydrol
42:15-55
9. Bran+LuebbeAnalyzingTechnologies(1986) Orthophosphate
in waterandseawater.Industrial
MethodNo. 812-86T
10. Bran+LuebbeAnalyzing Technologies (1986) Ammonia in water and seawater. Industrial
MethodNo. 804-86T
11. Bran+LuebbeAnalyzingTechnologies(1987) Nitrate/nitrite
in water and seawater.Industrial
MethodNo. 818-87T
12. Bran+LuebbeAnalyzing Technologies(1987) Silicates in water and wastewater.Industrial
MethodNo. 785-86T
13. BurnsCW, StocknerJG (1991) Picoplanktonin six New Zealandlakes:abundancein relationto
seasonandtrophicstate.IntRev Ges Hydrobiol76:523-536
14. CaronD (1991) Evolving role of protozoain aquaticnutrientcycles. In: Reid PC et al (eds)
Protozoaand their role in marineprocesses. (NATO-ASISeries, vol G25) Springer-Verlag,
Heidelberg
15. CoveneyMF, WetzelRG(1992)Effectsof nutrientson specificgrowthrateof bacterioplankton
in
oligotrophiclakewatercultures.ApplEnvironMicrobiol58:150-156
16. CurrieDJ, BentzenE, Kalff J (1986) Does algal-bacterial
phosphoruspartitioningvaryamong
lakes?A comparativestudyof orthophosphate
uptakeandalkalinephosphataseactivityin freshwater.CanJ FishAquatSci 43:311-318
16
J.D. Wehret al.
17. DoddsWK, PriscuJC, Ellis BK (1991) Seasonaluptakeandregenerationof inorganicnitrogen
andphosphorusin a largeoligotrophiclake:size-fractionation
andantibiotictreatment.J Plankton
Res 13:1339-1358
18. GoldmanJC(1984)Conceptualrolesfor microaggregates
in pelagicwaters.Bull MarSci 35:462476
19. GloverHE (1985) The physiologyandecology of the marinecyanobacterial
genusSynechococcus. Adv AquatMicrobiol3:49-107
20. HargraveBT, Burns NM (1979) Assessmentof sedimenttrap collection efficiency. Limnol
Oceanogr24:1124-1136
21. Hessen DO, AndersenT (1992) The grazer-algaeinterface:feedbackmechanismslinked to
elementalratiosandnutrientcycling. ArchHydrobiolBeih ErgebnLimnol35:111-120
22. HobbieJE, DaleyJ, JasperS (1977) Use of nucleporefiltersforcountingbacteriaby fluorescence
microscopy.ApplEnvironMicrobiol33:1225-1228
23. HopcraftRR, Roff JC, Berges JA (1990) Size-fractionatedsedimentationin a tropicalneritic
ecosystemnearKingston,Jamaica.ContShelfRes 10:795-806
24. KirchnerWB (1975) An evaluationof sedimenttrapmethodology.LimnolOceanogr20:657-660
25. Le J, WehrJD, CampbellL (1993) Uncouplingof inorganicnutrient-limited
bacterioplankton
growthfromphytoplankton.
Submittedto ApplEnvironMicrobiol
26. Lewis MR, WarnockRE, PlattT (1986) Photosyntheticresponseof marinepicoplanktonat low
photonflux. In: PlattT, Li WKW(eds) Photosyntheticpicoplankton.Can Bull Fish AquatSci
214:235-250
27. LorenzenCJ (1967) Determination
of chlorophyllandpheo-pigments:spectrophotometric
equations. LimnolOceanogr12:343-346
28. MazumderA, TaylorWD, McQueenDJ, LeanDRS (1989) Effectsof fertilizationandplanktivorousfish on epilimneticphosphorusandphosphorussedimentation
in largeenclosures.CanJ Fish
AquatSci 46:1735-1742
29. MenzelDW, CorwinN (1965) The measurement
of totalphosphorusin seawaterbaseduponthe
liberationof organicallyboundfractionsof persulfateoxidation.LimnolOceanogr10:280-282
30. OlsenY, JensenA, ReinertsenH, B0rsheimKY, HeldalM, LangelandA (1986) Dependenceof
the rateof releaseof phosphorusby zooplanktonon the P:Cratioin the food supply,as calculated
by a recyclingmodel. LimnolOceanogr31:34 44
31. PorterKG, Feig YS (1980) The use of DAPI for identifyingand countingaquaticmicroflora.
LimnolOceanogr25:943-948
32. SchindlerDW (1977) Evolutionof phosphoruslimitationin lakes. Science 195:260-262
33. SherrEB, SherrBF (1991) Planktonicmicrobes:tiny cells at the base of the ocean'sfood webs.
TrendsEcol Evol 6:50-54
34. SieburthJMcN, SmetacekV, LenzJ (1978) Pelagicecosystemstructure:heterotrophic
compartmentsof theplanktonandtheirrelationshipto planktonsize fractions.LimnolOceanogr23:12561263
35. StocknerJG, Antia NJ (1986) Algal picoplanktonfrom marineand freshwaterecosystems:a
multidisciplinary
perspective.CanJ Fish AquatSci 43:2472-2503
36. StocknerJG, ShortreedKS (1988) Responseof AnabaenaandSynechococcusto manipulation
of
nitrogen:phosphorus
ratiosin a lakefertilizationexperiment.LimnolOceanogr33:1348-1361
37. StoneL, WeisburdSJ(1992)Positivefeedbackin aquaticecosytems.TrendsEcolEvol7:263-267
38. SuttleCA, HarrisonPJ (1988) Ammoniumand phosphateuptakerates, N:P supplyratios, and
evidencefor N andP limitationin some oligotrophiclakes. LimnolOceanogr33:186-202
39. ThingstadTF (1987) Utilizationof N, P, andorganicC by heterotrophic
bacteria.1. Outlineof a
chemostattheorywith a consistentconceptof "maintenance"
metabolism.MarEcol ProgrSer
35:99-109
40. Toolan T, Wehr JD, FindlayS (1991) Inorganicphosphorusstimulationof bacterioplankton
productionin a meso-eutrophic
lake. ApplEnvironMicrobiol57:2074-2078
41. WehrJD (1989) Experimental
tests of nutrientlimitationin freshwaterpicoplankton.ApplEnvironMicrobiol55:1605-1611
42. Wehr JD (1990) Predominanceof picoplanktonand nanoplanktonin eutrophicCalderLake.
Hydrobiologia203:35-44
MicrobialLoop Efficiency
17
effects on picoplanktonand size structurein
43. Wehr JD (1991) Nutrientand grazer-mediated
communities.IntRev Ges Hydrobiol76:643-656
phytoplankton
of lightandphosphorussupplyon compe44. WehrJD (1993)Effectsof experimentalmanipulations
tition among picoplanktonand nanoplanktonin an oligotrophiclake. Can J Fish Aquat Sci
50:936-945
45. WetzelRG (1983) Limnology.SaundersCollegePublishing,Philadelphia,Pennsylvania
46. WilkinsonL (1988) SYSTAT:The systemfor statistics.Systat,Inc., Evanston,Illinois