to Hydroponic and Aquaponic Water Quality Condit

16 Pages·2017·0.97 MB·English

horticulturae
Article
Growth and Tissue Elemental Composition Response
of Spinach (Spinacia oleracea) to Hydroponic and
Aquaponic Water Quality Conditions
DanielA.Vandam1,TylerS.Anderson1,DaviddeVilliers2andMichaelB.Timmons1,*
1 DepartmentofBiologicalandEnvironmentalEngineering,CornellUniversity,Ithaca,NY14853,USA;
[email protected](D.A.V.);[email protected](T.S.A.)
2 DepartmentofHorticulture,CornellUniversity,Ithaca,NY14853,USA;[email protected]
* Correspondence:[email protected]
AcademicEditor:DouglasD.Archbold
Received:30December2016;Accepted:5May2017;Published:9May2017
Abstract: Spinach(Spinaciaoleraceacv. Carmel)wasgrowninaconventionalglassgreenhouseunder
threedifferentnutrientsolutiontreatments. Lightingandtemperatureconditionswereidentical. Six
growingsystemswereusedtoprovideaduplicatetroughsystemforeachofthesethreetreatments.
Sixtrialswereharvestedfromeachsystemoveratwomonthtimeperiod. Twotreatmentsreceived
hydroponicnutrientinputs,withonetreatmentatpH7.0(referredtoasH7)andtheotheratpH5.8
(H5), andthethirdtreatmentwasaquaponic(A7), receivingallofitsnutrientsfromasinglefish
tankwithkoi(Cyprinuscarpio)exceptforchelatediron. SystempHwasregulatedbyaddingK CO
2 3
toaquaponicsystemsandKOHtohydroponicsystems. Comparisonsmadebetweentreatments
were total yield, leaf surface area, tissue elemental content, and dry weight to fresh weight ratio.
Dryweightbiomassyieldvalueswerenotdifferentinpairwisecomparisonsbetweentreatments
(A7 vs. H5: p = 0.59 fresh weight, p = 0.42 dry weight). Similarly, surface area results were not
differentbetweentreatments. TheimportantcomparisonwasthatA7achievedthesamegrowthas
H5,theconventionalpHwithacompleteinorganicnutrientsolution,despiteunbalancedandless
than“ideal”nutrientconcentrationsintheA7condition.
Keywords: hydroponics; aquaponics; spinach; pH; biomass; nutrient analysis; tissue
analysis;temperature
1. Introduction
Hydroponicsisamethodofgrowingplantsusingmineralnutrientliquidsolutionwithoutsoil.
The method of application of the nutrient solution to the roots varies widely [1]. Aquaponics is a
method of growing plants hydroponically using nutrients provided from an aquaculture system.
Aquaponics makes multiple uses of resources such as water and nutrients [2,3]. Hydroponics is
an increasingly important field as the demand increases for more food and sustainably produced
products[4]. Withurbanagricultureontherise[5],greenhousesaregrowingfoodonrooftopsandin
decayingbuildingsandabandonedwarehouses[4]. Hydroponicsusesinorganicnutrientfertilizers,
whileaquaponictreatmentsrelyonfishwaste,whichhasthepotentialtobeatlessormorethanideal
concentrationsforplants. Aquaponicsusesfishrecirculatingwatersormineralizedwastedischargeto
generatethenutrientsneededbyplants,meaningthenutrientcompositionisnotformulatedtoexact
concentrationsandcanbelessstable[6]. Growingplantsaquaponicallyusesnutrientsthatwould
otherwise be lost from a fish system, which has an added value in marketing to a certain class of
consumers. Theplantsalsohelpfilterthefishwaterandhelptoreducenitratewhichcanbetoxicto
somefishsalmonidspeciesatelevatedlevels,e.g.,greaterthan40mg/L[3].
Horticulturae2017,3,32;doi:10.3390/horticulturae3020032 www.mdpi.com/journal/horticulturae
Horticulturae2017,3,32 2of16
Theobjectiveofthisstudywastocompareaquaponicandhydroponicspinachyield,andlook
fordifferencesinleafsurfaceareaandelementalcompositionbetweentreatments. Twohydroponic
treatmentswerechosen,pH5.8andpH7.0,andoneaquaponictreatmentwasusedatpH7.0,alower
practicallimitforfishproduction. Theaquaponictreatmentreceivednutrientsfromfishrecirculating
water,withtheexceptionofchelatediron. Fishwastehasnoavailableironinitforplants,sincethe
iron would be in the ferric form. As a result, chelated iron is commonly added to the water of an
aquaponicsystem;theamountofironbeingabsorbedbythefishdirectlyfromthewaterisconsidered
smallandnon-harmful[2].
Thesolutionformulaemployedforthehydroponicspinachwasaformuladerivedforlettuce
by Sonneveld and Straver [7]. In earlier research, the lettuce formula was as effective at half the
concentration recommended by Sonneveld and Straver as at full concentration [8]. Half strength
SonneveldandStraversolutionhasanelectroconductivityof1300microsiemens/cm. Moderatelylow
pH,around5.8,keepsmostionsavailableinsolutionwhilehigherpH,around6.5andhigher,can
causenutrientdeprivationbecauseofnutrientprecipitation[9]. TheSonneveldandStraversolution
usedinthisresearchwasoriginallydesignedforusewithapHaround5.8,andbothBothetal.[8]and
Bugbee[9]usedthispHforlettucetomaximizenutrientavailability.
An added constraint in this research was that our aquaponic treatment needed to be
kept at the same low root zone temperature as the two hydroponic treatments to combat
Pythium (Pythium aphanadermatum) root disease, which is a major hazard in spinach production.
DeVilliersetal.[10]havediscussedgrowingmethodsandstrategiestoaddressthisdisease,which
weemployedinourexperiment,e.g.,lowrootzonetemperature,harvestingafter13daysinchannel
water, and spraying 70% ethanol on all potentially contaminated surfaces during planting and
solutionsampling.
Amajordifferenceofaquaponicscomparedtohydroponicsisitspotentialfornon-uniformity
inwaternutrientconditions; thesystemdoesnothaveafixednutrientcomposition,andisreliant
upon the fish water to provide all nutrients at all times with the exception of chelated iron. This
non-uniformityispossiblyoneofthereasonsthataquaponicssometimeshashadloweryieldsthan
hydroponics [11]. Nutrient levels are highly dependent on the fish activity, fish number, and fish
species[12,13]. Evenifthefishwasteprovidesadequatenutrientstosuccessfullygrowhighyielding
plants,thehydrodynamicsoftheaquaponicssystemflowcanbedetrimentaltotheplantsideofthe
system. Forexample,theflowrateandrateofrecirculationinthesystemcanreduceyieldiftheyare
tooslowortoofastwhichcausesrootstress[14]. Theoverallobjectiveofthisstudywastocompare
yieldsinanaquaponicssystem(A7)toyieldsobtainedfromaconventionalhydroponicssystemat
pH5.8(H5). AhydroponicconditionofpH7(H7)wasalsoincludedforcomparisontootherresearch
andpossiblyhelpexplainanydifferencesdetectedbetweenH5andA7.
2. MaterialsandMethods
Seedsweregerminatedathighhumidityinatemperaturecontrolledgrowthchamberforthree
days (additional details of growth chamber provided below). After germination, flats of spinach
were grown for 13 additional days using deep-flow troughs/channels housed in a conventional
glassgreenhouseunderthreedifferenttreatmentswhichconsistedofthreedifferentnutrientsolution
conditions. Two treatments received hydroponic nutrient inputs, with one treatment at pH 5.8
(referredtoasH5)andtheotheratpH7.0(referredtoasH7). Reverseosmosis(RO)waterwasusedto
makeupthenutrientsolutions. Growingconditionsmimickedconventionaldeep-flowgrowponds
andplantsweregrownusingamodifiedSonneveldnutrientsolutiondesignedforlettuce(see2.8.
Nutrients). Thethirdtreatmentwasaquaponic(referredtoasA7), receivingitsnutrientsfromthe
aquaculturerecirculatingwaterthatcontainednutrientsexcreteddirectlybythefish(nitrogenous
wasteasammonia,nitriteornitrate)orasnutrientsbeingreleasedfromthemineralizationofwaste
solidscapturedbythesystembeadfilter. Theonlyextraneousnutrientaddedwaschelatediron,which
wasusedtoraisetheinitialFeconcentrationto3mg/L).Theaquaponicsourcewaterwastapwater
Horticulturae2017,3,32 3of16
thatwasinitiallyhighinalkalinity. Thiscarbonfilteredwaterhadaveragemacro-elementalcontents
of50mg/LCa,13mg/LMg,5.5mg/LS,andanECof450µS/cm. Theconcentrationsofthemakeup
waterforCa,MgandSvariedslightlyseasonally. AdjustmentsinpHwereinitiallymadetoachieve
targHeotrtvicualltuurease 2a0n17d, 3t,h 3e2 nmadedailybyadding1MK2CO3totheaquaponicsystemor1MKOH3 otf o16t he
hydroponicsystems. Priortotheexperiment,initialpHadjustmentsweremadeusingHNO orthe
3
treaatcmhieenvte’s tarergspete cvtaivlueebs aasneda sthjeuns tmmaednet idoanielyd .bIyn aitdiadlinang d1 fiMn aKl2wCaOt3e rtoe ltehme eanqtuaalpvoanliuce ssyfsotremth eorn 1u tMrie nt
KOH to the hydroponic systems. Prior to the experiment, initial pH adjustments were made 
growingconditionsaregiveninTable7forallthreetreatments.
usTinhge HspNinOa3c ohr ctrhoep tsreiantmalelntrteʹsa rtemspenecttsivwee breasger aosw jnusitn mtheentsioanmeed.g Irneietniahl oaunsde fainnadl aweartiearl sepleamceenutnald er
values for the nutrient growing conditions are given in Table 7 for all three treatments. 
thesamelightingandtemperatureconditions,withperiodicharvestofoldandinsertionofnewtrials
The spinach crops in all treatments were grown in the same greenhouse and aerial space under 
intohydroponicgrowingchannels(describedfurtherbelow,seeFigure1forageneraloverview). Six
the same lighting and temperature conditions, with periodic harvest of old and insertion of new trials 
trialswereconductedfromJanuary17toFebruary19, 2016, withapreliminarytrialconductedto
into hydroponic growing channels (described further below, see Figure 1 for a general overview). Six 
validateprocedures. Rootzonetemperaturewasmatchedacrossalltreatmentsat18◦C.Alltreatments
trials were conducted from January 17 to February 19, 2016, with a preliminary trial conducted to 
weregrownfor13daysonceaflatwasfloatedinitsrespectivechannel. Eachofthethreetreatments
validate procedures. Root zone temperature was matched across all treatments at 18 °C. All 
wastraeaptpmlieendtsi nweared ugrpolwicnat feorg r1o3w dainygs osnycsete am fl,arte wsualst ifnlogatiends iinx igtsr orewspinegctisvyes ctehmansn.eTl.h Eeatcwh oofh tyhed rthorpeoen ic
treatrtemaetmntesnthsa wdafso uaprpcloiemd pinle ate dlyupinlidcaetpee gnrdoewnitngre scyirscteumla,t irnesgusltyinstge min ssiixn cglruodwiningg tshyesitremsosu. rTchee wtwaote rs,
whhilyedtrhoepwonaitce trrefraotmmetnhtes hfiasdh ftoaunrk cofomrpthleetetlwy oinadqeupaenpdoennict rseycsitrecmulastwinags sycostnetminsu ionuclsuldyibnegi tnhgeimr sioxuedrc.eA  n
indwivaidteursa,l wtrhiaillee tnhdee wdawteirth frtohme htharev feissht otafnakll fsoixr tflhaet stwofos apqinuaapchonfrico msysetaecmhsg wroaws icnogntsiynsutoeums.lyE baecihngtr ial
wasmoixffesde.t Abny itnhdrieveidduaayls t;riaadl denitdioedn awlihtha rtvhee shtadrveetasti losfa arlel spixr oflvaitds eodf sbpeinloawch. fSrtoymro efaocahm grfloowaitnsgc soyvsetreemd. all
watEearcshu trrfiaacl ewsatos oreffdsuetc ebya ltghareeeg droawyst;h a,dmdiintiiomniazl eheavrvaepsot rdaetitoanil,s aanred pbrloovckidseodl abrelroawdi.a Sttioynro,fwoahmic hflowaotsu ld
havceovdeersetdro aylle dwathteer isruornfaccheesl taot orre.duce algae growth, minimize evaporation, and block solar radiation, 
which would have destroyed the iron chelator.  

FigFuirgeu1re. F1l. oFoloroprl panlano fogf rgereenenhhoouusese..F Fiisshh ttaannkk iiss sseeppaarraatteedd ffrroomm ssppininaachch chcahnannenles lbsyb ay tahitnh ignregerneheonuhsoeu se
parptiatriotintiowna wll.alCl.h Cahnannenleslas raeren nuummbbeereredd1 1t thhrroouugghh 66.. AA.. RReesseerrvvooirisr swwitiht hcoclodl dfinfignegrse r(she(hate eaxtcehxacnhgaenrgs)e rs)
useudsetod ctoo oclooanl adnfidl ftielrteirn idnidviivdiudualalh hyyddrrooppoonniicc cchhaannnneellss;; BB.. DDuuppllicicaatete chchanannenlesl osfo HfH5 t5retaretmatemnte n(5t a(5nda nd
6);6C);. CD.u Dpulipcalitceatceh cahnannenleslso fofH H77t rtereaatmtmeenntt ((33 aanndd 44));; DD.. SShhaarreedd aaqquuaapponoinci crerseesrevroviro iwrawteart,e wr,itwhi tahn an
independent sump pump to return water to the fish system; E. Channels of A7 treatment (1 and 2), 
independentsumppumptoreturnwatertothefishsystem;E.ChannelsofA7treatment(1and2),
not duplicate because of shared water; F. Bead filter for nitrification and capture of fish solid waste 
notduplicatebecauseofsharedwater;F.Beadfilterfornitrificationandcaptureoffishsolidwaste(in
(in series with sump (H) and fish tank (G); G. Fish tank; H. Sump pump and settling tank for 
serieswithsump(H)andfishtank(G);G.Fishtank;H.Sumppumpandsettlingtankforrecirculating
recirculating fish system; flow continuously recycles with fish tank (G); 25% of sump pump flow is 
fishsystem;flowcontinuouslyrecycleswithfishtank(G);25%ofsumppumpflowisshort-circuitedto
short‐circuited to fill reservoir water tank for aquaponic channels (E). 
fillreservoirwatertankforaquaponicchannels(E).
Horticulturae2017,3,32 4of16
2.1. TheGreenhouse
The spinach was grown in the middle section of a multi-sectioned glass greenhouse built in
1953,withdimensions9m×11m×7mhightotheridge,orientedeastwest(Figure1). AnArgus
monitoringandcontrolsystem(Surrey,BC,CanadaV3S7A4)loggedCO ,humidity,aerialtemperature,
2
andlightlevel,andalsocontrolledaerialtemperatureanddailylightintegral(DLI).Therewasno
carbondioxidesupplementation. Twoidenticalwater-to-airheatexchangersonoppositesidesofthe
greenhouseratedat115,000kJ/husedfanstomoveairthroughradiatorsthenacrossthegreenhouse,
providingairmixingandrapidadjustmentofairtemperaturestotargetvalues.
A target DLI of 17 mol/m2/day was used for all trials. The DLI is the quantity of
photosyntheticallyactiveradiation(PAR,inunitsofmoles/m2/day)achievedbycontrolleduseofa
lightingarraytosupplementnaturallight.Theenvironmentalparametersweresampledapproximately
everytwosecondsanddataqueuesaveragedandloggedevery2min. DLIwascontrolledtoitstarget
valuebysupplementingnaturallightusinganarrayof20highpressuresodium(HPS)lights(GE,400
wattclearS51/O,MogulBaseratedED18HSP,LU400/H/ECO;GeneralElectric, Cleveland, OH,
USA). The DLI was reset daily at 6:00 am. The greenhouse had a heating set point of 24◦C, and a
coolingsetpointof25◦C.Thequantityofbothnaturalandsupplementallightingreceivedatplantlevel
wasrecordedusingaLiCorquantumsensor(Li-Cor,Lincoln,NE,USA).TheaverageDLIfortrials
wasconsistentlybetween17.0and17.1mol/m2/dayforalltrials. Theaveragenaturallightintegrals
were4.0,4.1,4.7,4.6,5.4,and5.4mol/m2/dayfortrials1–6respectively. Theaveragesupplementary
lightintegralswere13.0,12.9,12.3,12.4,11.6,and11.6mol/m2/dayfortrials1–6respectively.
2.2. TheChannels
Flats of spinach were floated in six channels raised 1.27 m above the floor. The channels
were made of 2 × 12 lumber, which provided insulation on the sides and ends. They were also
insulated at the bottom with 25 mm polystyrene and 12 mm of plywood, and carefully lined
with 0.006 mil plastic to make a functioning pond. The outside dimensions of the channels were
29cmhigh×42.5cmwide×244cmlong. Internaldimensionswere26cmdeep×35cmwide×
236 cm long. Each channel was accompanied by a reservoir at floor level (volume 50 L). Nutrient
solutionwascooledinthereservoir,pumpeduptothefarendofthechannel,thendrainedbackto
thereservoirbygravitywithdepthcontrolledbyastandpipeatthedownstreamendofthechannel.
Thechannelswerefilledtoroughly24cmwith2cmoffreeboard,whichprovidedatotalvolumeof
200Lperchannelplusreservoirvolume. Thetopsofthefloatingflatswere1.27mabovethefloor
and1.30mbelowthelightfixturestomaximizelightuniformity(naturalandsupplemental)among
allchannels. Atthewaterlevelheightsmaintained, thetopofthefloatsclearedthechannelsides.
Thechannelswerelabeled1through6andweregroupedintothreeblocks: channels1and2were
block1(A7treatment),channels3and4wereblock2(H7treatment),andchannels5and6wereblock
3(H5treatment)(seeFigures1and2,Table1).
Horticulturae2017,3,32 5of16
Horticulturae 2017, 3, 32  5 of 16 

Figure 2. Photograph of channels and general experimental setup. The two channels on the left are 
Figure2.Photographofchannelsandgeneralexperimentalsetup.Thetwochannelsontheleftarethe
the hydroponic pH 5.8 treatment. The two channels in the middle are the hydroponic pH 7.0 
hydroponicpH5.8treatment.ThetwochannelsinthemiddlearethehydroponicpH7.0treatment.
treatment. The two channels on the right are the aquaponic treatment. The insulated PVC pipe coming 
Thetwochannelsontherightaretheaquaponictreatment.TheinsulatedPVCpipecominginfrom
in from the top right is the inflow and the outflow from the fish tank in an adjacent greenhouse section 
thetoprightistheinflowandtheoutflowfromthefishtankinanadjacentgreenhousesectiontothe
to the right, outside of picture. 
right,outsideofpicture.
Table 1. Fresh weight biomass results (grams/plant) from a separate experiment conducted to 
Table1.Freshweightbiomassresults(grams/plant)fromaseparateexperimentconductedtomeasure
measure and test any potential differences between channels, in order to justify the decision not to 
andtestanypotentialdifferencesbetweenchannels, inordertojustifythedecisionnottoreverse
reverse treatment positions; the major factor of concern was light distribution throughout the 
treagtrmeeennhtopuoses it(iuonnps;utbhlieshmeda jodraftaac, toDreoVfilclioenrsc,e rDn.,w  Aasnldigerhstond,i stTr.i;b uCtoiornnetllh rUoungivheorsuittyt,h eCgorneteronlhleodu se
(unEpnuvbilriosnhmedendta Gtar,oDupe)V. illiers,D.,Anderson,T.;CornellUniversity,ControlledEnvironmentGroup).
    Channel       
Channel
TrialTrial  AverAavgeer age
1  2  3  4  5  6 
1 2 3 4 5 6
1  3.64  3.48  3.98  3.88  3.97  3.61  3.76 
1 3.64 3.48 3.98 3.88 3.97 3.61 3.76
2 2  2.822.82  2.820.80  3.336.36  2.828.88  33.1.122  3.32.02 0 3.03 3.03
3 3  3.883.88  3.639.69  4.040.00  3.737.77  33.6.644  3.34.74 7 3.74 3.74
4 3.24 3.08 3.63 3.49 2.87 3.53 3.31
4  3.24  3.08  3.63  3.49  2.87  3.53  3.31 
5 2.19 2.20 2.14 2.19 1.94 2.15 2.14
5  2.19  2.20  2.14  2.19  1.94  2.15  2.14 
6 2.53 2.75 2.74 2.79 2.53 2.54 2.65
Average6  3.052.53  3.020.75  3.321.74  3.127.79  32.0.513  2.35.40 8 2.65 3.10
Average  3.05  3.00  3.31  3.17  3.01  3.08  3.10 
2.3. SeedingandFloating
2.3. Seeding and Floating 
Theseedingprotocolinvolvedhand-seedingspinach(Spinaciaoleracea,cv. Carmel)seedsinto
The seeding protocol involved hand‐seeding spinach (Spinacia oleracea, cv. Carmel) seeds into 
eachcellofeachflat. Duringtrials1and2,seedsweredoubleseededineachcell,butduetosomewhat
each cell of each flat. During trials 1 and 2, seeds were double seeded in each cell, but due to 
lowgerminationintheflats,trials3to6weretripleseeded. Aftergermination,standswerethinned
somewhat low germination in the flats, trials 3 to 6 were triple seeded. After germination, stands 
toonlyoneplantpercellpriortofloatingtheplantsinthechannels(seeStandcorrectionbelowfor
were thinned to only one plant per cell prior to floating the plants in the channels (see Stand correction 
moredetails).
below for more details). 
Wehavefoundthenotoriousandhistoricaldifficultyingerminatingspinachlargelydisappearsif
We have found the notorious and historical difficulty in germinating spinach largely disappears 
seedingisintopre-moistenedmediumandthemoisturecontentofthemediumiscontrolled. Potting
if seeding is into pre‐moistened medium and the moisture content of the medium is controlled. 
mePdoiattiunsge dmewdaias uLsMed-1 wgaesr LmMin‐1a tgieornmminiaxti(obnle mnidx o(bflfiennde -ofifb fienree‐dfibCearneda dCiaannasdpiahna gspnhuamgnpuema tpmeaot smsoaslos ng
witahlofinnge wveitrhm fiincue lviteermanicduplieter laitned), pbeyrlLitaem), bbyer Lta(Qmubeerbte (cQ,Cueabneacd, aC)a.nPardea-m). oPirset‐emnoeidstmeneeddi ummedwiuams cwreaast ed
bycardedatiendg b1yL aodfdrinevg e1r sLe oofs rmevoesrissew oasmterosfiosr wevateerry fo1rk egveorfyL 1M k-g1 opfo LttMin‐g1 mpoetdtiinag, tmoeadciha,i etvo eaachmieovies tau re
conmteonisttoufrer ocuongthelnyt 3ofp raorutsghwlya t3e rpatortso nweatpear rttod ornye mpaarttt edrry(t hmeapttoert t(itnhge mpoetdtiinagi smreoduiag hisl yrohuaglhflwy ahtaelrf as
recewivateedr) a.sP roetcteinivgedm).e Pdoiattwinags mscerdeiean weads tshcrroeuengehda th6rmoumghs aie 6v emimnt osiemvue litni-tcoe mlluSlttyi‐rcoeflol aSmtyrfloafotsa.mD felapttsh. of
seeDdienpgthw oafs secoednitnrogl wledasb cyonatrwoolloedd ebny da iwbboloedretnh adtibcoblmerp tahcatte cdotmhpeamcteeddi uthme mtoeadisuemtd teop at hse.tW deepimth.p Wroev ed
Horticulturae2017,3,32 6of16
ourgerminationsuccessbydiscardinganyseedsthatlookedunhealthy,e.g.,misshapen,discolored,or
oddlysized. Thegoalofseedselectionwastocreatethemostuniformplantstandpossible.
After seeding, the trays were sealed in 15 L white plastic bags, to control humidity to near
saturation,andgerminatedinagrowthchambershieldedfromlight,foreightyhours.Thegermination
chambersweremaintainedat24◦C.Wedeterminedthateightyhourswasthebesttimetofloatthe
flats,asthatwasthetimewhenmostplantshadrecentlyemergedbutminimalgrowthorstretching
hadoccurred. Theflatswere6.3cmthickby34.5cmwide×31cmlengthandcomprised132cellsin
an11by12matrix. Onlyplantsfromthecentral56doubly-guardedcellswereharvested(theouter
tworowswerenotused,whichleavesa7×8rowmatrix),wherethecelldensitywas1300cellsper
m2,adensityusedbycommercialgrowers.
Afterfloating,theplantsweregrowninthechannelsforanadditional14daysunderfulllighting.
Theseeding,floating,andgrowingprocedureswerethesameforalltrialsandtreatmentsexceptthe
increasedseedingaspreviouslydescribed. Trialsoverlappedwitheachotherintheirchannels,since
flatswereaddedevery3daysasanewtrial,soittookfivesuccessivetrialplacements(trials)before
thefirsttrialwasharvested;thustrial1and6werecompletelyindependentinallways,including
theirwaterenvironments.
2.4. StandCorrection
Anattemptwasmadetocontrolforthevariationingerminationamongflats. Theprocedure
entailedadjustingplantstandstoasetnumberofhealthy,equivalent,plantsintheinteriorofeachof
theflatsforeachparticulartrial. Thenumberwasdeterminedbytheworstgerminationofthesixflats
inagiventrialafterhavingremovedanydeformedorverylateemergingplants. Fromtheremaining
plants,additionalhealthyplantswereremovedsothateachtrialhadthesamenumberofplantsper
flat. Oncethenumberofplantstoremovefromeachflatwasdetermined,theseplantswererandomly
selectedandremovedblindlytoavoidbias.
Eventhoughtrialswerestandcorrectedtobethesamewithintrial,thereweresmalldifferences
between trials since each trial had a different number of healthy plants at the time of floating.
The number of plants varied from 44 to 51 out of the 56 potential guarded cells that were to be
harvestedfordataanalysis.
2.5. TheHydroponicSystems
The hydroponic channels were closed-loop recirculating water systems, with each channel
independentandunconnectedtoothers. Identicalcirculatingpumps(24L/min)wereusedinallsix
channelsandmixedthewaterinthechannelsatarateequaltoahydraulicretentiontime(HRT)of
8min. Thisflowratemaintainedgoodmixinginthechannelsandrootsappearedtobekeptunder
gentle movement, which we did to avoid possible problems of root stress previously mentioned
by Shete et al. [14]. A crucial part of the hydroponic channels was the nutrient solution reservoir
(madewithmodificationstoacoolerchest). Eachchannel’sflowwentthroughitsreservoir,inwhich
temperaturewascomputer-controlledbyactivationofcoolingcoilsinwhichwatercooledto7 ◦C
waspassedwhennecessary. Twohundredlitersofnutrientsolutionwereaddedtoeachchannelto
begintheexperiment. Nutrientsolutionwasaddedonanas-neededbasistoreplacewaterlossby
evaporationortranspiration. Foreachgiventrial,oneflatwasfloatedineachchannelatthesametime
tocommencethatparticulartrial.
2.6. TheAquaponicSystem
Theaquaponicsystemcontainedkoi(Cyprinuscarpio)separatelyhousedinanadjacentsectionof
theclimate-controlledgreenhouserange,asshowninFigure1. Koiwerechosenasthefishbecausethe
commoncarpisahardyfishandcantolerateawiderangeofwatertemperature,andkoiretainthat
resilience. Thefishtankwas1000Landhadbeenincontinuousoperationfortwoyears. Thesystem
continuallyrecirculatedwaterwithminimalwaterdischargeandusedabeadfiltertocaptureand
Horticulturae2017,3,32 7of16
mineralize solids. The return flow from the fish sump was split to go to the bead filter or the A7
reservoir(designatedasHinFigure1)fortheaquaponicchannels. Aseparatesumppumpinthe
A7reservoirreturnedwaterdirectlytothefishtank. Thebeadfilterflowwasreturnedtothefish
tankviaanexternalspraybartoprovideaeration. Thebeadfilterwasback-flushedonceperweek
withanetlossofwaterof100Ltoremoveretainedsolids,whichallowedsignificanttimeforsolids
mineralization. The flow to the aquaponics channels was the same 24 L/min per channel as the
hydroponicchannels.
The pH of the system was regulated daily to a pH of 7.0 by adding 1M K CO . Due to fish
2 3
nitrificationandfishrespiration,thepHwasstable,sotherewasnoneedforpHloweringchemicals
suchasacid. FishwerefedasyntheticmanufacturedKoifeed(BlackwaterCreek’sMaxGrowthKoi
Food“PremiumKoiandGoldfishFood”). Thefishwerehandfedtwicedailytoapparentsatiation
onweekdays(totalof90g),and60gonweekends(oncedaily),whichwasapproximately1.5%body
weightperdayonweekdays. Thekoiwereapproximately200–400ginsize(tankdensityof~7kg/m3)
andtheirbodyconditionfactorwasconsideredtypicalandindicativeofappropriatefeedinglevels.
Thelowerfeedinglevelonweekendswastoreduceworkrequirementsandreducepotentialsystem
problemsduringlow-staffperiodsontheweekend. SeeTable2fortheelementalanalysisofthefish
feedusedasdeterminedbytheCornellNutrientAnalysisLab(CNAL).Thefeedusedwasastandard
commercialdietprovidedbyareputablefeeddealerforornamentalkoi.
Table2.Elementalanalysisobtainedfromaciddigestionmethodofcommercialfeedthatwasusedfor
aquaponicsystem(MaxGrowthformulafromBlackwaterCreekFarm).
Element Content Units
TotalC 40 %
TotalN 6.3 %
TotalH 6.3 %
Ca 4.1 %
P 2.2 %
K 1.2 %
S 5623 mg/kg
Na 4137 mg/kg
Mg 3545 mg/kg
Fe 789 mg/kg
Al 283 mg/kg
Sr 269 mg/kg
Zn 226 mg/kg
Mn 88 mg/kg
B 28 mg/kg
Cu 16 mg/kg
Ba 15 mg/kg
As 4.2 mg/kg
Cr 1.7 mg/kg
V 1.2 mg/kg
Pb 1.1 mg/kg
Ni 1.0 mg/kg
Cd 0.7 mg/kg
Mo 0.5 mg/kg
Co 0.5 mg/kg
Ti 0.0 mg/kg
Theaquaponictreatmentchannelswerenotindependentwatersystemsfromoneanother. Water
frombothsystemscamefromandwasreturnedtothefishtank,andthesystemssharedareservoir
whichwascooledinthesamewayasthehydroponicchannels. Thefishtankwasseparatedfromthe
spinachchannelsbyaglassgreenhousepartitionwall,whichhadtheadvantageofallowingthefish
tanktobekeptatacolderairtemperature. Sincespinachrootzonetemperaturesneededtobekeptat
Horticulturae2017,3,32 8of16
18◦C,wemaintainedthefishwaternear20◦Candtherestofthechillingwasaccomplishedbythe
chillerpreviouslydescribed.
2.7. Measurements
AlkalinitywasmeasuredbytitratingtoanendpointofpH4.5using0.01M(0.02N)sulfuricacid.
Theresultingequilibriumalkalinitieswereapproximately20mg/LCaCO fortheH5treatmentsand
3
40mg/LCaCO forboththeH7treatmentandtheaquaponictreatment. Thealkalinitymeasurement
3
hadanaccuracyof±4mg/LasCaCO .
3
Electroconductivity(EC)wasmeasuredbetween1200to1500µS/cm. ECwasnotcontrolledin
the treatments, but due to the differences among treatments, slightly different EC ranges resulted.
Intheaquaponictreatment,theaverageECwas1400–1500µS/cm. IntheH5andH7treatments,the
averageECwas1200–1300µS/cmand1300–1400µS/cm,respectively. Typically,therewasverylow
variabilityinthenutrientsolutionECintheH5treatment. TheadditionalionsintheA7treatment
from the tapwater resulted in the EC stabilizing at the highest values. The adjustment of the pH
upwardintheH7treatmentfromitsnaturalstartingpointofpH5.8resultedintheH7treatment
havinghigherECvaluesthantheH5treatment.
2.8. Nutrients
Thehydroponictreatmentsreceivedacustom-madeinorganichydroponicfertilizer. Tosetupthe
hydroponicexperiment,eachofthefourhydroponicchannelswasfilledwith200Lofthemodified
SonneveldandStraverlettucesolution[7]. Tocreate200L,onelitereachoftwoconcentrates,known
asStockAandStockBbycommonpractioners,weremixedinsuccessively. Thenutrientscontained
ineachStocksolutionareshowninTable3(seeTable7forresultinginitialconcentrations).
Table3.NutrientcontentsofStocksolutions;seeTable7forresultinginitialconcentrations.
Stock A B
Nutrients Calciumnitrate(Ca(NO ) 3H O Potassiumnitrate(KNO )(67%ofK)
3 2 2 3
Chelatediron(Sprint330,Fe-DTPA) Epsomsalts(MgSO ·7H O)
4 2
Ammoniumnitrate(NH NO ) Manganesesulfate(MnSO ·1H O)
4 3 4 2
Potassiumnitrate(KNO )(33%ofK) Boricacid(H BO )
3 3 3
Ammoniummolybdate
(NH ) Mo O ·4H O)
4 6 7 24 2
Zincsulfate(ZnSO ·7H O)
4 2
Coppersulfate(CuSO ·5H O)
4 2
Potassiumsulfate(K SO )
2 4
Thetubswerefirstfilledwithwater,thenStockAandBconcentrateswereaddedona1:1ratio
sequentiallywhilevigorouslystirringbetweenadditions. AdditionalmodifiedSonneveldandStraver
solutionswerecreatedin200LquantitiesinbarrelsatthetwodifferentpH’s,usingROwaterandthen
usedforreplenishmentoflosttubwateroverthecourseofeachtrial.
Tosetuptheaquaponicexperiment,eachofthetwoaquaponicchannelswasfilledwith200L
offishwater,duringatimewhenthefishtankwasroutinelyreplenishedwithtapwater(toreplace
waterlostduringsolidsfiltercleaning). Theaquaponicchannelsrequiredtheadditionofiron,since
ironisarequiredelementforplantssoweaddedchelatedirontotheaquaponicsystemintheformof
Fe-DTPA(Sprint-330). Thechelatedironwasinaformwhichisnottakenupbyfishbutisavailableto
thespinach.
Uponcompletionoftheexperiment,tissuedataanalyseswereperformedbytheCornellNutrient
AnalysisLaboratoryusinghotplateaciddigestionplusICP-AESmetalanalysisandcombustionash
analysisforcarbonandnitrogen. Nutrientsolutionanalysiswasdoneimmediatelybeforeandafter
theentireexperiment. Onesamplewastakenimmediatelypriortotheadditionofthefirstplants,and
Horticulturae2017,3,32 9of16
theothersamplewastakenaftertrial6washarvested. Thenutrientsolutionanalysiswasalsorunby
theCornellNutrientAnalysisLaboratory. ElementalanalysisresultsareshowninTables5and6.
2.9. Harvest
Plantswereharvestedafter14daysofgrow-outinthechannels,atatypicalmarketablesizefor
baby-leafspinach. Duringharvest,flatsofplantswereremovedfromthechannelsoneatatimeand
takenoutofthegreenhouse. Plantswereharvestedatnightbecausetheyaregrowingveryfastinthe
day,sonightharvestingminimizedanyadvantageaparticularflatcouldhavereceivedbyitsorderof
harvest. Ifharvestedduringtheday,flatsharvestedlaterwouldhavearelativeyieldadvantage.
Eachflathad132cellsforpossibleplants,butthetwoouterperimeterrowsofplantswereableto
spreadoutbeyondtheirflatandthereforenotconsideredtypical(wouldreceivemorelight)andwere
thusdiscarded.Afterremovingthetwoperimeterrows,eachflathadtheplantsfrominterior/guarded
cellsforanalysis. Duringharvest,plantsweresnippedwherethestememergedfromthemedium,i.e.,
throughthehypocotyl,andthenumberofplantsperrowwerecounted. Allplantswithinthesame
rowweregrouped,andstemsandleaveswereseparatedineachrow. Finally,totalstemsandleaves
perrowwereweighedwithanaccuracyof0.01grams. Thesedatawasusedtocalculatebothtotalflat
massandaveragemassperplant.
Afterseparation,stemsandleaveswereplacedinlabeledbrownpaperbagsanddriedinovens
maintainedat70◦Cforthreedays. Weconsiderdryweighttobeapurerepresentationofbiomass
yield,sincewaterconcentrationcanmakeinterpretationdifficult. Wealsopresentfreshweightdataas
beingequallyimportantbecausefreshweightismarketableweight.
2.10. LeafSurfaceArea
Inordertodetermineandcalculatesurfacearea,leaveswerecutafterweighingandpressedflat
formeasurementwithaclearscratch-resistantacrylicpolycarbonateplexiglass. Eachrowwouldallfit
underneatha25cm×50cmpieceoftheplexiglass. Imagesofthepressedleaveswerethenusedto
determinetotalleafsurfacearea.
SurfaceareawasmeasuredusingthecomputersoftwarecalledImageJ.ImageJisanopen-source
imageprocessingprogram,withtheintentofbeingusedforscientificmultidimensionalimages. Touse
thesoftware,apicturewasloaded,andthebrightnessandhuethresholdoftheimagewaschanged
tothepointwhereonlygreencolorwasrecognized. Surfaceareaswereextractedusingareference
lengthwithinthepicture.
2.11. StatisticalAnalysis
Threetreatmentconditions(H5,H7,andA7)wereevaluatedbasedprimarilyuponfreshanddry
matterresponses.WecollecteddataforsixtrialsfromJanuary13toFebruary19,2016,correspondingto
atotaldaylengthincreaseof84min(9h19minto10h43min,ora15%increaseinnaturaldaylength).
Wefirstevaluatediftherewasatrialeffectonbiomassresponseacrossalltrialsbygroupingthethree
treatmentsforeachtrial,sinceresponsemayhavebeenimpactedbychangingdaylength. Wealso
evaluatedtrialeffectonindividualplantresponsesinceourstandcorrectionproceduregaveusthe
samenumberofplantsforeachflatbutresultedinadifferentnumberofplantsperflatbytrialandthe
naturaldaylenthincreasedforeachtrial. Afterevaluatingforbothpossiblesourcesofatrialeffect,
datawascombinedamongtrialstoprovideincreaseddegreesoffreedomforstatisticalanalysisof
treatmenteffects(n=84,from6trials,2channelspertreatment,and7rowsperflatpertreatment).
Aftercombiningdatabytreatmentacrosstrialsandpairedacrossreplicatedchannels,treatmentswere
comparedusingpairedt-tests. Forflatresponses(Table4c),thereare6flatsorresponsespertreatment
andtworeplicatedchannels,son=12.
Horticulturae2017,3,32 10of16
Table4.Averageplantfreshweight(a)anddryweight(b)dataforallchannelsandtrials,ingrams
perplant.Standarddeviations(SD)givenforaveragevaluesinparentheses.Totalflatbiomass(c)is
shownforfreshweightdata,ingrams. Channel1and2areaquaponics(A7),Channel3and4are
hydroponicsatpH7(H7),Channel5and6arehydroponicsatpH5.8(H5).Superscriptlettersidentify
thatsignificantdifferencesoccurbetweenvalueswithdifferentsuperscriptlettersforagivenbiomass.
(a) Channel
Average(SD)
Trial 1 2 3 4 5 6
1 3.01 3.09 3.21 3.18 3.16 3.11 3.13(0.07)
2 2.85 2.99 3.08 2.90 3.00 2.94 2.96(0.08)
3 2.76 3.23 2.92 2.99 2.62 2.87 2.90(0.21)
4 2.59 2.62 2.93 3.03 2.87 2.82 2.81(0.17)
5 2.96 2.86 3.10 3.07 2.96 3.02 2.99(0.09)
6 2.72 2.73 2.71 2.82 2.68 2.84 2.75(0.06)
Average 2.82b 2.92b 2.99a 3.00a 2.88b 2.93b
SD (0.16) (0.23) (0.18) (0.13) (0.20) (0.11)
(b) Channel
Average(SD)
Trial 1 2 3 4 5 6
1 0.152 0.157 0.161 0.163 0.163 0.161 0.159(0.004)
2 0.144 0.160 0.156 0.154 0.160 0.152 0.154(0.006)
3 0.162 0.170 0.156 0.160 0.147 0.154 0.158(0.008)
4 0.145 0.154 0.158 0.161 0.170 0.149 0.156(0.009)
5 0.159 0.156 0.163 0.154 0.165 0.157 0.159(0.004)
6 0.155 0.150 0.147 0.154 0.157 0.157 0.153(0.004)
Average 0.153a 0.158a 0.157a 0.158a 0.160a 0.155a
SD (0.007) (0.007) (0.006) (0.004) (0.008) (0.004)
(c) Channel
Average(SD)
Trial 1 2 3 4 5 6
1 108 111 110 118 109 115 111(3.8)
2 124 120 120 130 128 120 123(4.4)
3 121 117 116 120 115 100 114(7.5)
4 105 103 125 114 120 106 112(7.6)
5 126 141 140 143 130 144 137(6.2)
6 130 142 134 142 136 140 137(3.0)
Average 119b 122b 124a 127a 123b 120b
SD (11.9) (13.0) (11.2) (11.9) (10.3) (17.0)
3. Results
3.1. TrialEffect
We found no statistical difference in biomass responses for wet or dry weights within each
treatmentbetweentrials(p=0.29,n=84). Inspectionofthedataforplantsize(Table4a)showeda
trendofslightlydecreasingindividualplantsizeastrialsprogressed. Thismayhavebeenduetothe
increasingnaturallightaspartofafixedDLItarget,butitmayhavebeenalsorelatedtothenumberof
plantsperflatincreasingslightlyoverthetrialsconducted. WhiletheDLIwascontrolledtoaconstant
valueof17mol/m2/day,successivespinachtrialsreceivedanincreasingpercentageofnaturallight
foreachsuccessivetrial(seeTheGreenhouseSection2.1). Additionally,amuchlargernumberofplants
inanindividualflatwouldresultinsmallerindividualplants. Probablythemostimportantresponse
variableforcommercialinterestisthetotalfreshweight(biomass)producedfromanindividualflat
(Table4c). Herewealsosawnosignificanteffectwithineachtreatmentbetweentrials(p=0.25,n=12).
3.2. BiomassResults
Wefoundnostatisticaldifferencesinourbiomassresults,whichwerenormallydistributedas
shown in Tables 4 and 5, between the A7 and H5 treatments, (A7 vs. H5: p = 0.59 fresh weight,
p=0.42dryweight,n=84)evenwhentotalflatmasswasmeasured(Table4c). Similarly,leafsurface
arearesultswerenotdifferentbetweentreatments.