Duckweed as a future food - USIM Research Repository System

ContentslistsavailableatScienceDirect

Future Foods

journalhomepage:www.elsevier.com/locate/fufo

Duckweed as a future food: Evidence from metabolite profile, nutritional and microbial analyses

Nazariyah Yahaya

^∗

, Nabila Huda Hamdan , Atiqah Ruqayyah Zabidi , Ammar Mirza Mohamad , Mohammad Luqman Hakim Suhaimi, Muhammad Azhan Azfar Md Johari, Hanis Nadia Yahya, Hafiza Yahya

Food Biotechnology Programme, Faculty of Science and Technology, Universiti Sains Islam Malaysia (USIM), 71800 Bandar Baru Nilai, Negeri Sembilan, Malaysia

a r t i c le i n f o

Keywords:

Duckweed

GC-MS-based metabolomics Lemna minor

Wolffia globosa

a b s t r a ct

Duckweedspeciesarenutritionallymeaninglessplantwhichgrowwildlyinunattendedareas.Therefore,un- derstandingthemetabolitescontentinduckweedspeciesisessentialfordesigningafuturefoodproducts.Here, wereportanuntargetedGasChromatography-MassSpectrometry(GC-MS)-basedmetabolomicsapproachfor comprehensivelydiscriminatingbetweenLemnaminorandWolffiaglobosaofduckweedsspeciesusingprincipal componentanalysis(PCA)andpartialleastsquares-discriminantanalysis(PLS-DA).Tendifferentialmetabolites levelsweretentativelyidentifiedbetweenL.minorandW.globosa.RelativetoW.globosa,L.minorappearedto enrichwith5-Hydroxyl-L-tryptophan,Tocopherylacetate,Naringenin,𝛼-linolenicacidandglutamicacid.Fur- thermore,thenutritionalandmicrobialanalysesoficecreamformulatedwithdriedL.minorwereinvestigated.

Thenutritionalanalysisresultsshowthatrelativetocontrol,theicecreamwith2%driedL.minorhadsignif- icantlyincreasedprotein,fiberandashcontent.Inaddition,totalplatecount(TPC)formicrobialanalysisof duckweedicecreamwasperformed.Theresultsuggestedthatthesmallamountofbacteria(3.82cfu/g)was tracedinformulatedicecreamwith2%driedL.minor.Overall,themetabolitesprofile,nutritionalandmicrobial analysesoffoodusedL.minorplantindicatethatduckweedisagoodcandidateforfuturefood.

1. Introduction

Duckweedcanbeconsideredasaplant-basedingredientforfuture foodproductsandasustainablealternativesourceofproteinthathas thepotentialofreplacinganimalmeat,healthierandmoreaffordable protein(Appenrothetal.,2018).Duckweedisanaquaticgreenplant, tinyandknownasfloatingfreelyplantsthatcancommonlybefound in slowly-moving bodies of water like lakes andponds (Van Hoeck etal.2015; Sreeetal.2016).Itis thesmallestfloweringplantglob- allyandconsistsoflessthanthreeleaveswithasingleroothangingin thewater(Sreeetal.2016).However,leavesofduckweedcanbemul- tipliedbyvegetativemethodtoresultina50%increaseoftheyieldper day.

Duckweednutrientcontentandmetabolitecompositionhavegained extensiveattention,particularlyintheanimalfeedindustry,aquacul- ture,healthsupplement,biofertilizer,biofuelandemergingfoodprod- ucts for humans (Anthonius et al. 2018; deBeukelaar et al. 2019; Naseem et al. 2020). It has been reported that duckweed contains 20% to30% of protein,which is higher thancereal (deBeukelaar

∗Correspondingauthorat:FoodBiotechnologyProgramme,FacultyofScienceandTechnology,UniversitiSainsIslamMalaysia(USIM),71800BandarBaruNilai, NegeriSembilan,Malaysia.

E-mailaddress:nazariyah@usim.edu.my(N.Yahaya).

etal.2019).Themainproteininduckweedisribulose-1,5-bisphosphate carboxylase(RuBisCO),whichisagoodsourceofessentialaminoacids.

Moreover, RuBisCOprotein isa goodcandidateasafunctionalfood due to its nutritional value, in vitro digestibility and non-allergenic (Chakrabartietal.2018).Inaddition,RuBisCOproteinhasgoodgelling andemulsification properties,highfoamingcapacity, andhighsolu- bility(Di Stefanoetal.2018).Duckweedalsocontains4%to7%of fat,4%to10%ofstarch,carotenoidsandpolyphenolslikeflavonoids andanthocyanins (Appenroth etal.,2018).Thefat contentin duck- weedconsistsofpolyunsaturatedfattyacids,60%to63%oftotalfatty acidsand41%to47%of𝛼-linolenicacid(Chakrabartietal.2018).The starchofduckweedcomprises35.7%amyloseand26.5%amylopectin (Leeetal.2016).

Lemnaminor,oneoftheduckweedsspeciesinthefamilyLemnaceae, showsellipticalform,regularspeciessizewith1.7mmdiameteronav- erage,2-4frondscoloniesanddevelopedrootssystem,whichwasde- scribedextensivelybySaveriandFornasiero(1983).L.minorusesboth rootsandfrondsfortakingupnutrients(CedergreenandMadsen,2002), whileWolffiaglobosa,whichisarootlessplant,usesthefrondssurfaceto

https://doi.org/10.1016/j.fufo.2022.100128

Received22December2021;Receivedinrevisedform3February2022;Accepted7February2022

2666-8335/© 2022TheAuthor(s).PublishedbyElsevierB.V.ThisisanopenaccessarticleundertheCCBY-NC-NDlicense (http://creativecommons.org/licenses/by-nc-nd/4.0/)

takeupthenutrientfromthegrowthmedium.L.minorandLemnagibba arecommonlyusedforresearch(Appenrothetal.,2018;Xieetal.2019; Alkiminetal.2019),whereasWolffiellaandWolffia,whichgrowfast andhashighstarchcontent,aremoreusefulinrenewablebiorefinery feedstock(Anetal.2018).Additionally,Wolffiahasbeenrecognized asarichsourceof proteinandistraditionallyemployedasanatural foodsourceinSoutheastAsia.TherearemanysubspeciesintheWolf- fiagenus,andthemostcommononeisknownasWolffiaglobosa. W.

globosaisfamiliaraswatermealorKhaiNaminThailand.RawWolffia isutilizedtomakecertainmealssuchassalads,omelettesorvegetable curries.

Duckweedfullydepends onoptimumtemperaturerangebetween 17.5 and 30°C and sunlight to grow. It also requires nitrogen, phosphorus and potassium as supplemental nutrients (Hasan and Chakrabarti,2009).However,sinceduckweedis aphytoremediation plant that extracts metals, radionuclides and other pollutants from wastewaterandaccumulatesthoseintheirtissues,thesafetyofcon- sumingthisaquaticplanthasbecomeaconcern(Landesmanetal.2005; Kamyabetal.2017;Radulovicetal.2019).Inaddition,ahighamount ofpathogenssuchasEscherichiacoli,Clostridiumbotulinum,Salmonella spp.,fungiandmicroscopicinvertebratecouldcontaminateduckweed collectedfromwastewateroropengreenhousepools(Moyoetal.2003; Islametal.2004;Ansaetal.2012;Markouetal.2018).Therefore,to produceduckweedasafoodsourceforhumans,itmustbegrownina sustainableenvironmentsuchasaxenicculture,whichcouldproduce anabundantamountofduckweedundercontrolenvironmentwithlow bacteriaabundance andlesscontaminant.However,tomass-produce duckweedforfoodproduction,thelaboratorysetisextensivelyexpen- sive.Thus,anopen-airsystemwithdirectsunlightandminimumnu- trientusageisrequiredtocommercializeduckweedasafuturefoodto low-incomecommunities.

Inthisstudy,weinitiallyinvestigatedmetabolitecontentsinL.minor andW.globosabyusingGC-MS.Thedatawasintegratedwithprinciple componentanalysis(PCA)andpartialleastsquares–discriminantanal- ysis(PLS-DA).Wealsoinvestigatedprotein,fat,fiber,ashandmoisture contentinbothduckweedspeciesandtheirminimumrequirementfor growth.Asapartofthefoodproductdevelopment,wefocusedonL.

minorbyinvestigatingthenutritionalcompositionandmicrobialanal- ysisofduckweedicecream,whichwererelevantforhumanconsump- tionandsafety.Hence,ourstudywastorevealduckweedasapotential futurefoodintheaspectsofmetaboliteprofile,nutritionalvaluesand microbialanalysisinduckweeddevelopedfoodproduct.

2. Materialsandmethods

2.1. Biologicalmaterialsandgrowthcondition

Twospeciesofduckweed,L.minorandW.globosa,wereusedinthis experiment.InitialmaterialofL.minorandW.globosawerepurchased fromMegauppyAquaticSupplies,MalaysiaandYurenAquaticSdn.Bhd.

Store,Malaysia,respectively.

Bothduckweedspeciesweremaintainedin300mLofwaterwith500 gmultipurposecompostand300mLNitrogen:Phosphate:Potassium (N:P:K)solutionbymixing1.52gN:P:K(12:12;17,BajaTaiping) pelletinto1litreofdistilledwaterin theoutsideenvironmentunder shieldwithtemperaturearound29°Cday/12hoursand24°Catnight/12 hours.

Forlarge scalepreparation of duckweed growthtodevelopfood products,duckweedwasgrownina70Lcontainerwithsurfacearea 55cmX88cm,undershieldwith1kgofmultipurposecompostand5L ofN:P:Kfertilizer(perweek)insimilarenvironmentmentionedabove.

Duckweedwasharvestedeverytwoweeks.

2.2. MetaboliteextractionandGC-MS

MetaboliteextractionofL.minorandW.globosawasconductedac- cordingtothemethoddescribedbyGonzalez-Penaetal.(2017)with modification.Twoduckweedspeciesweredriedintheovendryer(65°C for24hours)andgroundintofinepowder.Inthisexperiment,100mg ofdriedduckweedpowderwasmixedwith1mLofmethanolforeach replicate.Themixturewasvortexedfor10s,leftonicefor10minand latercentrifugedat17000gfor2minatroomtemperature.Thesuper- natantwascollectedina1.5mLcentrifugedtubeandstoredat80°Cfor GC-MSanalysis.InGC-MSanalysis,threereplicateswereperformedfor eachduckweedspecies.

Followingmetaboliteextraction,sampleswereanalyzedusingAgi- lentJandWGasChromatographyColumnwithTripleQuadrupolemass spectrometryoperatedat70eV.Analiquotofa1.0𝜇lsamplewasin- jectedintotheDB-35MS(30mlengthx0.250mmdiameterx0.25𝜇m filmthickness)column.Heliumwasusedasacarriergas,andthescan rangewassetto40to500Da.Theinitialoventemperaturewassetto 60°Cfor10minandwasincreasedby40°C/minto280°C,thenheld for10minandincreasedby40°C/minuntil310°C.Bothinjectorand transfertemperaturesweresetto250°Cwhilethesourcetemperature wasadjustedto300°C.Thefullscanrangewasacquiredafter30min withasplitratioof10:1(Carryetal.2018).Eachrecordedspectrum wascomparedwiththestandardmassspectralibraryoftheNational InstituteofStandardsandTechnology(NIST).

2.3. Metaboliteprofilingbymultivariateanalysis

The raw data from GC-MS was converted to Microsoft Excel in .csv format and processed by the MetaboAnalyst 4.0 website (https://www.metaboanalyst.ca).Adata integritycheck andnormal- izationbysum,logtransformationanddatascalingandmeancentring wereperformedbeforebeingsubjectedtoprincipalcomponentanalysis (PCA)andpartialleastsquare-discriminantanalysis(PLS-DA).Finally, predictiveability(Q²)andgoodness-of-fit(R²)parametersassessedthe qualityoftheresultantPLS-DAmodels.

2.4. Nutritionalanalysisofrawmaterialsandfoodproduct

Thenutritionalanalysiswasdeterminedbymeasuringtheproximate contentofbothduckweedspeciesaccordingtotheAssociationofOffi- cialAnalyticalChemists(AOAC,1985)methods.Amongtheparameters measuredwereproteincontent,fatcontent,fibercontent,ashcontent andmoisturecontent.Allanalysisofproximatecompositionwasper- formedwiththreesamplesforeachtypeofanalysis.

ProteinanalysiswascarriedoutusingtheKjeldahlmethod,which isbasedonthedeterminationofnitrogencontent.Theprocedurecan bedividedintothreesteps,digestion,distillationandtitration.Atthe endoftheprocess,thepercentageofNitrogenpresentinthesamplewas calculatedusingthefollowingequation1.

%𝑁𝑖𝑡𝑟𝑜𝑔𝑒𝑛= 1.4×𝑐× (𝑉 −𝑉𝑏)

𝑆𝑎𝑚𝑝𝑙𝑒𝑤𝑒𝑖𝑔ℎ𝑡(𝑔), (1)

Wherec:Concentrationofthestandard-acidsolution:Hydrochloric acid0.1Norc=0.1mol/L;Alternative:sulfuricacid0.1Norc=0.05 mol/L,whichN=Normalityofstandardacid.

V:Consumptionofthestandardacidinml(Sample) Vb:Consumptionofthestandardacidinml(BlankSample) EquivalentweightofNH₃is17g=14gNitrogen.Factor1.4iscal- culatedfromthe14gofNitrogendividedby1000mlof1Nacidand multiplywith100.

The percentage of protein (% raw protein) in the sample need to calculate by multiply percentage of N which calculated from equation1with6.25(%N^∗6.25).Conversionfactor6.25isequiva- lentto0.16gNitrogenpergramofprotein.

Followingproteinanalysis,fatcontentwasdeterminedusingtheau- tomatedSoxthermmethod.Initially,2gofdriedsamplewasweighedon

crudefilterpaperandplacedintoextractionthimbleinthebeakerwith 3-5boilingstones.Next,130mLofpetroleumetherwasaddedintothe beakeraheadoftheextractionofthesampleinautomatedSoxtherm.

Aftertheextraction,thebeakerscontainingtheexudatearedriedinthe dryingovenforanhourat105°C.Then,theextractionbeakerswere transferredtoadesiccatortocooldown.Finally,theextractedsamples wereweighed.Thesamplewaslefttodryforanother30minutesbefore weighedagaintodeterminetheconsistencyoftheweight.Attheendof theprocess,thetotalfatcontentinthesamplewascalculatedusingthe followingequation2.

𝑤= 𝑚2−𝑚1× 100 𝑚0

, (2)

Where

m₁:Massoftheemptyextractionbeakerwithboilingstonesing m₂:Massoftheextractionbeakerwithfatafterdryinging m₀:Weightofthedriedduckweedsampleatthestartoftheanalysis ing

Thecrude fiberwas extracted andanalyzed using theFiber Bag method.Inthisanalysis,1gofsamplewasputintotheFiberBag,andthe masswasweighedwith1mgpreciseness.Afterwards,theglassspencer wasinsertedintoFiberBag,andthenthebagwasaddedtothecarousel.

Next,theanalysiswascontinuedbyde-fattingthesamplebyimmersing thecarouselthreetimesinarowinto100mlof40/60petroleumether.

Shortlyafter,thedrainedFiberBagwastakenoutandtransferredinto thecrucible.Next,thecruciblewasplacedinthedryingovenovernight at105°C.Finally,thecrucibleandresidueleftweretransferredintoa desiccatortoletitcoolbeforeascertainingthefinalweightbyusing equation3.

%𝐶𝑟𝑢𝑑𝑒𝐹𝑖𝑏𝑒𝑟=((𝐶−𝐴)−(𝐷−𝐸))

𝐵 × 100 (3)

BlankValueE=D– F A=MassFiberBaging

B=MassSampleweighting(hastobeadjustedaccordingtodry content)

C=MassCrucibleanddriedFiberBagafterdigestioning D=MassCrucibleandAshing

E=BlankValueoftheemptyFiberBaging F=MassCrucibleing

Ashcontentwasmeasuredbytaking3gofsampleandburnedonthe electrichotplateuntiltheresiduehadceasedsmoking.Later,thecru- ciblewasignitedat550°Covernightinthemufflefurnace.Thesample wastransferredintothedesiccatorbeforemeasuringthefinalweight andrecordedusingequation4.

%𝑎𝑠ℎ𝑐𝑜𝑛𝑡𝑒𝑛𝑡= 𝑤𝑒𝑖𝑔ℎ𝑡𝑎𝑓𝑡𝑒𝑟𝑎𝑠ℎ𝑖𝑛𝑔(𝑔)

𝑤𝑒𝑖𝑔ℎ𝑡𝑏𝑒𝑓𝑜𝑟𝑒𝑎𝑠ℎ𝑖𝑛𝑔(𝑔)× 100 (4) Todeterminemoisturecontent,1gofsamplewasplacedonthepan surfaceinthemoistureanalyzerandletrunforafewminutesbeforeit shutoff automatically,whichsignalledthatthemeasurementwascom- pleted.Then,thedatawastakenandrecordedfromthereadingofthe moistureanalyzer.

2.5. Duckweedicecreamformulationanddevelopment

Theduckweedwas driedusingoven dryingat65°Cfor 24hours andgroundusingadryblender.Thepowderthenwassievedusinga strainertoobtainafinerpowder.Controlicecreamandduckweedice creamwithspecificformulationswereprepared.Forcontrol,theformu- lationwas75%offullcreammilk,9%non-fatmilksolids,5%butter, 10%Castorsugar,1%ovaletteand0.4%vanillaessence.Induckweed icecream,2%ofduckweedpowderwasaddedintothecontrolformula- tion.First,fullcreammilkwasheatedat55–56°Cfor5min.Afterthat, butterandovaletteweremeltedtogetherinthepot.Then,allingredi- entswerecombinedtogetherintoastainless-steelcontainerandmixed usingahandmixerataspeedof3for2min.Themixturewasstirred

for15minunderafreezingtemperatureturningintosofticecream.All theicecreamswerestoredinafreezerat-20°C.Thenutritionalanal- ysis oftheicecream wasconductedusingthemethod mentionedin section2.4.Theproteincontent,fatcontent,fibercontent,ashcontent andmoisturecontentwereanalyzedevery10dayswithin30dayswith threereplicates.

2.6. Microbialanalysisonformulatedicecream

Forthemicrobialanalysisoftheformulatedicecream(withoutduck- weedand2%driedduckweed),thetotalplatecount(TPC)methodwas conductedbymixingthesampleintopeptonewaterinaratioof1:7 usingastomacherbag.Thedilutedmixturewithafactor10⁻¹to10⁻⁶ waspreparedbyadding1mlof themixturefromthestomacherbag into9mlofpeptonewaterinthefirsttube,vortexandlabeleditasthe solutionwithdilutionfactor10⁻¹.Then,1mlofthemixturefromdilu- tionfactor10⁻¹wastransferredintothesecondtube(10⁻²dilutionfac- tor)andmixedhomogeneouslyusingavortex.Thestepswererepeated until10⁻⁶dilutionfactor.Afterallofthesamplesweremixedhomoge- neouslyusingavortex,0.1mLofsampleculturefromdilutionfactor 10⁻¹to10⁻⁶weredispensedontothesurfaceofTotalPlateCountagar (TPC)andspreadthesampleevenlyusingthesterilespreader.Theagar plateswereinvertedandincubatedat35°Cfor24hours.Thenumberof thebacteriagrowthontheplatewascalculatedusingequation5.The calculatednumberofcolonieswasnormalizedbylog₁₀transformation.

𝐶𝑜𝑢𝑛𝑡(𝐶𝐹𝑈∕𝑔)= 𝑎𝑣𝑒𝑟𝑎𝑔𝑒𝑛𝑢𝑚𝑏𝑒𝑟𝑜𝑓 𝑐𝑜𝑙𝑜𝑛𝑖𝑒𝑠𝑓𝑟𝑜𝑚𝑝𝑙𝑎𝑡𝑒𝑠

𝑣𝑜𝑙𝑢𝑚𝑒𝑝𝑙𝑎𝑡𝑒𝑠𝑋𝑑𝑖𝑙𝑢𝑡𝑖𝑜𝑛𝑓𝑎𝑐𝑡𝑜𝑟 (5) 3. Results

3.1. OverviewofmetabolitescontentinL.minorandW.globosa

Twogeneraofduckweeds(LemnaminorandWolffiaglobosa)were investigatedconcerningthecontentoftheirmetabolitesbyusingGC- MS.GC-MSprofilingoftheL.minorandW.globosarevealedatotalof 67and74identifiedpeaks,respectively.Ninechemicalcompoundsin L.minorand13chemicalcompoundsinW.globosawithapeakareaof morethan1%arepresentedinTable1.Themajorcompoundspresent in L.minorandW.globosa withthehighestpercentageof peakarea included𝛾-Sitosterol,StigmasterolandCampesterol.

3.2. MetabolitesdifferencesbetweenL.minorandW.globosa

ThemorphologicalleveldifferencebetweenL.minor(Fig.1a)andW.

globosa(Fig.1b)couldreflectthemetabolitesprofileinbothduckweed species. Themorphologyoftwoduckweedgenus wasrecordedusing microscopicanalysis.MicroscopicimageofL.minorshowedsuborbicu- lartoelliptic-obovateintheoutlineoffrondwhichnearlysymmetrical, whereasW.globosamicroscopicimageshowedovalinoutline(Fig.1a).

ComparedtoL.minor,W.globosaisatinyfloatingrootlessplantwhose sizeislessthan1mminanydimension.

Next,thevariationwithinthemetabolicprofilescorrespondstotwo duckweedgenususingprincipalcomponentanalysis(PCA)(Fig.1c)and partialleastsquares–discriminantanalysis(PLS-DA)(Fig.1d).Theanal- ysiswasperformedonthemetabolitesdatasetofL.minorandW.globosa frommethanolicextractsoftheirfronds.TheoutcomesofPCAclearly discriminatetheL.minorandW.globosaplantswhichindicatethatthese twogenerahavedifferentcomponentprofiles(Fig.1c).Theprofileof L.minorclusteredin aPC1-negativedirection,whileW.globosaclus- teredinaPC1-positivedirection.PCAanalysisshowedbothPC1and PC2accountedfor77.4%ofthetotalaccumulativevariance.

ThenthemetabolitesdatasetwassubjectedtoPLS-DA.PLS-DAis asupervisedmethod,whichusedtobuildmodelsthatdiscriminatebe- tweendata.Fig.1dshowsPLS-DAmodels(Component2plottedagainst Component1)ontherecordeddataofGC-MSanalysis.InPLS-DA,Com- ponent1discriminatesL.minorandW.globosa;theprofileoftheformer

Table1

MetabolitescontentinL.minorandW.globosadetectedbyusingGC-MS.

Name of the Compound Retention Time (RT) Mass (DB) Peak Area (%) L. minor

Neophytadiene 15.904 278.3 1.453

9,12,15-Octadecatrienoic acid, methyl ester, (Z,Z,Z)- 18.509 292.2 1.509

Phytol 18.621 296.3 6.800

Squalene 24.297 410.0 2.674

𝛼-Tocopheryl acetate 26.694 472.4 6.372

Campesterol 27.782 400.4 9.137

Stigmasterol 28.149 412.4 25.260

𝛾-Sitosterol 28.775 414.4 10.049

W. globosa

Neophytadiene 15.904 278.3 2.418

Hexadecanoic acid 17.309 256 1.215

Linolenate 18.506 292 1.362

Phytol 18.618 296.3 3.881

9,12-Octadecadienoic acid (Z,Z)- 19.043 280.2 1.280

Squalene 24.300 410.0 6.584

𝛼-Tocopheryl acetate 26.688 472.4 5.006

Campesterol 27.22 400.4 11.059

Stigmasterol 28.116 412.4 15.505

𝛾-Sitosterol 28.809 414.4 22.535

𝛽-Amyrin 29.283 426.4 3.436

Epiligulyl oxide 29.847 230 6.934

Fig. 1.Morphology of (a)L. minor and(b) W.globasaundermicroscopeobservationand scoreplotofbetweenselectedprinciplecompo- nentsobtainedfromGC-MSdataseton(c)PCA and(d)PLS-DAfromthreebiologicalreplicates ofduckweedplantsforeachspecies.

Fig.2. ImportantfeatureswithVariableImportantinProjec- tion(VIP)scoresbetween1and3wereobtainedfromapplying theGC-MSdatasetonPLS-DA.Thecoloredboxesontheright indicatetherelativeintensityofthecorrespondingmetabolites betweenL.minor(boxattheleft)andW.globosa(boxatthe right).

Fig.3.ThegrowthrateofduckweedspeciesL.minorinM3 Compost,DistilledwaterandN:P:Kfertilizer.

plantclusteredinanegativedirection,whilethelatterplantclustered inapositivedirection(Fig.1c).

Furthermore, followingthe PLS-DA, metabolites pattern foreach metabolitewithvariableimportanceinprojection(VIP)scoresbetween 1and3forL.minorandW.globosawereshowninFig.2.Tendiffer- entialmetaboliteslevelsweretentativelyidentifiedbetweenL.minor andW.globosa.RelativetoW.globosa,L.minorappearedtoenrichwith 5-Hydroxyl-L-tryptophan,𝛼-Tocopherylacetate,Naringenin,𝛼-linolenic acidandglutamicacid(Fig.2).

3.3. NutritionalcomponentinL.minorandW.globosa

Initially,theobservationofL.minorgrowthinmultipurposecom- post,N:P:Kfertilizeranddistilledwaterwereconductedbycounting thefrondnumber.EarlyobservationshowedthatN:P:Kmediumwas thebestproliferativemediathathadproducedahighamountofduck- weed.However,multipurposecompostmediumproliferated ahigher amountofduckweedinthelong-term,whiledistilledwaterseemedto achievetheequilibriumphase(Fig.3)nearly.Inthisstudy,bycombin- ingmultipurposecompostandN:P:Kmediuminthecontainerwith surfacearea55cmX88cm,undertheshield,estimatearound17.25g

ofwetweight,whichisequivalentto12.5gofdryweightand3gof proteinfromL.minorwasobtainedwithintwoweeks(Fig.4).

TofollowthemetabolitescontentinL.minorandW.globosa,thenu- tritionalqualitywasincludedinthisinvestigation.Theproteincontent ofthetwoduckweedspecieswasbetweenapproximately26.38%and 29.91%.FatcontentwasundetectableinL.minor,andapproximately 1.25%wasdetectedinW.globosa.RelativetoL.minor,fiberandmois- turecontentsinW.globasaislow,whichmakeitdigestedeasilywhen itisintegratedintoanimalfeed.Incontrast,ashcontentissignificantly higherinW.globasawhencomparedwithL.minor(Fig.4).

3.4. NutritionalcomponentsandmicrobialanalysisinL.minorandits foodproduct

Weused2%ofdriedL.minorasanadditionalingredientinicecream formulationtofurtherourstudy.Nutritionalanalysisofformulatedice creamwithdriedL.minorwascomparedwithicecreamwithoutL.minor within30days.Theresultofthenutritionalanalysisshowsthatrelative totheicecreamwithoutdriedL.minor(0%),theicewith2%driedL.

minorhadasignificantincreaseofprotein,fiberandashcontent,and fatcontentwassignificantlyreducedthroughouttheentireexperiment (Fig.5).Itindicatesthaticecreamcontaining2%driedL.minorhasa

Fig.4. Determinationprotein,fat,fibre,ashandmoisturein twospeciesofduckweed(L.minorandW.globosa).Alldata aremean±standarddeviations(n=3).^∗showsasignificant difference(P<0.05)betweentwosamples.

Fig.5. Determination(a)protein,(b)fat,(c)fiber,(d)moistureand€ashinicecreamformulatedwith0%and2%ofdriedL.minor,while(f)isthetotalnutrient in1gofdriedL.minor.Alldataaremean±standarddeviations(n=3).^∗showsasignificantdifference(P<0.05)betweentwosamples.

Table2

Totalplatecount(TPC)(cfu/g)inicecreamformulatedwithoutdriedduckweed (Control)andicecreamformulatedwith2%ofdriedduckweed.Thedatashows meanofcolonieswasnormalizedbylog₁₀transformation.

Time Point Week 1 Week 2 Week 3 Week 4

Samples Mean Log 10(cfu/g)

Ice cream (0% dried duckweed) < 0 < 0 < 0 < 0 Ice cream (2% dried duckweed) 3.82 3.70 3.82 3.82 Note:DescriptivestatisticsofTPC-MeanLog₁₀.<0showsnocolonieswasdetectedat cfug⁻¹as<1timesatdilutionfactor10⁻⁵.TheTPCwasconductedeverysevendays withinfourweekswithadilutionfactorof10^−5,andthetotalvolumeoftheplate was10mL.

highproteincontentwithessentialaminoacidsandlowfat,whichare suitableforhumannutritionalneeds.

Thetotalplatecount(TPC)isatraditionalmethodusedtoassessthe qualityandlevelofhygieneofformulatedicecreamwiththepresence ofdriedL.minor.Ahighlevelof bacteriaindicatesa generallypoor conditioninthegrowingandcleaningofduckweed.Inthisstudy,the smallamountofbacteriawastracedinformulatedicecreamwith2%

ofdriedL.minor(Table2).

4. Discussion

Inthis study, the detectedmetabolites are mostly related tothe secondarymetabolismpathwayandcreatedbytheenzymaticprocess in plantsystems.Forinstance,𝛾-sitosterol,stigmasterol andcampes- terol,whichweredetectedinGC-MSin bothduckweedsspecies,are typesofphytosterolthatnaturallyoccurredinplantleaves.𝛾-Sitosterol isbelieved tohaveantidiabeticproperties(Balamuruganetal.2011; Tripathietal.2013),whereasstigmasterol actsasaprecursorinthe synthesisofprogesteroneandisalsoknownasanintermediateprod- uct inbiosynthesisof vitaminD3, estrogen,androgenandcorticoids (Chaudharyetal.2011;Gabayetal.2010).Estimatearound200-400mg ofsitosterolandcampesterolhavebeenconsumedinthedailywestern diet,andtheintestineabsorptionofcampesterolishigherthansitosterol (Behrman andGopalan, 2005).However,the differences in metabo- liteprofilebetweenL.minorandW.globosaarenotwellunderstood.

Ithasonlybeenreportedthatdifferentduckweedspeciesareinvolved intheapplicationofwastewatertreatmentsystemsandtoxicologytests (Zhaoetal.,2014;Alkiminetal.2019).

Intermsofduckweedgrowthconditions,ithasbeenreportedthat witha smalltrace ofNandPandhightemperature, duckweed can growrapidly.However,theplantwilldieifthewatertemperaturerises to35°C(HasanandChakrabarti,2009).Inaddition,recent research showedthatN:P:Kfertilizerenrichedwithmolassesandtraceelements wasthemostpromisingmediumofstimulatingduckweedgrowthand, inthemeantime,suppressingmicroalgaegrowth(Satyaetal.2020).On thecontrary,whenL.minorisgrowninahighconcentrationofammo- nium(NH₄⁺),itshowstoxicitysymptomssuchasgrowthinhibition,cell deathinductionandtheaccumulationofreactiveoxygenspecies(ROS) (Wang etal.2016).Ingeneral, theplant requiresNtocreateamino acidsforgrowthanddevelopment,whilePisimportantinenergystor- ageandtransfer,andKplaysaroleasanenzymeactivatortopromote metabolism(Uchida,2000).Ncanbeobtainedeitherinorganicssuch asfromanimalmanureslikefishandpoultryorinorganicform.Inte- grationduckweedgrowthwithfishesinonepondorusedwastewater fromtheagricultureindustrycouldreducetheusageoftheinorganic formofN(Landesmanetal.2005;Yaoetal.2017;Kamyabetal.2017; HossainandAlam,2020).Fortherapidgrowthofduckweed,asmall quantityofPisessentialafterN,andthesensitivityofduckweedtoPor Kisdecreasedafteritreachestheadequatethreshold.Thesmalltraceof mineralssuchasK,Calcium(Ca),Magnesium(Mg),Sodium(Na),Chlo- rine(Cl),Sulfate(S)andBicarbonate(HCO₃)inwaterareessentialfor supportingthesurvivalofduckweed(HasanandChakrabarti,2009).

In general, duckweed contains about 20-35% protein of its dry weight(Appenrothetal.,2018;Herawatietal.2020)which aligned withourfindings.Leucine,isoleucineandvalineconstitutedofmajor- ityessentialaminoacidsinduckweedspecieswhicharerequiredinan- imalfeeds(Chakrabartietal.2018).Thisevidencealsoconcludedthat thepotentialityofusingduckweedasanimalfeedswasobviousdueto therichcrudeproteincontentandrapidaccumulationofbiomassofthis genusincomparisontootherterrestrialplants.Ithasbeenreportedthat duckweedcontainsapproximately1-5%fatofitsdryweightanddom- inantlybypolyunsaturatedfats(PUFA),which𝛼-linolenicacidmainly andfollowedbylinoleicacid(Chakrabartietal.2018).Despitethefact thatfatcompositioninduckweedismainlylow,thevalueoffattyacid profileinL.minorandW.globosashouldbesufficienttomeetthehuman diet’sdemand(Appenrothetal.,2018).

Inthisstudy,squaleneand𝛼-Tocopherylacetate(Table1),which have antioxidant activity, were detectedin L. minor andW. globosa metabolitesprofiles.However,thesqualenecompoundis threetimes higherin W.globosathanL.minor.Squaleneisacompoundthathas anantioxidantactivitytoprotectthecellfromoxidativedamageand inhibittumorgrowthinhumantissue.Thehumanbodycouldalsobe secreted0.3-0.5mg/gofsqualenebythesebaceousglandsforskinpro- tection(Lozano-Grandeetal.2018).Ithasbeenreportedthatfreshvir- ginoliveoil,whichisapartofthehumandiet,containsahighamount ofsqualeneandaremorestableafterthefirstfryingprocesswhencom- paredwithotherplantoilsuchassunfloweroil,cottonseedoil,cornoil, etc.(KalogeropoulosandAndrikopoulos,2004;Tsimidouetal.,2010).

Metaboliteidentifiedas𝛼-Tocopherylacetate,whichwasrelatively higherinL.minorthanW.globosa,isknownasaprimaryformofvitamin E,whichisnaturallypresentinthemembraneofgreenphotosynthetic leavesandiscommonlypresentinthemembraneusedasafoodaddi- tive.Inplant,𝛼-Tocopherylacetate,synthesizedinthechloroplast,plays aroleinstresstoleranceandphoto-protectiveandinducesplantresis- tanceagainstabioticstressessuchasmetaltoxicity(Sadiqetal.2019).

Apreviousstudyshowedthat𝛼-Tocopherylacetatewasincreasedafter duckweedwasexposedtozincandcadmium(Artetxeetal.2002).In human,𝛼-Tocopherylactsasanantioxidantagainstfreeradicalsanden- hancehumanimmunefunction(PekmezciandLitwack,2011).Thepres- enceofchemicalspropertiesthathaveantioxidantactivityinduckweeds plantsshowsthisplantcanbebeneficialtohumanhealth.Therefore,it canbeapartoffoodsupplementformulationsandasasourceofbioac- tivecompounds.However,thefood-friendlyprocessoftheduckweed plantneedstobeestablishedtoproducehighnutritionalfoodwithfree food-bornemicroorganisms.Accordingtomicrobiologicalstandardsin FoodAct1983(Regulation39),TPCinicecreammustbebelow5×10⁴ cfu/g.Itshowsthatmicrobialanalysisoficecreamformulatedwith2%

driedduckweedfollowstheMalaysianregulationoffood-safestandards.

Inrecentyears,thepublishedsurveyonduckweedacceptabilityas humanfoodamongconsumershasallowedabetterknowledgeofhuman preferencesonparticularfittingmealsthatcouldbeappliedwithduck- weedplants.Thesurveyshowedmostparticipantsprefertoeatduck- weedasasalad,andfewerparticipantsacceptduckweedplantsadded intheircookies,creamsauce,burritoorevenchickenbreast(deBeuke-

laaretal.2019).However,thepublishedstudywasconductedamong westernconsumersonly.Morestudyonthehumanpreferenceofduck- weedintegratedfoodproductsshouldbeconductedamongawiderange ofhumanages,includingchildrenwhopreferthetasteandlookrather thannutritionalvalue.

5. Conclusion

Overall,thepresentedoutcomesofthestudyappeartosuggestthat L.minorandW.globosacontainnutritiousmetabolitesthatcoulden- hancethequalityoffuturefood.However,thehighfibrecontentinL.

minorrelativetoW.globosasuggeststhatL.minorwasmoresuitablefor humanfood,whichisgoodforthehumandigestionsystem.However, furtherresearchshouldbeperformedinmoredepthtoinvestigatethe capacityofduckweedasasustainableandcheapestresourceofhealth supplementwithhighantioxidantcontent.

Conflictofinterest

Discloseanypotentialconflictofinterestappropriately.

Theauthorsdeclarenoconflictofinterest.

Acknowledgments

Author contribution to this paper: Nazariyah Yahaya: Writing- originaldraftpreparation,editing,supervisionandprojectadministra- tion,NabilaHudaHamdan^,AtiqahRuqayyahZabidi,AmmarMirzaMo- hamad,MohammadLuqmanHakim Suhaimi,Muhammad AzhanAz- farMd Johari: Investigation, Hanis Nadia Yahyaand Hafiza Yahya:

Reviewing andediting. This research is supportedby USIM Innova- tion Development Grant (grant code: PPP/GPI/FST/051014/60419) anda partof thisresearch is supported byMinistry of Higher Edu- cation,Malaysia underResearch ExcellenceConsortium (grant code:

USIM/KKP-S03/IFFAH/FST/LUAR-K/44220).

Supplementarymaterials

Supplementarymaterialassociatedwiththisarticlecanbefound,in theonlineversion,atdoi:10.1016/j.fufo.2022.100128.

References

Alkimin, G.D., Daniel, D., Frankenbach, S., Serôdio, J., Soares, A.M.V.M., Barata, C., Nunes, B, 2019. Evaluation of pharmaceutical toxic effects of non-standard endpoints on the macrophyte species Lemna minor and Lemna gibba . Sc. Total Environ. 657, 926–

937. doi: 10.1016/j.scitotenv.2018.12.002 .

An, D., Li, C., Zhou, Y., Wu, Y., Wang, W., 2018. Genomes and transcriptomes of duck- weeds. Front. Chem. 6 (230). doi: 10.3389/fchem.2018.00230 .

Ansa, E.D.O., Lubberding, H.J., Ampofo, J., Amegbe, G.B., Gijzen, H., 2012. Attach- ment of faecal coliform and macro-invertebrate activity in the removal of faecal coliform in domestic wastewater treatment pond systems. Eco. Eng. 42, 35–41.

doi: 10.1016/j.ecoleng.2012.01.018 .

Anthonius, C., Yong, A.S.K., Fui, C.F, 2018. Supplementation of duckweed diet and citric acid on growth performance, feed utilization, digestibility and phosphorus utilization of TGGG hybrid grouper ( Epinephelus fuscoguttatus x Epinephelus lanceolatus ) juvenile.

Songklanakarin J. Sci. Technol. 40 (5), 1009–1016. doi: 10.14456/sjst-psu.2018.123 . Appenroth, K.-J., Sree, K.S., Bog, M., Ecker, J., Seeliger, C., Bohm, V., Lorkowski, S., Jahreis, G., 2018. Nutritional value of the duckweed species of the genus Wolffia (Lemnaceae) as human food. Front. Chem. 6 (483). doi: 10.3389/fchem.2018.00483 . Artetxe, U, García-Plazaola, J.I, Hern´ande, z A., Becerril, J.M, 2002. Low light-grown duckweed plants are more protected against the toxicity induced by Zn and Cd. Plant Physiol. Biochem 40 (10), 859–863. doi: 10.1016/S0981-9428(02)01446-8 . Balamurugan, R., Duraipandiyan, V., Ignacimuthu, S., 2011. Antidiabetic activity of 𝛾-

sitosterol isolated from Lippia nodiflora L. in streptozotocin-induced diabetic rats. Eur.

J. Pharmacol. 667 (1–3), 410–418. doi: 10.1016/j.ejphar.2011.05.025 .

Behrman, E., Gopalan, V., 2005. Cholesterol and plants. J. Chem. Educ. 82 (12), 1791.

doi: 10.1021/ed082p1791 .

Carry, E., Zhao, D., Mogno, I., Faith, J., Ho, L., Villani, T., Wu, Q., 2018. Targeted analysis of microbial-generated phenolic acid metabolites derived from grape flavanols by gas chromatography-triple quadrupole mass spectrometry. J. Pharm. Biomed. 159, 374–

383. doi: 10.1016/j.jpba.2018.06.034 .

Cedergreen, N., Madsen, V.T, 2002. Nitrogen uptake by the floating macrophyte Lemna minor . New Phytol 155, 285–292. doi: 10.1046/j.1469-8137.2002.00463.x .

Chakrabarti, R., Clark, W.D., Sharma, J.G., Goswami, R.K., Shrivastav, A.K., Tocher, D.R, 2018. Mass production of Lemna minor and its amino acid and fatty acid profiles.

Front. Chem. 6 (479). doi: 10.3389/fchem.2018.00479 .

Chaudhary, J., Jain, A., Kaur, N., Kishore, L., 2011. Stigmasterol: A comprehensive review. Int. J. Pharm. Sci. Res. 2 (9), 2259–2265.

doi: 10.13040/IJPSR.0975-8232.2(9).2259-65 .

de Beukelaar, M.F.A., Zeinstra, G.G., Mes, J.J., Fischer, A.R.H, 2019. Duckweed as human food. The influence of meal context and information on duckweed acceptability of Dutch consumers. Food Qual. Prefer. 71, 76–86. doi: 10.1016/j.foodqual.2018.06.005 . Di Stefano, E., Agyei, D., Njoku, E.N., Udenigwe, C.C, 2018. Plant RuBisCO: An under- utilized protein for food applications. J. Am. Oil Chem. Soc. 95 (8), 1063–1074.

doi: 10.1002/aocs.12104 .

Gabay, O., Sanchez, C., Salvat, C., Chevy, F., Breton, M., Nourissat, G., Berenbaum, F., 2010. Stigmasterol: A phytosterol with potential anti-osteoarthritic properties. Os- teoarthr. Cartil. 18 (1), 106–116. doi: 10.1016/j.joca.2009.08.019 .

González-Peña, D., Dudzik, D., García, A., de Ancos, B., Barbas, C., Sánchez-Moreno, C., 2017. Metabolomic fingerprinting in the comprehensive study of liver changes asso- ciated with onion supplementation in Hypercholesterolemic Wistar rats. Int. J. Mol.

Sci. 18 (2), 267. doi: 10.3390/ijms18020267 .

Hasan, M.R. , Chakrabarti, R. , 2009. Floating aquatic macrophytes-Duckweeds. In: Use of algae and aquatic macrophytes as feed-in small-scale aquaculture- A review. FAO Fish.

Tech. Pap. 531, 29–51 .

Herawati, V.E., Pinandoyo, Darmanto, Y.S., Rismaningsih, N., Windarto, S., Radjasa, O.K, 2020. The effect of fermented duckweed ( Lemna minor ) in feed on growth and nu- tritional quality of tilapia ( Oreochromis niloticus ). Biodiversitas 21 (7), 3350–3358.

doi: 10.13057/biodiv/d210759 .

Hossain, M.S. , Alam, M.J. , 2020. Integration of fish culture in hydroponic agriculture in flood-prone areas. Sains Malays 49 (8), 1799–1808 .

Islam, M.S., Kabir, M.S., Khan, S.I., Ekramullah, M., Nair, G.B., Sack, R.B., Sack, D.A., 2004. Wastewater-grown duckweed may be safely used as fish feed. Can. J. Microbiol.

50 (1), 51–56. doi: 10.1139/w03-102 .

Kalogeropoulos, N., Andrikopoulos, N.K., 2004. Squalene in oils and fats from domes- tic and commercial fryings of potatoes. Int. J. Food Sci. Nutr. 55 (2), 125–129.

doi: 10.1080/09637480410001666531 .

Kamyab, H., Chelliapan, S., Din, M.F.M., Shahbazian-Yassar, R., Rezania, S., Khademi, T., Azimi, M., 2017. Evaluation of Lemna minor and Chlamydomonas to treat palm oil mill effluent and fertilizer production. J. Water Process. Eng. 17, 229–236.

doi: 10.1016/j.jwpe.2017.04.007 .

Landesman, L. , Parker, N.C. , Fedler, C. , Konikoff, M. , 2005. Modeling duckweed growth in wastewater treatment systems. Livest. Res. Rural. Dev. 17 (6) .

Lee, C.J., Yangcheng, H., Cheng, J.J., Jane, J.L, 2016. Starch characterization and ethanol production of duckweed and corn kernel. Starch/Staerke 68 (3–4), 348–354.

doi: 10.1002/star.201500126 .

Lozano-Grande, M.A., Gorinstein, S., Espitia-Rangel, E., Davila-Ortiz, G., Martinez- Ayala, A.L., 2018. Plant sources, extraction methods, and uses of squalene. Int. J.

Agron., 1829160 doi: 10.1155/2018/1829160 .

Markou, G., Wang, L., Ye, J., Unc, A., 2018. Using agro-industrial wastes for the cultiva- tion of microalgae and duckweeds: Contamination risks and biomass safety concerns.

Biotechnol. Adv. 36 (4), 1238–1254. doi: 10.1016/j.biotechadv.2018.04.003 . Moyo, S., Dalu, J.M., Ndamba, J., 2003. The microbiological safety of duckweed fed chick-

ens: a risk assessment of using duckweed reared on domestic wastewater as a pro- tein source in broiler chickens. Phys. Chem. Earth , Parts A/B/C 28 (20), 1125–1129.

doi: 10.1016/j.pce.2003.08.021 .

Naseem, S., Bhat, S.U., Gani, A., Bhat, F.A, 2020. Perspectives on utilization of macro- phytes as a feed ingredient for fish in future aquaculture. Rev. Aquac. 13 (1), 282–300.

doi: 10.1111/raq.12475 .

Pekmezci, D. , Litwack, G. , 2011. Chapter eight - Vitamin E and immunity. Vitamins and Hormones 86, 179–215 Academic Press .

Radulovic, O. , Petric, M. , Raspor, M. , Stanojevic, O. , Janakiev, T. , Tadic, V. , Stankovic, S.A , 2019. Culture-dependent analysis of 16S rRNA sequences associated with the rhizosphere of Lemna minor and assessment of bacterial phenol-resistance:

Plant/bacteria system for potential bioremediation. Pol. J. Environ. Stud. 28 (2), 811–822 .

Sadiq, M., Akram, N.A., Ashraf, M., Al-Qurainy, F., Ahmad, P., 2019. Alpha- tocopherol-induced regulation of growth and metabolism in plants under non- stress and stress conditions. J. Plant Growth Regul. 38 (4), 1325–1340.

doi: 10.1007/s00344-019-09936-7 .

Satya, A., Satya, I.A., Chrismadha, T., 2020. Nitrogen uptake competition between minute duckweed ( Lemna perpusillaTorr ) and microalgae under various nutrient compositions.

IOP Conf. Ser. Earth Environ. Sci. 535 (1). doi: 10.1088/1755-1315/535/1/012027 . Saveri, A., Fornasiero, B.R, 1983. Morphological variations in Lemna minor L.

and possible relationships with abscisic acid. Caryologia 36 (1), 57–64.

doi: 10.1080/00087114.1983.10797644 .

Sree, K.S., Bog, M., Appenroth, K.J., 2016. Taxonomy of duckweeds (Lem- naceae), potential new crop plants. Emir. J. Food Agric. 28 (5), 291–302.

doi: 10.9755/ejfa.2016-01-038 .

Tripathi, N., Kumar, S., Singh, R., Singh, P., Varshney, W.K, 2013. Isolation and identifica- tion of 𝛾- sitosterol by GC-MS from the leaves of Girardinia heterophylla (Decne). The Open Bioactive Compounds Journal 4, 25–27. doi: 10.2174/1874847301004010025 . Tsimidou, M.Z. , Preedy, V.R. , Watson, R.R , 2010. Chapter 61- Squalene and tocopherols in olive oil: importance and methods of analysis. In: Olives and Olive Oil in Health and Disease Prevention. Academic Press, San Diego, pp. 561–567 .

Uchida, R. , 2000. Essential nutrients for plant growth: nutrient functions and deficiency symptoms. Plant Nutrient Management in Hawaii’s Soils, Approaches for Tropical and Subtropical Agriculture: College of Tropical Agriculture and Human Resources.

the University of Hawaii at Manoa .

Van Hoeck, A., Horemans, N., Monsieurs, P., Cao, H.X., Vandenhove, H., Blust, R., 2015.

The first draft genome of the aquatic model plant Lemna minor opens the route for future stress physiology research and biotechnological applications. Biotechnol. Bio- fuels 8 (1), 188. doi: 10.1186/s13068-015-0381-1 .

Wang, W., Li, R., Zhu, Q., Tang, X., Zhao, Q., 2016. Transcriptomic and physiological analysis of common duckweed Lemna minor responses to NH 4( ⁺) toxicity. BMC Plant Biol 16. doi: 10.1186/s12870-016-0774-8 , 92-92 .

Xie, L., Solhaug, K.A.r., Song, Y., Brede, D.A., Lind, O.C., Salbu, B., Tollefsen, K.E, 2019.

Modes of action and adverse effects of gamma radiation in an aquatic macrophyte Lemna minor . Sci. Total Environ. 680, 23–34. doi: 10.1016/j.scitotenv.2019.05.016 .

Yao, Y., Zhang, M., Tian, Y., Zhao, M., Zhang, B., Zhao, M., Yin, B., 2017. Duckweed ( Spirodela polyrhiza ) as green manure for increasing yield and reducing nitrogen loss in rice production. Field Crops Res 214, 273–282. doi: 10.1016/j.fcr.2017.09.021 . Zhao, Y., Fang, Y., Jin, Y., Huang, J., Bao, S., Fu, T., Zhao, H., 2014. Poten-

tial of duckweed in the conversion of wastewater nutrients to valuable biomass:

A pilot-scale comparison with water hyacinth. Bioresour. Technol. 163, 82–91.

doi: 10.1016/j.biortech.2014.04.018 .