impact of environmental conditions on biomass yield, … of life science & food technology,...

AQUACULTURE ENVIRONMENT INTERACTIONSAquacult Environ Interact

Vol 8 619ndash636 2016doi 103354aei00200

Published November 8sect

INTRODUCTION

Cultivation of macroalgae is a rapidly growing in -dustry in a global perspective (FAO 2016) The maindriver is the establishment of a production of marine-

based biomass for food energy protein and biomole-cules (Bruton et al 2009 Kraan 2013 Wei et al 2013)but also exploitation of the bio-mitigation capacity ofthe algae is in focus (Troell et al 1999 Castine et al2013 Marinho et al 2015a) The non-use value eco-

copy The authors 2016 Open Access under Creative Commons byAttribution Licence Use distribution and reproduction areunrestricted Authors and original publication must be credited

Publisher Inter-Research middot wwwint-rescom

Corresponding author anbrbiosaudk

Impact of environmental conditions on biomass yield quality and bio-mitigation

capacity of Saccharina latissima

Annette Bruhn1 Ditte Bruunshoslashj Toslashrring2 Marianne Thomsen3 Paula Canal-Vergeacutes2 Mette Moslashller Nielsen12 Michael Bo Rasmussen1

Karin Loft Eybye5 Martin Moslashrk Larsen4 Thorsten Johannes Skovbjerg Balsby1Jens Kjerulf Petersen2

1Department of Bioscience Aarhus University Vejlsoslashvej 25 8600 Silkeborg Denmark2Danish Shellfish Centre Institute of Aquatic Resources Technical University of Denmark DTU-Aqua Oslashroddevej 80

7900 Nykoslashbing Mors Denmark3Department of Environmental Sciences Aarhus University Frederiksborgvej 399 4000 Roskilde Denmark

4Department of Bioscience Aarhus University Frederiksborgvej 399 4000 Roskilde Denmark5Division of Life Science amp Food Technology Danish Technological Institute Kongsvang Alleacute 29 8000 Aarhus C Denmark

ABSTRACT Seaweeds are attractive as a sustainable aquaculture crop for food feed bioenergyand biomolecules Further the non-value ecosystem services of seaweed cultivation (ie nutrientrecapture) are gaining interest as an instrument towards sustainable aquaculture and for fulfillingthe aims of the EU Marine Strategy Framework Directive Environmental factors determine theyield and quality of the cultivated seaweed biomass and in return the seaweed aquacultureaffects the marine environment by nutrient assimilation Consequently site selection is critical forobtaining optimal biomass yield and quality and for successful bio-mitigation In this study 5 sitesfor cultivation of Saccharina latissima were selected within a eutrophic water body to guide siteselection for future kelp cultivation activities Results were coupled to marine monitoring data toexplore the relationship between environmental conditions and cultivation success The biomassyields fluctuated 10-fold between sites due to local variations in light and nutrient availabilityYields were generally low ie up to 510 g fresh weight (FW) per meter seeded line however thedry matter contents of protein and high-value pigments were high (up to 17 protein and 01fucoxanthin) Growth performance biomass quality and bio-mitigation potential was restricted bylow availability of light and bioavailable phosphorus and biofouling through juvenile suspensionfeeders was a critical factor at all cultivation sites At specific sites the tissue metal contents (Pband Hg) exceeded the limit values for feed or food Our results emphasize the importance of care-ful site selection before establishing large-scale cultivation and stress the challenges and benefitsof kelp cultivation in eutrophic waters

KEY WORDS Eutrophication middot Limfjorden middot Seaweed farming middot Metals middot Nitrogen middot Phosphorus middotSite quality middot Ecosystem service

OPENPEN ACCESSCCESS

sectCorrections were made after publication For details seewwwint-res comarticlesaei20179q009p019pdfThis version January 23 2017

Aquacult Environ Interact 8 619ndash636 2016

system service (Daly 1998) provided by cultivatedalgae in terms of recapturing nutrients in coastalareas is of commercial and societal interest mdash as com-pensation for increased aqua culture activities (San -derson et al 2012 Handaring et al 2013 Smale et al2013 Holdt amp Edwards 2014 Marin ho et al 2015a) oras a potential instrument for circular nutrient man-agement improving the eco logical status of eutro -phic marine areas (Seghetta et al 2016) in line withthe EU Marine Strategy Framework Directive (EU2008a 2014)

In Europe the effort concerning cultivation of largebrown algae (Laminariales) in particular is increas-ing The most commonly cultivated brown algae spe-cies in Europe Saccharina latissima ((Linnaeus) CELane C Mayes Druehl amp GW Saunders) has beencultivated on a smaller or larger scale in IrelandScotland Germany Holland Spain Norway FaroeIslands and Denmark (ie Buck amp Buchholz 20042005 Buck et al 2008 Werner et al 2009 Wegeberg2010 Edwards amp Watson 2011 Forbord et al 2012Handaring et al 2013 Peteiro amp Freire 2013b Wegeberget al 2013 Marinho et al 2015a) The achieved bio-mass yields and the biochemical composition of thebiomass vary considerably seasonally and spatiallyprimarily because of different environmental condi-tions (Edwards amp Watson 2011 Handaring et al 2013Peteiro amp Freire 2013ab Marinho et al 2015a) Inreturn the algae production also exerts an impact onthe environmental conditions through the removal ofnutrients (Troell et al 1999 Stephens et al 2014Marinho et al 2015a) Thus algae cultivation sitesshould be carefully selected for optimizing biomassproduction biomass quality as well as the non-valueecosystem services (Kerrison et al 2015) Further theseasonal timing of the deployment and harvest of thealgae needs to be optimized according to local envi-ronmental conditions The focus of the optimizationie high protein yield or high carbohydrate yield willdepend on the final application of the biomassSporophytes of Lami naria species store nutrients forlength growth during periods when environmentalnutrients concentrations are high (Bartsch et al2008) Thus high environmental nutrient concentra-tions favour high tissue nitrogen (N) concentrationsin wild S latissima up to 35 N of dry matter (DM)(Gevaert et al 2001 Nielsen et al 2014) up to 50N of DM when cultivated in close proximity to fishaquaculture (Handaring et al 2013 Marinho et al 2015a)and even up to 67 N of DM when cultivated underhighly eu trophic conditions (Nielsen 2015) Themolar Nphosphorus (P) ratio is commonly in therange of 9minus251 (Atkinson amp Smith 1983) and P con-

centrations of up to 08 of DM are reported in nutri-ent-rich waters (Marinho et al 2015a) High tissue Nconcentrations reflect a correspondingly high con-tent of proteins (Manns et al 2014 Marinho et al2015b Angell et al 2016) Consequently both thebiomass quality and the bio-mitigation capacity of theproduced algae increase in nutrient-rich waters in -creasing the value of the biomass as well as im -proving the environmental condition of the waterbody through harvest and thus removal of nutrients

Of the 21 Danish water bodies Limfjorden receivesthe highest annual net supply of nutrients (82 t N and030 t P kmminus2 yminus1 Seghetta et al 2016) These high nu-trient loadings have caused a regime shift in the fjordfrom benthic to pelagic primary production (Krause-Jensen et al 2012) The high pelagic prima ry produc-tion supports a substantial stock of benthic suspensionfeeders including blue mussels Mytilus edulis L sup-porting a local mussel fishery (Maar et al 2010 Tim-mermann et al 2014) Mussel farming has been suc-cessfully tested as an instrument to recapture nutrientsand improve the ecological status of Limfjorden (Pe-tersen et al 2014) and farming of long-line blue mus-sels is an emerging business in Limfjorden Alongwith the development of the mussel farming industryinterest in macroalgae cultivation is increasing partlybecause the 2 crops may be cultivated using the samestructures (Nielsen 2015) Due to the high environ-mental nutrient concentrations cultivation of largebrown algae in a water body like Limfjorden wouldtheoretically hold a potential for the production of aSaccharina biomass with high protein content repre-senting a higher value for the food or feed market Atthe same time the potential of seaweed cultivation asan instrument for circular nutrient managementwould be maximized Despite the relatively small sizeof Limfjorden (1500 km2) local environmental condi-tions differ considerably between the different basins(Maar et al 2010) Cultivation of S latissima has todate been documented only once at 1 site in Lim -fjorden indicating a potential for cultivation of Slatissima This study however also demonstrates theneed for investigating optimal timing of cultivationand harvest in order to maximize biomass yield andavoid biofouling (Wegeberg 2010)

Testing and evaluating the interactions betweenlocal environmental conditions and bio mass yieldquality and potential for bio-mitigation throughnutrient recapture of cultivated kelps in coastalwaters is needed before im plementing cultivation ona larger scale This applies not only to Limfjordenbut to any water body where macroalgae cultivationis intended

620

Bruhn et al Environmental effects on S latissima yield and quality

The aim of this study was to compare thebiomass yield bio-mitigation capacity andnutritional quality for food and feed ofS latissima cultivated at 5 sites in Limfjor-den as well as to explore the influence oflocal environmental conditions on theseparameters with the purpose of guidingsite selection and timing of harvest The 5selected cultivation sites each representedtheir basin in Limfjorden with the basinscharacterized by different environmentalconditions regarding salinity turbiditynutrient availability and sediment metalconcentrations

MATERIALS AND METHODS

Study area and cultivation sites

Limfjorden is a shallow semi-enclo sedestuary located between the North Seaand the Kattegat (Fig 1) The total surfacearea of the fjord is ~1500 km2 and theaverage depth is 46 m The total catch-ment area is 7587 km2 and is predomi-nantly agricultural land Despite a small tidal ampli-tude tidal forces and wind are the drivers of theannual net flow of 68 km3 of water from the NorthSea via the Thyboroslashn channel in the west throughLimfjorden to the Kattegat Limfjorden consists ofseveral relatively shallow water basins connected bynarrow and deep sounds The big broads have waterdepths of 5minus8 m whereas the sounds have depths of18minus22 m the deepest point being Oddesund (28 m)The average salinity varies from 32minus34 in the west-ern part to 19minus25 in the central and eastern part (Lyngby et al 1999 Markager et al 2006 Krause-Jensen et al 2012 Timmermann et al 2014)

Five existing mussel farms were selected as exper-imental cultivation sites (Fig 1 Table 1) for the

follow ing reasons (1) they were each located in a dis-tinct basin of Limfjorden (2) aquaculture licenseswere already active (3) the mussel cultivation struc-tures could be used for the seaweed cultivation and(4) the 5 basins were covered by the Danish NationalMonitoring and Assessment Program for the Aquaticand Terrestrial Environment (NOVANA)

Environmental data

For each of the 5 cultivation sites the data on bio-mass yield and quality were coupled to environmen-tal data from an environmental monitoring stationlocated centrally within each basin (Fig 1 Table 2)

621

Site Position Basin Size Depth Sea bed Degree of exposureLatitude Longitude (m times m) (m) classification

(degN) (degE)

Odby Bay 56577 8570 NissumKaas Broad 300 times 300 4 Soft mud Exposed to winds from E and SLysen Broad 56692 8841 Sallingsund 250 times 500 2minus5 Fine sand with clay ProtectedFur Sund 56816 8968 Fur Sund 250 times 750 5 Rockysandy Exposed to strong currentsFaeligrker Vig 56834 9073 Loslashgstoslashr Broad 300 times 300 4 Hard sandstone ProtectedRiisgaarde 56736 9151 Riisgaarde Broad 250 times 500 10 Soft mud Exposed to winds from N E SBroad Skive Fjord

Table 1 Location size and characteristics of the 5 Saccharina latissima cultivation sites in Limfjorden Denmark N north E east S south W west

Fig 1 Location of Limfjorden in Denmark and the 5 Saccharina latissimacultivation sites (filled circles) and 4 environmental monitoring stations(open circles) Stns VIB3702 VIB3708 and VIB 3727 are pelagic stationsfor monitoring water quality Stns 3702 3705 3708-1 and 3727 are

stations for monitoring benthic metal concentrations


Data from the monitoring stations were retrievedfrom NOVANA through the National Database forMarine Data (ODAM) (Fig 1 Table 1)

Data on water temperature salinity turbidity andconcentrations of oxygen chlorophyll a (chl a) in -organic nutrients (dissolved inorganic N [DIN =NO2

minus-N NO3minus-N NH4

+-N] dissolved inorganic bio -available P [ortho-P]) and sediment metals were col-lected and analysed using standard methods accord-ing to the current national Technical Instructions forMarine Monitoring (Markager 2004 Pedersen et al2004 Larsen 2013 Markager amp Fossing 2013 Vang2013 Vang amp Hansen 2013) Sampling was per-formed on average every 2minus3 wk Sampling of sedi-ment was performed every 1minus5 yr By trapezoidalintegration all pelagic environmental data were cal-culated into weighted averages over 2 periods up tothe time point of each biomass sampling mdash earlyspring the period of detectable growth from 1 Feb -ruary 2012 to Sampling 1 11 April 2012 and latespring the last part of the grow-out period from Sampling 1 (11 April 2012) to Sampling 2 (25 May or12 June 2012) (see next section and Table 2)

Data regarding temperature salinity and turbiditywere differentiated according to the actual cultiva-tion depths (15 and 25 m respectively) Data re gar -ding nutrients oxygen and chl a were only availablefrom 1 m of depth but no significant stratificationprevailed during the cultivation period Sedimentmetal concentration data were averaged for each sta-tion over a period covering the preceding 10 yr(2003minus2012) Data on local incoming light was sup-plied from the Danish Meteorological Institute

Ideally cultivation sites and monitoring stationscould have been geographically closer However thedata from the environmental monitoring stations wasconsidered as being representative for the cultivationsites despite the distances of 8minus20 km between mon-itoring station and cultivation site for a number of

reasons (1) other studies correlating monitoring da taand macrovegetation performance in Limfjordengenerally achieve good correlations (eg Krause-Jensen et al 2012) (2) the experimental period fromwinter to early summer is a period of maximal wind-driven circulation (Wiles et al 2006) and absence ofvertical stratification (Christiansen et al 2006) (3)mixing was confirmed as no stratification was ob -served during the experimental period and (4) sitesand stations were located in the more open parts ofthe basins in proximity to point sources of run-offfrom land Coupling of biomass yield and quality toenvironmental data for the cultivation site at LysenBroad was not possible as only data on sedimentchemistry was available from the environmentalmonitoring station in this basin

Cultivation and sampling of Saccharina latissima

Two batches of S latissima seeded lines were usedin the cultivation experiment (Table 2) Batch 1 con-sisted of 500 m of ready-made seeded line (diameter6 mm) produced by direct sporulation (Wegeberg2010) at Blue Food AS Denmark This batch wasdelivered to the Danish Shellfish Centre on 5 Decem-ber 2011 kept in running seawater overnight anddeployed the following day at 4 sites Odby BayLysen Broad Fur Sund and Riisgaarde BroadBatch 2 was deployed at Faeligrker Vig and consisted of125 m of seeded line (diameter 6 mm) also producedthrough direct sporulation but at the Danish Shell-fish Centre during August 2011 Both batches wereproduced from fertile material from a S latissimapopulation in the Danish Belt Sea and visual inspec-tion of the lines upon deployment did not reveal anydifference between the 2 batches in quality densityor size of the juvenile sporophytes Length of theseedlings at deployment was ~1 mm All lines were

622

Site Deployment Sampling 1 Sampling 2 Batch Pelagic Sediment Distancedate date date station station (km)

Odby Bay Dec 6 2011 Apr 11 2012 Jun 12 2012 1 VIB3702 3702 14Lysen Broad Dec 6 2011 Apr 11 2012 Jun 12 2012 1 minusa 3705 85Fur Sund Dec 6 2011 Apr 11 2012 Jun 12 2012 1 VIB3708 3708-1 20Faeligrker Vig Oct 28 2011 Apr 11 2012 May 25 2012 2 VIB3708 3708-1 14Riisgarde Broad Dec 6 2011 Apr 11 2012 Jun 12 2012 1 VIB3727 3727 14aNo pelagic monitoring station was within proximity to the cultivation site at Lysen Broad

Table 2 Deployment and sampling dates at the 5 Saccharina latissima cultivation sites in Limfjorden batches of seeded linesas well as identification numbers of and distance to the environmental monitoring stations (pelagic and sediment stations see

Fig 1) from which data were used for analyses


deployed as vertical droppers each 25 m long at -tached to a horizontal long-line with a 50 cm tether-ing line The droppers were interspaced by 40 cmalong the horizontal long-line During the grow-outperiod the horizontal long-lines were kept 50 cm be -low the water surface to avoid disturbance by float-ing ice and heavy storms Consequently the seededlines were positioned between 1 and 35 m depth

Sampling of biomass was performed twice by ran-dom selection of 3 droppers from each site Samp -ling 1 on 11 April 2012 and Sampling 2 on either25 May or 12 June 2012 (Table 2) The lines werebrought to the laboratory where the upper 2 m ofeach line was divided into 2 sections the upper sec-tion represented the seeded line hanging in 1minus2 mdepth (average 15 m) and the lower section repre-senting the seeded line hanging at a depth of 2minus3 m(average 25 m) The remaining 50 cm of each seededline with the attached bottom weight was discardeddue to lack of biomass The following parameterswere recorded for both sections of the lines totalweight of sample (seeded line + algae + epiphytes)weight of seeded line weight of algae weight andtaxonomy of dominating biofouling epiphytic organ-isms and finally average sporophyte frond lengthbased on 15 randomly selected sporophytes Aftersampling tissue samples were stored at minus20degC untilbiochemical analyses were performed Due toincreasingly heavy biofouling by epiphytic organ-isms over time algae material harvested from lateMay and onwards (Sampling 2) was fully coveredwith epiphytic organisms such as ascidians and juve-nile mussels and thus considered unsuitable for foodor feed applications Therefore only algae materialsampled in April (Sampling 1) was used in the bio-chemical analyses Due to very limited biomass har-vested from Fur Sund at 25 m in April only pigmentanalyses were performed on this biomass

Calculations of growth rates and biomass yields

Specific growth rates (SGRs) were calculated frommeasurements of the fresh weight (FW) per runningmeter of seeded line as

(1)

where FW0 and FWt corresponded to the freshweight of S latissima per m of seeded line at time 0and after t days of cultivation respectively Biomassyields were reported as g FW per m of seeded line (g

FW mminus1) The average frond length of the S latissimasporophytes was calculated as an average length plusmnSE of the 15 randomly selected sporophytes fromeach sample

Saccharina latissima tissue biochemistry

DM ash carbon (C) N and P Algae samples werefreeze-dried at minus40degC and homogenized by drymilling DM content was calculated as percentage ofFW A known amount of dry algae was combusted at550degC for 2 h and the ash fraction was calculated aspercentage of DM Concentrations of C and N in thefreeze-dried algae tissue were analysed by Pregl- Dumas ignition in pure oxygen atmosphere followedby chromatographic separation of C and N with de -tection of the individual elements by thermal conduc-tivity (Culmo 2010) Total P content of the algae bio-mass was as analysed spectrophotometrically ac cor -ding to standard methods (Grasshoff et al 1983) Priorto analysis the dried and homogenized samples wereheated at 550degC for 2 h autoclaved with 2 M hydro -gen chloride (HCl) (20 mg DM for 7 ml acid) and fi-nally filtered through GFF filters (Whatman)

Metals Metal concentrations (As Cd Hg Pb) weredetermined by inductively coupled plasma-massSpectrometry (ICP-MS) In short a 02 g dry sub-sample was digested in a closed vessel microwaveoven using 5 ml of nitric acid (7 M) and 1 ml of hydro-gen peroxide then diluted to 50 ml with milliQ waterfollowed by ICP-MS determination using internalstandards of Rh Ir and Ge to correct for drift (seeNielsen et al 2012) Certified reference material ofmacroalgae from IAEA-140 (Coquery et al 2000) wasused for quality assurance

Pigments Pigment concentrations (chl a fuco -xanthin violaxanthin and β-carotene) were deter-mined using acetone extraction and quantification byHPLC as described in Boderskov et al (2016) Pig-ment standards were obtained from DHI LaboratoryProducts

Crude protein and amino acids (AAs) Crude pro-tein and AA composition were analysed only for sam-ples from Faeligrker Vig Total organic bound crudeprotein was determined by the Kjeldahl principleaccording to Nordic Committee on Food Analysis(2005) Protein content was calculated by multiplyingthe amount of N by a factor of 5 and expressed as per-cent of DM (Angell et al 2016) The determination ofAAs was done by HPLC according to EU 1522009(A) and ISO 139032005 AA contents were expressedas percentage of DM

t

t

SGR() 100ln

FWFW0= times

⎛⎝⎜

⎞⎠⎟

623


Data analysis

For comparing growth performance and biomassquality between sites and depths 2-way ANOVA(using Tukeyrsquos post hoc analysis) and linear regres-sion analyses were performed using JMP 100 (SASInstitute) Explorative data analysis was performedto identify significant correlation patterns betweenmacro algae growth and environmental parametersData were log transformed in order to obtain normaldistribution and homogeneity of variance for theresiduals of the models Multivariate data analysis(MVDA) was performed to guide model selection ofvariables to be tested using general linear models(GLM) Partial least square regression (PLS-R) wasused as explorative technique for pattern recognitionusing the Unscrambler v102 (CAMO Software) Bio-mass yield and biofouling in early and late spring aswell as bio-mitigation capacity ie N and P contentin the harvested seaweed biomass were selec ted asY-variables in the PLS-R models and modelled usingenvironmental parameters characterising the mar-ine growth environment surrounding the individualcultivation sites as original explanatory variables(data not shown) GLMs were used to assess theeffect of light salinity availability of ortho-P temper-ature and environmental NP ratio (NP_E) on growthperformance biofouling and biomass quality Theenvironmental parameters were selected as inde-pendent variables based on the indicative impact onthe dependent variable (biomass growth parametersand quality) as observed from MVDA (data notshown) As several of the independent variablesshowed strong correlations (Pearson Table S1 in theSupplement at wwwint-rescomarticlessuppl q008p619_supppdf) the independent variables were splitinto 2 models to avoid issues with collinearity mdashModel 1 light salinity and ortho-P Model 2 tem-perature salinity and NP_E These analyses wereperformed in SAS 93 (SAS Institute) using the Procmixed function with cultivation site as a random fac-tor The level of significance applied was 005 unlessmentioned otherwise

RESULTS

Environmental conditions

The environmental conditions differed among thebasins of Limfjorden (Fig 2 Table 1) Differenceswere most pronounced with regard to salinity lightand concentrations of inorganic nutrients and chl a

Salinity

The salinity in the different basins decreased withincreasing distance from the North Sea NissumBroad 290minus319 Loslashgstoslashr Broad 260minus289 andSkive Fjord 235minus265 In Nissum Broad the salinityincreased slightly over the grow-out period whereasin Loslashgstoslashr Broad and Skive Fjord the salinitydecreased over the period reflecting a stronger influ-ence of run-off from land (Fig 2A) No pronouncedstratification of the water column was observed fromthe monitoring data during the grow-out period atany of the stations (data not shown)

Temperature

Generally the differences in temperature amongstations were minor (lt1degC) and even less betweenthe 2 cultivation depths at any station The tempera-tures experienced during the full grow-out periodranged from minimum temperatures in all basinsmeasured on 1 February (between ndash02 and 15degC) tomaximum temperatures in June (138minus141degC) (Fig 2B)

Inorganic nutrients

The average concentrations of DIN from deploymentto April ranged between 20 and 40 microM however withconcentrations up to 58 microM in Skive Fjord in winterand early spring (Fig 2C) In late spring betweenApril and June the DIN concentrations decreasedlt2 microM in Nissum Broad but remained high between10 and 20 microM in the other basins In all periods thehighest DIN concentrations were measured nearSkive Fjord and the lowest in Nissum Broad Concen-trations of ortho-P were high during the winter period(04minus09 microM) but decreased below 01 microM during thespring bloom from February to April (Fig 2D)

Pelagic chl a

In early spring February and March the phyto-plankton concentrations peaked with 12 and 16 microgchl a lminus1 in Loslashgstoslashr and Nissum Broad respectively(Fig 2E) In Skive Fjord the highest chl a concentra-tions were measured in early June (14 microg chl a lminus1)

Light

The photon flux density generally decreased by~50 from 15 to 25 m emphasizing the high turbidityof Limfjorden (Fig 2F) The algae at 15 m experiencedan average of 400minus700 micromol photons mminus2 sminus1 in late

624


spring whereas the algae at 25 m only experienced upto 400 micromol photons mminus2 sminus1 in the same period

Overall a high degree of inter-correlation be tweenthe key environmental parameters was ob served(Table S1 in the Supplement) In early spring (Febru-ary to April) the concentration of phytoplankton bio-mass (chl a) correlated strongly to the concentrationsof dissolved inorganic nutrients During early springthe concentrations of pelagic chl a correlated posi-tively to ortho-P and negatively to DIN concentra-tions whereas light availability correlated negativelyto ortho-P concentrations The DIN concentrationswere negatively correlated to salinity In late springthe pelagic phytoplankton biomass was negativelycorrelated to salinity and positively to temperature

Saccharina latissima growth performance

Biomass yield frond length SGR and biofouling

At all cultivation sites the biomass yields andfrond lengths were higher at 15 m than at 25 mdepth (Fig 3AB Table 3) The highest biomassyield in April (mean plusmn SE 510 plusmn 66 g FW mminus1) aswell as the longest fronds in April and June (409 plusmn37 cm in April and 337 plusmn 90 cm in June) wereachieved at Faeligrker Vig at 15 m (Fig 3ABTable 4) In June the biomass yield in Odby Bayand Faeligrker Vig at 15 m was significantly higherthan at the remaining 3 sites (Table 4) At 25 m thehighest biomass yield in June was ob tained in Odby

625

Fig 2 Seasonal pattern of selected environmental parameters at the 3 pelagic stations (see Table 2) during the grow-out pe-riod of Saccharina latissima (OctoberminusDecember 2011 to June 2012) (A) salinity (B) temperature (C) dissolved inorganic nitrogen (DIN) (D) ortho-phosphate (E) chlorophyll a and (F) photosynthetically active radiation (PAR) estimated at the

cultivation depths (15 and 25 m) Data represent the actual measured values


Bay whereas the longest fronds were found inFaeligrker Vig (Fig 3AB Table 4)

The SGR (in the period from deployment to April)reflected the same pattern as the biomass yield at15 m depth Faeligrker Vig (38 dminus1) gt Odby Bay(37 dminus1) gt Riisgaarde Broad (31 dminus1) gt Fur Sund(24 dminus1) gt Lysen Broad (22 dminus1) and all with sig-nificantly higher SGRs at 15 m as compared to 25 m(Fig 3C Table 3) However from April to June theSGR de creased for the algae nearest to the surface(15 m) at Odby Bay Faeligrker Vig and RiisgaardeBroad and at the 2 latter sites to negative values AtLysen Broad and Fur Sund the SGR of the algae nearthe surface was constant throughout the full grow-out period Regarding the algae growing at 25 mfrom April to June diverging trends were observedat 3 cultivation sites (Odby Bay Lysen Broad andFaeligrker Vig) the SGRs exceeded the SGRs at 15 m inthe early growth period whereas at the other 2 sites(Fur Sund and Riisgaarde Broad) the SGRs decreasedto around or below zero

The degree of biofouling increased dramatically atall sites from April to June and was in June signifi-cantly higher at 25 m than at 15 m depth with theone exception of Faeligrker Vig (Fig 3D) In June thebiomass yield of biofouling organisms (predomi-nantly hydroids juvenile M edulis and ascidians)exceeded the biomass yields of S latissima at 3 sites(Lysen Broad Fur Sund and Riisgaarde Broad) atboth depths (Fig 3AD)

In early spring the growth performance (biomassincrease [Fig 4A] length growth and SGR) was pos-itively correlated to the light availability with alsosalinity and ortho-P availability being positively cor-related to length growth and SGR (Fig 4B statisticsare provided in Table S2 in the Supplement) respec-tively The total biomass yield in June was negativelycorrelated to the degree of biofouling in late spring(linear regression p = 0003 R2 = 028) (Fig 4C) Thebiofouling in late spring was positively correlated tothe sea temperature at the cultivation depth betweenApril and June (Fig 4D Table S2)

626

Fig 3 Growth performance and biofouling of Saccharina latissima at the 5 cultivation sites (see Fig 1) (A) biomass yields (B)average frond lengths (n = 15) (C) specific growth rates (SGR) and (D) biofouling of S latissima at 15 and 25 m depths at the5 cultivation sites sampled in April (white bars) and June (grey bars) respectively Solid bars represent batch 1 of seeded lines

and crossed bars represent batch 2 Data represent means plusmn SE n = 3


Saccharina latissima biomass quality

DM tissue N and P

The DM content of the algae varied between 63and 168 of fresh weight (Fig 5A) The C contentgenerally ranged between 268 and 334 of DMexcept at Fur Sund where the C content was signifi-cantly lower (153minus205 of DM) At Odby Bay andFur Sund the tissue C concentrations were signifi-cantly higher in the biomass closest to the surfaceThis was not the case at the other sites (Fig 5BTable 3)

The tissue N concentration in April was signifi-cantly higher in biomass from Odby Bay (45 ofDM) than from any of the other cultivation sites(35minus40 of DM) (Fig 5C Table 4) Only at Riis-gaarde Broad was there a significantly higher N con-centration in the algae cultivated at 25 m than at15 m depth The tissue P content was significantlyhigher in the algae cultivated at 25 m than at 15 min Odby Bay and Riisgaarde Broad where the P con-tent in the algae from 25 m was up to 028 of DMcompared to 011 of DM at 15 m (Fig 5D)

The bio-mitigation capacity of N and P varied be -tween sites and depths from (mean plusmn SE) 002 plusmn 001to 184 plusmn 024 g N mminus1 and 0001 plusmn 00004 to 005 plusmn001 g P mminus1 respectively (Fig 5EF) reflecting pre-dominantly the large fluctuations in biomass yields(Fig 3A)

The environmental concentration of ortho-P waspositively related to the tissue DM and N contents(Fig 4B) while not related to the tissue P content

(Table S2) Temperature was positively correlated tothe tissue DM N and P contents (Table S2) Lightavailability correlated positively to the tissue C con-tent but negatively to P content (Table S2)

627

Site Yield Yield Frond Frond Biofou- Biofou- DM N P C Chl a Fuco Viola β-car(Apr) (Jun) length length ling ling

(Apr) (Jun) (Apr) (Jun)

15 m depthFaeligrkerVig A A A A A A B B A B AB B AB AOdby Bay B A B B B A AB A A A A A A ARiisgaarde Broad B B B B AB A A B A A A B B AFur Sund B B B B AB A C B A C B B ndash ALysen Broad B B B B AB A A B A A A AB AB A

25 m depthFaeligrker Vig A B A A A C B B B B BC ABC AB AOdby Bay A A BC B B BC A A A AB A A A ARiisgaarde Broad A B B B B B A A AB A AB AB AB AFur Sund A B BC B B A B C B C C C ALysen Broad A B C B B A ndash ndash ndash ndash BC BC B A

Table 4 Tukey post-hoc pairwise comparisons of Saccharina latissima growth performance in April and June and tissue bio-chemistry in April among cultivation sites at 15 and 25 m depth Different letters are assigned to significantly different

results Fuco fucoxanthin Viola violaxanthin β-car β-carotene

Parameter Cultivation Cultivation Depthsite times Depth site

Growth performanceYield (g mminus1) (A) 0064 lt0001 lt0001(minus)Yield (g mminus1) (J) 0336 lt0001 0004(minus)Length (cm) (A) 0014 lt0001 lt0001(minus)Length (cm) (J) 0398 lt0001 0036(minus)SGR ( dminus1) (A) 0535 0003 lt0001SGR ( dminus1) (J) 0057 0025 0118Biofouling (g mminus1) (A) 0417 lt0001 0097Biofouling (g mminus1) (J) 0937 0058 0003

Biomass quality (A)DM ( FW) 0021 lt0001 0643C ( DM) 0030 lt0001 lt0001(minus)N ( DM) 0001 lt0001 0959P ( DM) 0006 0008 lt0001Chl a (mg g DMminus1) 0029 0005 lt0001Fucoxanthin (mg g DMminus1) 0433 0006 lt0001Violaxanthin (mg g DMminus1) 0302 0003 0004Beta-carotene (mg g DMminus1) 0316 0026 0005As (mg kg DMminus1) 0249 lt0001 0003Hg (mg kg DMminus1) 0057 0050 0761Pb (mg kg DMminus1) 0003 lt0001 lt0001Cd (mg kg DMminus1) 0926 lt0001 0831

Table 3 Dependency of Saccharina latissima growth perform-ance and biomass quality on cultivation site and depth as well asthe interaction between the two p-values from 2-way ANOVAdata were log transformed prior to analysis Statistical signifi-cance (p gt 005) is indicated in bold A April J June (minus) desig-nates a negative correlation otherwise the correlation is positive


The average molar tissue ratios of CN and NPwere (mean plusmn SE) 90 plusmn 03 and 697 plusmn 48 respec-tively indicating strong P-limitation already in earlyspring (data not shown)

Protein and AAs

The content of crude protein in the S latissima bio-mass from Faeligrker Vig was 170 plusmn 02 and 160 plusmn01 of DM in the biomass at 15 m and 25 mrespectively The essential AAs (EAAs) constituted238 plusmn 02 and 27 plusmn 19 of the total AAs (TAAs) at15 m and 25 m depth respectively The specificEAA methionine constituted 125 plusmn 004 (15 m)and 137 plusmn 010 (25 m) of the TAAs whereasanother EAA lysine constituted 325 plusmn 007 (15 m)and 406 plusmn 051 of TAAs (25 m)

Pigments

The tissue pigment contents ranged from 119minus249 mg chl a g DMminus 1 062minus109 mg fucoxanthin gDMminus1 001minus004 mg violaxanthin g DMminus1 and001minus 003 mg β-carotene g DMminus1 (Fig 6) Highercontents of fucoxanthin violaxanthin and β-carotene were found in algae cultivated at 25 mdepth than at 15 m Also there was a significantdifference in the content of the 3 pigments amongsites (Table 3) with algae cultivated at Odby Bayyielding the highest concentrations Regardingchl a there was a significant interaction effectbetween site and depth with higher concentrationsof chl a at 25 m depth as compared to 15 m(Table 3) except at Lysen Broad where no signifi-cant difference in the chl a content between culti-vation depths was ob served (Fig 6A Table 3) The

628

Bio

mas

s in

crea

se (g

FW

mndash1

)B

iom

ass

yiel

d in

Jun

e (g

FW

mndash1

)

Biofouling (g FW mndash1) Average temperature (degC)

Average ortho-P concentration (microM)PAR (micromol photons mndash2 sndash1)

Bio

foul

ing

(g F

W m

ndash1)

A B

C D

Fig 4 Correlations between environmental parameters and Saccharina latissima biomass yields and quality (A) Biomass in-crease or decrease in each of the 2 periods (early and late spring) as a function of light availability (B) specific growth rate tis-sue N and dry matter (DM) as a function of P availability in early spring (C) biomass yield in June as a function of the biofoul-ing in June (linear regression p = 0003 R2 = 028) (D) biofouling in June as a function of average water temperature Datarepresent means plusmn SE n = 3 Datapoints represent values for 4 stations (2 depths each) Significant correlations are indicatedby solid lines non-significant relations by dotted lines Statistics for correlations in (ABD) are given in Table S2 in the

Supplement at wwwint-rescomarticlessupplq008p619_supppdf


tissue concentrations of all pigments were nega-tively related to light availability (Table S2)

Harmful metals

The tissue concentrations of the harmful metals AsPb and Cd showed significant differences betweensites andor cultivation depths (Tables 3 amp 5) withhigher concentration of Pb and lower concentrationsof As at Fur Sund as compared to the other sites No

significant differences were observed in tissue Hgconcentrations between sites and cultivation depths(Tables 3 amp 5)

Metal concentrations ranged between (mean plusmn SE)990 plusmn 093 and 3167 plusmn 107 mg As kg DMminus1 091 plusmn013 and 172 plusmn 008 mg Cd kg DMminus1 111 plusmn 020 and1760 plusmn 333 mg Pb kg DMminus1 and between 018 plusmn 001and 103 plusmn 040 mg Hg kg DMminus1 (Table 5) The tissueconcentrations of As were positively correlated toSGR (linear regression p = 0004 R2 = 0285 F =10556 df = 23 slope = 6207) whereas the tissue Cd

629

Fig 5 Tissue concentrations of (A) dry matter (DM of fresh weight) (B) carbon (C) (C) nitrogen (N) (D) phosphorus (P)as well as bio-mitigation capacity of (E) N and (F) P of cultivation of Saccharina latissima harvested in April at the 5 cultivationsites at 15 (white bars) and 25 m depth (grey bars) Solid bars represent batch 1 of seeded lines and crossed bars represent

batch 2 Data represent means plusmn SE n = 3


concentrations were positively correlated to sedi-ment concentrations of Cd (linear regression p lt00001 R2 = 0679 F = 51878 df = 23 slope = 2327)

DISCUSSION

Pelagic environment

The lack of correlation between the availability ofDIN and ortho-P indicated different origin of the 2nutrients The availability of DIN was negatively cor-related to salinity indicating input with freshwaterrun-off from the surrounding agricultural areas Theeffect of freshwater run-off was also observed inLoslashgstoslashr Broad and Skive Fjord as a decrease in salin-ity over the cultivation period

The positive correlation between ortho-P and chl aindicated that the availability of P was controlling theprimary production in Limfjorden in early springwith DIN concentrations being too high to be limit-ing P-limitation has previously been observed in

eutrophic coastal regions including parts of Limfjor-den (Lyngby 1990 Holmboe et al 1999 Lyngby et al1999 Pedersen et al 2010) as a consequence of amore efficient sewage treatment reducing the emis-sions of P as compared to N to the marine environ-ment (Conley et al 2000 Kronvang et al 2005) Inlate spring the lack of correlation between nutrientsand pelagic phytoplankton biomass indicated thatother factors came into play controlling phytoplank-ton biomass potentially grazing (Maar et al 2010) asalso indicated by the increasing density of biofoulingorganisms (filter-feeders)

The general inverse reflection of the pelagic phyto-plankton biomass (chl a) by the photon flux density atcultivation depth indicated a close coupling be tweenpelagic phytoplankton density and turbidity as iscommon for Limfjorden (Krause-Jensen et al 2012)However impaired light conditions were also ob -served in winter in particular in Nissum Broad andSkive Fjord most likely as a consequence of highwind speeds causing resuspension (Nissum) andorsoft sediment that is easily resuspended (Skive)

630

Fig 6 Tissue concentrations of the pigments (A) chlorophyll a (B) fucoxanthin (C) violaxanthin and (D) β-carotene in Saccharina latissima harvested in April at 15 (white bars) and 25 m depths (grey bars) Solid bars represent batch 1 of

seeded lines and crossed bars represent batch 2 Data represent means plusmn SE n = 3


Biomass yield and growth performance

The Saccharina latissima biomass yields and sporo-phyte lengths obtained at the 5 sites in Limfjordenwere generally low compared to values reportedfrom other cultivation trials in Europe (Peteiro ampFreire 2009 2013b Edwards amp Watson 2011 Forbordet al 2012 Handaring et al 2013) and in particular inDenmark (Marinho et al 2015a Nielsen 2015) Thefrond lengths were comparable to trials from Norway

with a shorter grow-out period (84 vs166 d at Faeligrker Vig) (Forbord et al2012) and the sporophyte lengths ob -tained at Lysen Broad were fully com-parable to earlier trials at the samesite (Wegeberg 2010) Only sporo-phytes obtained in the German BalticSea were smaller with maximumlengths of 20 cm obtained in a 1 yrgrow-out period (Roumlssner amp Krost2012) The low biomass yields wereex plai ned by several factors (1) lightlimitation (2) P-limitation reducingthe SGR and contents of DM and N inthe biomass and (3) the high degreeof biofouling forcing an early harvest

The higher yield observed at Faeligr -ker Vig was most likely a conse-quence of the combination of less tur-bid waters at this site during winterand early spring as well as the earlierdeployment (October instead of De -cem ber) which may have given thejuvenile sporophytes there a headstart in growth as has been docu-mented from trials in Spain and Ire-land (Peteiro amp Freire 2009 Edwardsamp Watson 2011) The fact that thesporophytes were derived from a dif-ferent batch of seeded lines andpotentially could have been of supe-rior quality was not supported by vi -sual inspection at deployment Com-paring only sporophytes from Batch 1the growth performance was best atOdby Bay where light conditions im -proved mar ked ly from April to June

The results in general support lightas a main controlling factor for growthof cultivated S latissima in Limfjordenin spring In early spring light waspositively correlated to growth andthe average PAR (100minus400 micromol pho-

tons mminus2 sminus1) reflected a photon flux density withinthe range reported to saturate photosynthesis of Slatissima (Esat 20minus500 micromol photons mminus2 sminus1 Bartschet al 2008) In contrast in late spring the availablePAR exceeded Esat and in this period the higheraverage PAR appeared to have a negative effecton growth since mainly the sporophytes from the deepest cultivation depth showed increased growthrates between April and June A positive correlationbetween frond length and salinity has previously

631

Cultivation site Depth As Cd Pb Hg(m)

Concentration in fresh biomass (mg kg FWminus1)Odby Bay 15 402 plusmn 018 016 plusmn 002 015 plusmn 003 002 plusmn 000

25 331 plusmn 021 019 plusmn 000 109 plusmn 003 008 plusmn 001Lysen Broad 15 234 plusmn 117 016 plusmn 008 031 plusmn 018 004 plusmn 002

25 nd nd nd ndFur Sund 15 067 plusmn 002 006 plusmn 000 111 plusmn 015 nd

25 042 plusmn 021 004 plusmn 002 068 plusmn 034 ndFaeligrker Vig 15 335 plusmn 007 011 plusmn 001 027 plusmn 008 004 plusmn 001

25 208 plusmn 060 008 plusmn 002 026 plusmn 004 002 plusmn 001Riisgaarde Broad 15 338 plusmn 018 026 plusmn 001 020 plusmn 002 016 plusmn 006

25 318 plusmn 051 027 plusmn 003 043 plusmn 002 008 plusmn 001

Limit values fresh biomassMussels ndash 1 15 05Fish meat ndash 005minus03 03 005minus1Food supplement ndash 1minus3a 3 01

Concentration in dry biomass (mg kg DMminus1)Odby Bay 15 3037 plusmn 038 119 plusmn 006 111 plusmn 020 018 plusmn 001






Limit values dry biomassFeed 40 (10b) 1minus2b 5bminus10Sludge 25c 08 120 08aFood supplement derived from seaweed bcomplete feed (As Cd for pet ani-mals based on seaweed) climit value only for use as fertilizer in private gardens

Table 5 Tissue metal concentrations of the biomass harvested in April expressedas ppm of fresh biomass (fresh weight) (mg kg FWminus1) and as ppm of dry matter (mgkg DMminus1) The concentrations are compared to the limit values of fresh biomass forfood and food supplement according to the EU food legislation (EU 2008b) to thelimit values of dry biomass for use in feed according to the EU feed legislation (EU2013) as well as to the limit values according to the Danish regulations on sludgeused as fertilizer (Danish Ministry of Environment 2006) Data are mean plusmn SE n =3 nd no data Underscored numbers indicate tissue concentrations exceedinglimit values for food supplement Numbers in bold indicate tissue concentra tionsexceeding limit values for feed and numbers in italics indicate tissue concentra-

tions exceeding limit values for use as fertilizer


been suggested (Nielsen et al 2014) and re ducedfrond length at lower salinities may be a conse-quence of increased allocation of energy to osmoreg-ulation at the expense of growth

The high turbidity of the waters in Limfjorden gen-erally limited the extent of the vertical productionpotential The turbidity in Limfjorden is primarily aconsequence of high nutrient loadings supporting ahigh pelagic primary production (Krause-Jensen etal 2012) Histo rically the primary production in mar-ine waters is considered to be controlled by N avail-ability (Ho warth 1988) Limfjorden however is anestuary with strong influence of freshwater run-offfrom agricultural land and in this study DIN wasavailable in the water column until late springearlysummer The limiting nutrient appeared to be Psince the bioavailable P disappeared with the onsetof the phytoplankton spring bloom in early springand subsequently P availability appeared to controlthe primary production That P rather than N avail-ability controlled the growth performance of S latis-sima in this study was supported by several observa-tions tissue NP ratios were already in early springalmost 3 times as high as other reports of kelp NPratios (9minus251) (Atkinson amp Smith 1983) the tissue Pcontents were generally below the P concentrationdefined as being critical for growth (022 P of DM694 micromol P gminus1 DM) as suggested by Pedersen et al(2010) whereas the tissue N concentrations were notbelow the concentrations critical for growth (NC)of 171 as sugges ted for brown algae (Pedersen ampBorum 1997) and 188 specifically for S latissima(Chapman et al 1978) and finally SGRs in earlyspring corresponded positively to the ortho-P con-centrations Phosphorus limitation of macroalgaegrowth has previously been observed (Pedersen etal 2010)

Biofouling of the biomass precluded a late summerharvest of the sporophytes and thus a biomass build-up over summer Devastation of biomass by biofoul-ing has been reported from cultivation trials in Nor-way (Handaring et al 2013) Spain (Peteiro amp Freire2013a) as well as from other trials in Denmark(Wegeberg 2010 Marinho et al 2015a Nielsen2015) and the phenomenon appears to be coupled torelatively sheltered locations with established natu-ral or cultured populations of suspension-feedersTemperature generally exerts positive control on thegrowth and development of juvenile filter-feeders(Widdows 1991 Nasrolahi et al 2013) and in thisstudy biofouling was positively correlated to temper-ature even within a very narrow range of tempera-ture differences In the eutrophic environment in

Limfjorden food (phytoplankton) is not a limitingfactor for the juvenile filter-feeders whereas suitablesubstrate for settling might be Thus any substrateintroduced in the water column including macro-algae sporophytes has the risk of becoming fullyovergrown In this study the degree of biofoulingwas most pronounced at the deeper cultivationdepths but did not correspond to the estimateddegree of exposure at the individual cultivation sitesThe negative correlation between biofouling andlength growth may indicate that heavily bio-fouledfronds did not grow well or that once the fronds weresufficiently long in early spring they were able toavoid the biofouling the latter partly being sup-ported by recent findings showing that dense naturalkelp canopies tend to be less prone to settling of epi-phytic organisms (Bennett et al 2015)

Biomass quality

If harvested before the onset of biofouling S latis-sima cultivated in Faeligrker Vig Limfjorden provideda rich source of protein essential AAs and pigmentswith bioactive properties suitable for food or feedpurposes Availability of ortho-P influenced the qual-ity of the biomass significantly increasing tissue DMand N content

As for growth performance the biochemical com-position of S latissima biomass showed large differ-ences among cultivations sites Tissue P concentra-tions were generally in the same range as reportedfrom cultivation trials in Kattegat Denmark (Marinhoet al 2015a) A doubling of tissue P concentrationsin macroalgae cultivated at 25 m depth at 2 sites(Odby Bay and Riisgaarde Broad) where the seabedwas characterized by soft mud indicated local differ-ences in resuspension events as also indicated by thepoorer light conditions at these sites during winterand early spring The N content of 3minus45 of DM inApril was high for this time of the year compared tonatural populations and cultivated biomass fromother locations in Denmark (Nielsen et al 2014 2016Marinho et al 2015a) and was more comparable to Ncontents obtained in close proximity to fish farms orin late autumnwinter months where environmentalN concentrations are naturally higher (Gevaert et al2001 Handaring et al 2013)

The high tissue N concentrations were indicative ofhigh tissue protein concentration in the range of160minus170 Compared to other cultivation trials inDenmark this protein content was high for April(Marinho et al 2015b) but comparable to what has

632


been reported elsewhere (Black 1950) The ratio ofEAAs and the content of methionine and lysine inthe biomass in Faeligrker Vig were higher than de -scribed from S latissima biomass cultivated in prox-imity to fish cages and thus the S latissima biomassfrom Limfjorden represented a biomass with an at -tractive profile for applications within food or feed(Marinho et al 2015b)

Light availability influenced biomass quality cor-relating positively to tissue C content but negativelyto the tissue concentrations of P and all pigmentsThe pigment contents in S latissima from the 5 sitesvaried by a factor of 2minus5 and were generally high dueto the turbid conditions in particular in the deepercultivated biomass The tissue contents of chl a andfucoxanthin in the biomass were up to 8 and 5 timeshigher respectively than the tissue contents in Slatissima fronds cultivated in autumn under low lightconditions in tanks (Boderskov et al 2016) Theantioxidant and other bioactive properties of fuco-xanthin have recently drawn attention as beingactive against obesity and diabetes (Miyashita et al2011 DrsquoOrazio et al 2012) Thus high contents ofthis pigment in kelp biomass are attractive for appli-cations in (functional) food and feed

The positive effects of temperature on DM N and Ptissue contents may in part be explained by in -creased activity of enzymes involved in nutrientassimilation over the range of temperatures experi-enced during early spring (Davison amp Davison 1987)

Only extreme levels of pollution are considered tocause significant reduction in production of marineplants (Sharp et al 1988) however tissue concentra-tions of specific metals (ie As Cd Hg and Pb) mayprevent the use of the produced biomass for foodfood supplement feed or fertilizer (Miljoslash styrelsen2006 EU 2008b 2013) In this study we only had access to sediment concentrations of selected metalsfrom the national environmental monitoring pro-gram as water concentrations are not monitored Forthis reason we had no basis for estimating the envi-ronmental metal concentrations experienced by thealgae and the potential direct consequential physio-logical impacts However through the sediment con-centrations we may get an indication of the locallevel of environmental pollution and an indication ofwhether this may be a predictive tool in future siteselection The tissue concentrations of As Pb and Hgfluctuated by a factor of 3minus5 between the 5 cultiva-tion sites whereas the tissue concentrations of Cdwere relatively constant The tissue metal concentra-tions in this study did not exceed the limit values setfor human consumption and only at one site (Riis-

gaarde Broad) would the tissue Hg concentrationsprevent the use of the biomass for food supplementsFor use in animal feed the Pb concentrations in thebiomass cultivated at Fur Sund and Odby Bay (25 m)exceeded limit values whereas the Cd concentra-tions would prevent the use for fertilizer of the bio-mass cultivated at any of the sites (limit value =08 mg kg DMminus1 Danish Ministry of Environment2006) The As concentrations found in this study didnot exceed limit values for use in food or feed andthey were generally lower than what has been foundin natural populations in more open Danish waters(Nielsen et al 2016) Since tissue As concentrationswere positively correlated to growth bioaccumu -lation may explain the higher As concentrationsfound in older individuals in natural populations ascompared to the 1-yr-old cultivated individuals inthis study The linear correlation be tween tissue andsediment Cd concentrations indicated that elevatedsediment concentrations of Cd may cause increasedavailability and hence uptake into the seaweed tis-sue At the 2 stations with the highest sediment Cdconcentrations (Riisgaarde 044 ppm and Lysen025 ppm) the seabed sediment and depth as well asthe degree of exposure diffe red At Riisgarde thesediment was soft and muddy and a high degree ofexposure increased the risk of resuspension of thesediment into the water column potentially increas-ing the availability of Cd to the seaweed Below thecultivation structures at the more sheltered and shal-low site in Lysen Broad the seabed consisted of finesand and clay and there the depth was lower Thusdespite diffe rent conditions regarding sedimentdepth and exposure the sediment concentration ofCd demonstrated a potential value as an instrumentin site selection

Bio-mitigation

The bio-mitigation capacity of S latissima in thisstudy proved to be relatively poor in comparison withother studies of Laminariales in Denmark (Marinhoet al 2015a) and Scotland (Sanderson et al 2012)where up to 4 and 14 times more N was removed permetre of seeded line respectively The low bio- mitigation capacity was primarily a consequence ofthe low biomass yields obtained due to turbid watersP-limitation and biofouling Thus in highly eutrophicwaters such as Limfjorden the pelagic primary pro-ductivity limits the efficiency of kelp cultivation as atool for bio-mitigation of N Consequently care shouldbe taken when extrapolating the bio- mitigation ca -

633


pacities described in the literature to any cultivationsite assuming high areal productivity (ie Holdt ampEdwards 2014) This study highlights the limitationsand challenges of kelp production for bio-mitigationpurposes in eutrophic waters where bio-mitigation isneeded the most

Site selection

Even within the relatively homogenous eutrophicLimfjorden production yields varied by a factor of 10between different basins Environmental monitoringdata proved useful as predictive instruments for siteselection Regarding the pelagic parameters gener-ally highly N-enriched sites with low light availabil-ity high pelagic NP ratios and high chl a concentra-tions should be avoided as they supported a lowerbiomass production and in conjunction with margin-ally higher temperatures during spring also presen -ted a higher risk of biofouling

Regarding sediment characteristics 2 recommen-dations for site selection are suggested (1) kelp culti-vation should be reconsidered in shallow areas dom-inated by soft muddy seabed as resuspension eventstend to increase turbidity and (2) sediment Cd con-centrations could be investigated as a part of siteselection High sediment Cd concentrations were re -flected as high Cd concentrations in seaweed bio-mass and depending on the post-harvest use of thebiomass high tissue Cd concentrations may have astrong negative impact on biomass value

CONCLUSIONS

Basin-scale differences in light and nutrient avail-ability seabed properties and sediment metal con-centrations cause pronounced local differences in thesuitability of an area for cultivation of Saccharinalatissima in terms of biomass yield and quality as wellas bio-mitigation and hence impact the profitabilityof potential seaweed production When selectingsites for cultivation of S latissima highly N-enrichedsites with low light availability high pelagic NPratios and chl a concentrations and high sedimentCd concentration should be avoided The highly N-enriched waters of Limfjorden appeared less suitablefor efficient biomass production of S latissima due toreduced light conditions and P-limitation in earlyspring and a high risk of devastating biofouling im -pairing growth performance bio-mitigation capacityas well as biomass quality However S latissima bio-

mass harvested in spring in Limfjorden had a highcontent of pigments and protein with a beneficialamino acid composition and proved highly suitablefor food or feed purposes

Acknowledgements The work behind this article was sup-ported by lsquoDe Lokale Dyderrsquo (The Market DevelopmentFund) the PEER project on improved resource flows be -tween human and natural systems the Macroalgae Biorefin-ery (MAB3) (Danish Council for Strategic Research) andfinally a grant supplied by the National Centre for Environ-ment and Energy (DCE) The authors thank Kristian Odder-shede Nielsen Helge Boesen Finn Bak and Pascal Barreaufor the field work Tanja Quottrup Egholm Kitte LindingGerlich Gitte Jacobsen Anne Marie Plejdrup and PeterKofoed for skillful lab work Ole Manscher and David Rytterfor extraction of data from ODAM Tinna Christensen forgraphical assistance and 3 anonymous reviewers for con-structive comments improving the manuscript

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Nasrolahi A Pansch C Lenz M Wahl M (2013) Temperatureand salinity interactively impact early juvenile develop-ment a bottleneck in barnacle ontogeny Mar Biol 160 1109minus1117

Neori A Chopin T Troell M Buschmann AH and others(2004) Integrated aquaculture rationale evolution andstate of the art emphasizing seaweed biofiltration inmodern mariculture Aquaculture 231 361minus391

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Nielsen MM Bruhn A Rasmussen MB Olesen B LarsenMM Moslashller HB (2012) Cultivation of Ulva lactuca withmanure for simultaneous bioremediation and biomassproduction J Appl Phycol 24 449minus458

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636

Editorial responsibility Alejandro Buschmann Puerto Montt Chile

Submitted April 4 2016 Accepted August 23 2016Proofs received from author(s) October 14 2016


system service (Daly 1998) provided by cultivatedalgae in terms of recapturing nutrients in coastalareas is of commercial and societal interest mdash as com-pensation for increased aqua culture activities (San -derson et al 2012 Handaring et al 2013 Smale et al2013 Holdt amp Edwards 2014 Marin ho et al 2015a) oras a potential instrument for circular nutrient man-agement improving the eco logical status of eutro -phic marine areas (Seghetta et al 2016) in line withthe EU Marine Strategy Framework Directive (EU2008a 2014)

In Europe the effort concerning cultivation of largebrown algae (Laminariales) in particular is increas-ing The most commonly cultivated brown algae spe-cies in Europe Saccharina latissima ((Linnaeus) CELane C Mayes Druehl amp GW Saunders) has beencultivated on a smaller or larger scale in IrelandScotland Germany Holland Spain Norway FaroeIslands and Denmark (ie Buck amp Buchholz 20042005 Buck et al 2008 Werner et al 2009 Wegeberg2010 Edwards amp Watson 2011 Forbord et al 2012Handaring et al 2013 Peteiro amp Freire 2013b Wegeberget al 2013 Marinho et al 2015a) The achieved bio-mass yields and the biochemical composition of thebiomass vary considerably seasonally and spatiallyprimarily because of different environmental condi-tions (Edwards amp Watson 2011 Handaring et al 2013Peteiro amp Freire 2013ab Marinho et al 2015a) Inreturn the algae production also exerts an impact onthe environmental conditions through the removal ofnutrients (Troell et al 1999 Stephens et al 2014Marinho et al 2015a) Thus algae cultivation sitesshould be carefully selected for optimizing biomassproduction biomass quality as well as the non-valueecosystem services (Kerrison et al 2015) Further theseasonal timing of the deployment and harvest of thealgae needs to be optimized according to local envi-ronmental conditions The focus of the optimizationie high protein yield or high carbohydrate yield willdepend on the final application of the biomassSporophytes of Lami naria species store nutrients forlength growth during periods when environmentalnutrients concentrations are high (Bartsch et al2008) Thus high environmental nutrient concentra-tions favour high tissue nitrogen (N) concentrationsin wild S latissima up to 35 N of dry matter (DM)(Gevaert et al 2001 Nielsen et al 2014) up to 50N of DM when cultivated in close proximity to fishaquaculture (Handaring et al 2013 Marinho et al 2015a)and even up to 67 N of DM when cultivated underhighly eu trophic conditions (Nielsen 2015) Themolar Nphosphorus (P) ratio is commonly in therange of 9minus251 (Atkinson amp Smith 1983) and P con-

centrations of up to 08 of DM are reported in nutri-ent-rich waters (Marinho et al 2015a) High tissue Nconcentrations reflect a correspondingly high con-tent of proteins (Manns et al 2014 Marinho et al2015b Angell et al 2016) Consequently both thebiomass quality and the bio-mitigation capacity of theproduced algae increase in nutrient-rich waters in -creasing the value of the biomass as well as im -proving the environmental condition of the waterbody through harvest and thus removal of nutrients

Of the 21 Danish water bodies Limfjorden receivesthe highest annual net supply of nutrients (82 t N and030 t P kmminus2 yminus1 Seghetta et al 2016) These high nu-trient loadings have caused a regime shift in the fjordfrom benthic to pelagic primary production (Krause-Jensen et al 2012) The high pelagic prima ry produc-tion supports a substantial stock of benthic suspensionfeeders including blue mussels Mytilus edulis L sup-porting a local mussel fishery (Maar et al 2010 Tim-mermann et al 2014) Mussel farming has been suc-cessfully tested as an instrument to recapture nutrientsand improve the ecological status of Limfjorden (Pe-tersen et al 2014) and farming of long-line blue mus-sels is an emerging business in Limfjorden Alongwith the development of the mussel farming industryinterest in macroalgae cultivation is increasing partlybecause the 2 crops may be cultivated using the samestructures (Nielsen 2015) Due to the high environ-mental nutrient concentrations cultivation of largebrown algae in a water body like Limfjorden wouldtheoretically hold a potential for the production of aSaccharina biomass with high protein content repre-senting a higher value for the food or feed market Atthe same time the potential of seaweed cultivation asan instrument for circular nutrient managementwould be maximized Despite the relatively small sizeof Limfjorden (1500 km2) local environmental condi-tions differ considerably between the different basins(Maar et al 2010) Cultivation of S latissima has todate been documented only once at 1 site in Lim -fjorden indicating a potential for cultivation of Slatissima This study however also demonstrates theneed for investigating optimal timing of cultivationand harvest in order to maximize biomass yield andavoid biofouling (Wegeberg 2010)

Testing and evaluating the interactions betweenlocal environmental conditions and bio mass yieldquality and potential for bio-mitigation throughnutrient recapture of cultivated kelps in coastalwaters is needed before im plementing cultivation ona larger scale This applies not only to Limfjordenbut to any water body where macroalgae cultivationis intended

620








Environmental data


621


(degN) (degE)















622










(1)








t

t

SGR() 100ln

FWFW0= times

⎛⎝⎜

⎞⎠⎟

623


Data analysis


RESULTS



Salinity


Temperature


Inorganic nutrients


Pelagic chl a


Light


624







625








626





DM tissue N and P






627













Protein and AAs


Pigments


628

Bio

mas

s in

crea

se (g

FW

mndash1

)B

iom

ass

yiel

d in

Jun

e (g

FW

mndash1

)



Bio

foul

ing

(g F

W m

ndash1)

A B

C D





Harmful metals




629





DISCUSSION

Pelagic environment





630










631























Biomass quality




632







Bio-mitigation


633



Site selection



CONCLUSIONS




LITERATURE CITED












634





































635


































636










Environmental data


621


(degN) (degE)















622










(1)








t

t

SGR() 100ln

FWFW0= times

⎛⎝⎜

⎞⎠⎟

623


Data analysis


RESULTS



Salinity


Temperature


Inorganic nutrients


Pelagic chl a


Light


624







625








626





DM tissue N and P






627













Protein and AAs


Pigments


628

Bio

mas

s in

crea

se (g

FW

mndash1

)B

iom

ass

yiel

d in

Jun

e (g

FW

mndash1

)



Bio

foul

ing

(g F

W m

ndash1)

A B

C D





Harmful metals




629





DISCUSSION

Pelagic environment





630










631























Biomass quality




632







Bio-mitigation


633



Site selection



CONCLUSIONS




LITERATURE CITED












634





































635


































636













622










(1)








t

t

SGR() 100ln

FWFW0= times

⎛⎝⎜

⎞⎠⎟

623


Data analysis


RESULTS



Salinity


Temperature


Inorganic nutrients


Pelagic chl a


Light


624







625








626





DM tissue N and P






627













Protein and AAs


Pigments


628

Bio

mas

s in

crea

se (g

FW

mndash1

)B

iom

ass

yiel

d in

Jun

e (g

FW

mndash1

)



Bio

foul

ing

(g F

W m

ndash1)

A B

C D





Harmful metals




629





DISCUSSION

Pelagic environment





630










631























Biomass quality




632







Bio-mitigation


633



Site selection



CONCLUSIONS




LITERATURE CITED












634





































635


































636








(1)








t

t

SGR() 100ln

FWFW0= times

⎛⎝⎜

⎞⎠⎟

623


Data analysis


RESULTS



Salinity


Temperature


Inorganic nutrients


Pelagic chl a


Light


624







625








626





DM tissue N and P






627













Protein and AAs


Pigments


628

Bio

mas

s in

crea

se (g

FW

mndash1

)B

iom

ass

yiel

d in

Jun

e (g

FW

mndash1

)



Bio

foul

ing

(g F

W m

ndash1)

A B

C D





Harmful metals




629





DISCUSSION

Pelagic environment





630










631























Biomass quality




632







Bio-mitigation


633



Site selection



CONCLUSIONS




LITERATURE CITED












634





































635


































636




Data analysis


RESULTS



Salinity


Temperature


Inorganic nutrients


Pelagic chl a


Light


624







625








626





DM tissue N and P






627













Protein and AAs


Pigments


628

Bio

mas

s in

crea

se (g

FW

mndash1

)B

iom

ass

yiel

d in

Jun

e (g

FW

mndash1

)



Bio

foul

ing

(g F

W m

ndash1)

A B

C D





Harmful metals




629





DISCUSSION

Pelagic environment





630










631























Biomass quality




632







Bio-mitigation


633



Site selection



CONCLUSIONS




LITERATURE CITED












634





































635


































636









625








626





DM tissue N and P






627













Protein and AAs


Pigments


628

Bio

mas

s in

crea

se (g

FW

mndash1

)B

iom

ass

yiel

d in

Jun

e (g

FW

mndash1

)



Bio

foul

ing

(g F

W m

ndash1)

A B

C D





Harmful metals




629





DISCUSSION

Pelagic environment





630










631























Biomass quality




632







Bio-mitigation


633



Site selection



CONCLUSIONS




LITERATURE CITED












634





































635


































636








626





DM tissue N and P






627













Protein and AAs


Pigments


628

Bio

mas

s in

crea

se (g

FW

mndash1

)B

iom

ass

yiel

d in

Jun

e (g

FW

mndash1

)



Bio

foul

ing

(g F

W m

ndash1)

A B

C D





Harmful metals




629





DISCUSSION

Pelagic environment





630










631























Biomass quality




632







Bio-mitigation


633



Site selection



CONCLUSIONS




LITERATURE CITED












634





































635


































636





DM tissue N and P






627













Protein and AAs


Pigments


628

Bio

mas

s in

crea

se (g

FW

mndash1

)B

iom

ass

yiel

d in

Jun

e (g

FW

mndash1

)



Bio

foul

ing

(g F

W m

ndash1)

A B

C D





Harmful metals




629





DISCUSSION

Pelagic environment





630










631























Biomass quality




632







Bio-mitigation


633



Site selection



CONCLUSIONS




LITERATURE CITED












634





































635


































636





Protein and AAs


Pigments


628

Bio

mas

s in

crea

se (g

FW

mndash1

)B

iom

ass

yiel

d in

Jun

e (g

FW

mndash1

)



Bio

foul

ing

(g F

W m

ndash1)

A B

C D





Harmful metals




629





DISCUSSION

Pelagic environment





630










631























Biomass quality




632







Bio-mitigation


633



Site selection



CONCLUSIONS




LITERATURE CITED












634





































635


































636





Harmful metals




629





DISCUSSION

Pelagic environment





630










631























Biomass quality




632







Bio-mitigation


633



Site selection



CONCLUSIONS




LITERATURE CITED












634





































635


































636





DISCUSSION

Pelagic environment





630










631























Biomass quality




632







Bio-mitigation


633



Site selection



CONCLUSIONS




LITERATURE CITED












634





































635


































636










631























Biomass quality




632







Bio-mitigation


633



Site selection



CONCLUSIONS




LITERATURE CITED












634





































635


































636








Biomass quality




632







Bio-mitigation


633



Site selection



CONCLUSIONS




LITERATURE CITED












634





































635


































636









Bio-mitigation


633



Site selection



CONCLUSIONS




LITERATURE CITED












634





































635


































636





Site selection



CONCLUSIONS




LITERATURE CITED












634





































635


































636







































635


































636




































636



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