Yun Hee Kang,Jae Ran Hwang,Ik Kyo Chung,and Sang Rul Park

1) Marine and Environmental Research Institute,Jeju National University,Jeju 609-756,Republic of Korea

2) Marine Environmental Research Division, National Fisheries Research and Development Institute,Busan 619-705, Republic of Korea

3) Division of Earth Environmental System, Pusan National University, Busan 609-735, Republic of Korea

4) School of Marine Biomedical Sciences, Jeju National University, Jeju 690-756, Republic of Korea

1 Introduction

Seaweed-based integrated aquaculture techniques serve environmental sustainability by integrating fed aquaculture (e.g.,finfish and shrimp) with inorganic and organic extractive aquacultures (e.g.,seaweed and shellfish) (Buschmannet al.,2001; Chopinet al.,2001).These techniques have been proposed as a means for developing environmentally sound aquaculture practices and resource management through a balanced coastal ecosystem approach,which removes excessive nutrients and contributes to economic diversification by producing additional value-added products (Chopinet al.,2008).Traditionally,seaweed-based integrated aquaculture techniques are used to provide a feed component for co-cultured fish in Korea(Fig.1).Additionally,the governments of several other countries have begun to emphasize the role of seaweed as a biofilter in estuarine and coastal areas,where it prevents nutrient enrichment from effluents (Buschmannet al.,2009).

The use of macroalgae as a biofilter material in seaweed-based integrated aquaculture has been proved to be an excellent example of ecotechnology (Neoriet al.,2004;Hernándezet al.,2006).Examples of such systems as reviewed by Troellet al.(2003),are integrated salmon-Gracilaria,integrated seabream-Ulva,integrated salmon-Laminaria(currentlysaccharina),and integrated fish-Pophyra.Thus,systematic and comprehensive data based on the ecophysiological characteristics of seaweeds are required for establishing guidelines or standards for the selection of optimal seaweed species (NFRDI,1994;Neoriet al.,2004; Kanget al.,2008).

The choice of seaweed species in seaweed-based integrated aquaculture should be made according to their ecophysiological characteristics and market value (Neoriet al.,2004).The ecophysiological characteristics such as biofiltration capacity,biochemical composition,and growth rate can be identified as the major limitations to successful macroalgae aquaculture (Santos,2006).Primarily,optimal seaweed species for IMTA should exhibit high uptake rates and uptake efficiency to remove nutrients from effluents (Chopinet al.,2008).In addition,they should show fast growth rates and be able to accumulate large amounts of nutrients,especially nitrogen (N).Seaweed should be less affected by the type of environmental stresses encountered during culture,and should maintain health for considerable periods of time.In addition,the ease of cultivation is a prerequisite when selecting seaweed species suitable for IMTA.

Fig.1 An example of fish-seaweed co-culture around Hawtae Island on the southern coast of Korea and nutrient uptake rate experiments in the laboratory.The brown alga Saccharina japonica is cultured using a line method around fish farms: A,a fish farm; B,seaweed around a fish farm; C,brown algae (S.japonica); and D,uptake rate experiments in the laboratory.

The economic value of seaweeds may be directly or indirectly related to their physiological health.The health of seaweeds should be monitored to prevent dramatic deterioration in their culture and to improve or maintain their quality.The biochemical composition and photosynthesis of seaweeds have been known to be powerful indices of physiological performance (Vergaraet al.,1993; Kanget al.,2009).Tissue N content may provide an efficient way for removing nutrients from eutrophic seawater (Heet al.,2008).In addition,responses in the growth and photosynthetic pigment concentrations of seaweeds are correlated with responses in tissue N content (Joneset al.,2002).Thus,it is necessary to evaluate the biochemical composition and photosynthesis of seaweeds when selecting optimal seaweed species.

The purpose of this study was to develop a seaweed species-selection index for selecting species suitable for seaweed-based integrated aquaculture.The species-selection index was modified based on the site-selection model developed by Shortet al.(2002).The index synthesizes available literature-based information,reference data,and physiological seaweed experiments to identify and prioritize species for this kind of aquaculture.The process for obtaining an index was divided into three steps: 1) the available physiological reference data were used to establish a preliminary seaweed-based integrated aquaculture suitability index (PSASI),which was then used to pre-screen and eliminate unsuitable seaweed species; 2) the PSASI-identified priority species were analyzed using laboratory experiments to determine nutrient uptake rate,nutrient reduction efficiency,tissue N content,and photosynthetic pigments; 3) based on the experimental results,a seaweed-based integrated aquaculture suitability index (SASI) score was calculated for each species.The species-selection index allowed us to reduce the necessary cost and effort on selecting seaweed suitable for given culture purposes (e.g.,seasonality and profitability).

2 Materials and Methods

Compilation of the species-selection index was carried out in three steps.Step I was to identify potential seaweed species according to available information,including previous physiological experimental data on seaweeds,yielding one of the scores that are incorporated into the calculation of the SASI.Step II involved the compilation of physiological characteristic data under various conditions for the highest-scoring species identified in Step I.Step III was the final calculation of the SASI based on the results of Steps I and II.

2.1 Step I: Identification of Potential Seaweed Species and PSASI I Rating

Step I involved reviewing available information on selecting potential seaweed genera for integrated multitrophic aquaculture (IMTA).The parameters of seaweed selection considered in Step I were economic value,ease of cultivation,and seaweed-based integrated aquaculture application.This information contributed to the PSASI,which was used as the first level of screening in optimal-species selection (Table 1).Information for the PSASI was collected from both published and unpublished literature.Each of the parameters in Step I received a rating (Table 1),and the PSASI I score was calculated as the product of these ratings.

Table 1 Data used in Step I of the preliminary seaweed-based integrated aquaculture suitability index (PSASI) for identifying potential seaweed species

The economic value of seaweed is an important consideration when selecting species for IMTA.Nutrients released from fish,shrimps,and bivalves are suitable for seaweed growth,and consequently,seaweed production can be dramatically increased in IMTA (Neoriet al.,2004).Thus,for profit maximization,it is important to select seaweeds with high economic value and good market standing.In this study,we gave seaweed a rating of 2 if it is used as a food source or in medical or industrial materials.Otherwise,the seaweed received a rating of 1.The economic value of seaweed was based on data from the FAO (2004) and Ohet al.(1990).

Even though seaweed has high market value and a high uptake rate,its cultivation must nonetheless be easy and efficient.Thus,if a seaweed genus could be cultivated easily and efficiently,it was rated 2.Otherwise,it was rated 1.Information on seaweed culture can be derived from the literature (e.g.,FAO,2004).Additionally,if a genus had been previously selected as a candidate for IMTA system application,it was rated 2.Otherwise,it was rated 1 (Troellet al.,2003).

2.2 Step Ⅱ: Physiological Characteristics of Seaweed Species

2.2.1 Seaweed collection

Six species of macroalgae from three divisions,Ulva compressa(Chlorophyta),Porphyra yezoensisandGracilaria incurvata(Rhodophyta),Ecklonia cava,andUndaria pinnatifida(Phaeophyta) were collected from the intertidal zone of a wave-exposed rocky shore at Geoje Island and a seaweed farm located in Ilkwang and Gamak bay,Korea.All thalli were rinsed to remove sediment,debris and epiphytes,and then acclimated in filtered seawater in a culture room (30 μmol photons m?2s?1; 12 : 12 light : dark photoperiod; 15 – 17 ℃) for 3– 4 d(Hanisak and Harlin 1978; Kanget al.,2008).The seawater was collected from the southwest part of the East Sea in October 2006.The average concentrations of nitrate (NO3–) and ammonium (NH4+) in seawater were less than 0.1 μmol L?1.The seawater was filtered first through Whatman glass-fiber filters (1.2 μm) and then through Advantec MFS membrane filters (0.2 μm).

2.2.2 PSASI Ⅱ experiment: ammonium uptake rate of seaweeds

The PSASI II experiment was conducted to measure the ammonium uptake rates of five species of seaweed.Specimens of 3.0 g fresh weight were put into a 500-mL corn bottle containing 300 mL of nutrient medium,which was prepared in triplicate.The initial NH4+concentrations of each medium were maintained at 0,10,50,100,200,300,500,and 750 μmol L?1(NH4Cl) with filtered seawater.The experiments were conducted in a controlledenvironment room with constant temperature (16℃ ±0.5 ℃) and light intensity (100– 150 μmol photons m?2s?1)for approximately 10 h.Control experiments were carried out without addition of algal materials.Water samples were collected from the chambers at consistent time intervals (1 ? 2 h) with NH4+concentrations analyzed immediately using the colorimetric technique of Parsonset al.(1984).At the end of the experiments,the dry weight(DW) of samples was measured by drying at 60 ℃ for 48 h.The uptake rate was plotted as a function of nutrient concentration,and NH4+uptake kinetics was derived using the Michaelis-Menten equation:

whereVis the NH4+uptake rate,Vmaxis the maximum uptake rate,Sis the NH4+concentration,andKmis the half-saturation value,that is,the nutrient concentration whereV=Vmax/ 2.The uptake rate was normalized to μmol NH4+g DW?1h?1.

Seaweeds with a highVmaxwere considered to be desirable for IMTA applications (Kraemeret al.,2004).Lobban and Harrison (1994) have reported that with low substrate concentrations,a high-affinity system operates,exhibiting a high degree of ion specificity and a low value ofKm,whereas with high substrate concentrations,a lowaffinity system is operative,exhibiting much less ion selectivity and a very high value forKm.Thus,the PSASI II ratings forVmaxwere assigned: 1 for < 100 μmol g?1DW h?1; 2 for ≥ 100 μmol g?1DW h?1.Considering that the NH4+levels in fish farm effluent were approximately 150 μmol L?1(Neoriet al.,1996; Carmonaet al.,2006),theKmof a seaweed was rated 1 if the level was less than 150μmol L?1and 2 if greater than or equal to 150 μmolL?1.

2.2.3 SASI experiments: NH4+ reduction efficiency,tissue N content,and chlorophyll a content

In the SASI experiments,NH4+reduction efficiency,tissue N content,and chlorophyllacontent of five seaweed species (U.compressa,P.yezoensis,G.incurvata,E.cava,andU.pinnatifida) were measured daily for 7 d at 17℃± 0.5 ℃ under 100μmol photons m?2s?1.Specimens of 5.0 g fresh weight were put into a 1000-mL flask containing 700 mL of medium (150 NH4+μmol L?1).The NH4+concentration of the media was determined based on the N concentration in fish farm effluent (Neoriet al.,1996; Carmonaet al.,2006).The culture medium was replaced daily during the experimental period.The NH4+reduction efficiency of seaweed was calculated using the difference between the initial and final NH4+concentrations (μmol L?1) in seawater according to the following equation

whereS0is the initial NH4+concentration,andStis the NH4+concentration aftertd.

For carbon (C) and N analysis,samples were taken before and after the experiments.The samples were dried for 48 h at 60 ℃,and then ground using a pestle and mo rtar.Approximately 2 – 3 mg of ground tissue was placed into a tin to determine the C and N contents using a CHN elemental analyzer (Vario-EL III),and the C : N molar ratios were calculated.

The chlorophyllacontent was extracted using N,N,dimethylformamide (DMF) following the methodology described by Wellburn (1994).The samples were examined spectrophotometrically after extraction with 4 mL of DMF for 24 h at 4 ℃ in the dark.

2.3 Step III: Calculating the SASI Score

At Step III,the SASI score was calculated,again as a multiplicative index,for each seaweed species (Table 2).The SASI was determined by combining the results of the PSASI I,PSASI II,and SASI,thus reflecting the results of the physiological tests on each seaweed.Table 2 shows the rating for each parameter.A PSASI rating of 1 was assigned if the PSASI score was ≤ 2 and 2 if the PSASI was over 2.The PSASI II rating was assigned as follows:1 if the PSASI score was < 2 and 2 if the PSASI was ≥ 2.

Table 2 Data from Step II in the calculation of the seaweedbased integrated aquaculture suitability index (SASI)

The SASI rating for reduction efficiency was based on NH4+concentrations in a water column observed on day 7.A score of 0 was assigned for < 30% reduction efficiency,1 for 30% – 75% reduction efficiency,and 2 for 75% reduction efficiency.The SASI ratings for the N content of seaweed were assigned as follows: 1 if the N content(mean ± SD) was less than the (mean – SD) of the initial pre-experiment samples or if data were not available for those parameters,and 2 if the N content was greater than or equal to the (mean – SD) of initial pre-experiment samples.The tissue N content is an optional SASI parameter that provides a finer resolution of the index.The chlorophyllacontent of the seaweed was rated 1 if the content(mean ± SD) was less than the (mean – SD) of the initial samples and 2 if it was greater than or equal to the (mean– SD) of the initial samples.

The SASI score was calculated with the equation:

3 Results and Discussion

3.1 PSASI I Data and Ratings: Characteristics of Seaweed Species Suitable for Application to IMTA

Genera were used for calculating the PSASI rating because seaweed species vary with region and local species should be used.The PSASI ratings,shown in Table 3,were derived from both published and unpublished literature.Five genera were collected and evaluated according to the three parameters employed in this study.All genera received a rating of 2 due to the economic values as food source or industrial/medical materials (Ohet al.,1990; FAO,2004),even though their values are not the same.It is possible that other genera,exceptEckloniaandUlva,can be relatively easily or industrially cultured.All genera exceptEckloniahave been found suitable for application to IMTA.Accordingly,Eckloniawas rated 1 for both cultivation and the application of seaweed-based aquaculture parameters (Troellet al.,2003; FAO,2004).In contrast,Ulvawas rated 1 for cultivation.Porphyra,Gracilaria, andUndariareceived a rating of 2 for both parameters.Based on the PSASI I scores for these ratings,Porphyra,Gracilaria, andUndariahad the highest PSASI I values (score = 8) of all tested genera,whereasEckloniahad the lowest PSASI I value (score = 2).Ulvahad PSASI I values of 4.

Table 3 Ratings of the PSASI I parameters and final PSASI I scores for seaweed species

3.2 PSASI II Data and Ratings: Ammonium Uptake Rates of Seaweeds

The NH4+uptake rates were determined for five seaweed species (U.pinnatifida,E.cava,P.yezoensis,G.incurvata,andU.compressa).In this study,theVmaxof the green and red algae was higher than that of the brown algae (Fig.2).Of all species,U.compressahad the highestVmax(140.3 μmol g?1DW h?1) and thus was assigned a rating of 2 in the PSASI II (Table 4 and Fig.2).Whereas theVmaxofP.yezoensis(111.5 μmol g?1DW h?1) was assigned a rating of 2 in the PSASI II,G.incurvatashowed a lowVmaxvalue (50.6 μmol g?1DW h?1) and received a rating of 1 (Table 4 and Fig.2).In particular,all brown algae (U.pinnatifidaandE.cava) showed very lowVmaxvalues (less than 30 μmol g?1DW h?1) and,accordingly,were assigned ratings of 1 in the PSASI II (Table 4 and Fig.2).

Fig.2 NH4+ uptake rates of seaweeds as a function of NH4+ concentration.Curves represent the best fits of the Michaelis-Menten equation; A, Undaria pinnatifida; B,Ecklonia cava; C, Gracilaria incurvata; D,Porphyra yezoensis; and E,Ulvacompressa.

In IMTA,the NH4+levels in fish farm effluent are relatively high,approximately 150 μmolL?1(Neoriet al.,1996; Carmonaet al.,2006).Therefore,seaweeds exhibiting highKmshould be utilized as biofilter material for NH4+effluent in IMTA,as the low-affinity system is operative and exhibits very high values ofKmat high concentrations (Lobban and Harrison,1994).Our results showed that theKmvalues of green and red algae were highest,whereas those of brown algae were relatively low.TheKmofP.yezoensisandU.compressawere 268.1 and 246.9 μmol L?1,respectively (Fig.2).G.incurvataalso showed a relatively highKm(151.7 μmol L?1,Fig.2).Therefore,these species were assigned ratings of 2.In contrast,the brown algae hadKmvalues below 100 μmol L?1and thus were assigned ratings of 1 (Fig.2).

Overall,U.compressaandP.yezoensishad the highest PSASI II scores (score = 4),followed byG.incurvata(score = 2,Table 4).U.pinnatifida, andE.cavashowed the lowest values (with PSASI II scores of 1) due to the low NH4+uptake rate.

Table 4 Ratings of the PSASI II parameters and final PSASI II scores for each species

3.3 SASI Data: Physiological Parameters of Seaweeds

To examine the physiological characteristics of sea-weed,tests of the NH4+uptake efficiency,tissue N content,and chlorophyllacontent were conducted on five species (U.pinnatifida,E.cava,P.yezoensis,G.incurveta,andU.compressa).Four species,excludingE.cava,showed NH4+uptake efficiencies higher than 75%.In particular,U.compressaabsorbed over 99% of the NH4+in the water column during 7 d.Generally,Ulvashowed high biofiltering effciency in IMTA system regardless of culture condition (Copertinoet al.,2009).Thus,four of the tested species were assigned ratings of 2,whereasE.cavareceived a rating of 1 due to its relatively low uptake rate (Table 5 and Fig.3).

Table 5 Ratings for each parameter are multiplied to determine the SASI score for seaweed species

Fig.3 NH4+ uptake efficiency of seaweeds during the 7-d experiment.Data are expressed as mean ± SE (n = 6);UND,Undaria pinnatifida; ECK,Ecklonia cava; POR,Porphyra yezoensis; GRA,Gracilaria incurvata; ULV,Ulvacompressa.

The tissue N content of seaweed is an optional parameter because it is not easy to generalize on the nutrient sequestration of seaweed (Neoriet al.,2004).Nevertheless,the tissue N content of seaweed increases with N enrichment in seawater until saturation (Neoriet al.,1991; Chopinet al.,1999; Davisonet al.,2007; Kanget al.,2011).Martinez and Rico (2002) reported that some N can be sequestered as photosynthetic pigments,free amino acids,and proteins.Additionally,N content ofGarcilariasteadily increases in various culture scale system for 1 – 2 months (Abreuet al.,2009,2011).Therefore,higher tissue N content or the ability of N accumulation in tissue could be an important factor in the selection of seaweed species for IMTA.In the present study,the mean tissue N contents ofU.pinnatifida,E.cava,P.yezoensis,andG.incurvataat the end of the experiment exceeded their initial values,whereas the mean tissue N content ofU.compressadecreased slightly compared with the initial value.This was despite lack of significant differences in tissue N content between the start and the end of the experiments.Because the tissue N contents (mean ± SD) of all species were equal to or higher than (mean – SD) of the initial pre-experiment samples,they were assigned ratings of 2 (Table 5 and Fig.4).

The chlorophyllacontent of seaweeds is dependent on the N concentration in the water column (Costanzoet al.,2000).It was found that macroalgae exposed to high N concentrations responded with increased chlorophyllaproduction within the 3-d incubation period.Our results showed that the chlorophyllacontents (mean ± SD) of four species (exceptU.pinnatifida) were lower than the(mean – SD) of the initial pre-experiment samples on day 7,and thus received ratings of 1 (Table 5 and Fig.5).However,U.pinnatifidareceived a rating of 2 because its chlorophylla(mean ± SD) was within the range of the(mean – SD) (Table 5 and Fig.5).

Fig.4 Tissue N contents of seaweeds at the beginning and the end of the experiment.Data are expressed as (mean –SD) and (mean ± SD) at the beginning and the end of the experiment,respectively (n = 6); UND,Undaria pinnatifida; ECK,Ecklonia cava; POR,Porphyra yezoensis;GRA,Gracilaria incurvata; and ULV,Ulvacompressa.

Fig.5 Chlorophyll a contents of seaweeds at the beginning and the end of the experiment.Data are expressed as(mean – SD) and (mean ± SD) at the beginning and the end of the experiment,respectively (n = 6); UND,Undaria pinnatifida; ECK,Ecklonia cava; POR,Porphyra yezoensis; GRA,Gracilaria incurvata; and ULV,Ulvacompressa.

3.4 Calculation of SASI Ratings

The PSASI I based on PSASI I score is the first SASI ratings parameter.In the PSASI I,U.pinnatifida,P.yezoensis,G.incurvata,andU.compressawere as- signed a rating of 2 because their PSASI I scores were 4 or 8 (Tables 3 and 5).However,E.cavawas assigned a rating of 1 for the PSASI I due to the low probability of its successful cultivation and application in seaweed- based aquaculture (Tables 3 and 5).For the PSASI II scores related to the NH4+uptake rate of each species,two species (P.yezoensisandU.compressa) were rated as 2,and the other species received ratings of 1.The final SASI scores for each species,as calculated by multiplying together the SASI ratings (PSASI I×PSASI II× SASI data)are shown in Table 5.Because five SASI parameters were measured,the maximum achievable SASI score was 32.Seaweed species with SASI scores ≥ 8 were considered priority species for IMTA.In the present study,U.pinnatifida,P.yezoensis,andU.compressareceived the highest SASI scores of 16.G.incurvatareceived a SASI score of 8.Accordingly,these four species were deemed suitable for IMTA.E.cavareceived the lowest SASI score and was considered unsuitable for IMTA due to its low removal efficiency and the difficulty in cultivation.

3.5 Application of the Species-Selection Model

The seaweed species receiving high scores using this index were those genera that have been applied to IMTA in previous literature (e.g.,Troellet al.,2003).Both this index and previous identification of seaweeds for IMTA have primarily focused on the nutrient removal efficiency of seaweed.Based on the present index,U.pinnatifidacan be selected as a good candidate seaweed species for IMTA.In fact,U.pinnatifidahas dominated culture production,constituting 46% of the total wet weight production in Korea (Yoon,2008),and has been co-cultured with fish in Korea for several decades.Few studies,however,have introducedU.pinnatifidaas a biofilter in IMTA.Thus,this index reflects the cultural conditions of local areas and is a useful index for species selection in a seaweed-based integrated aquaculture system.

The experiments used in developing the SASI index were conducted on a laboratory (i.e.,relatively small)scale.As the nutrient uptake rates of seaweeds depend on areal nutrient loads (Troellet al.,2003),we should confirm whether these results are reproducible in tank-based or land-based systems.At present,the experiments are being conducted in a large tank-based system using the seaweeds selected by the proposed index.So far,the seaweeds have shown increased nutrient uptake efficiencies up to 50% and slight increases in tissue N content and growth rate (Kang,unpublished data).

3.6 Supplementation of the Species-Selection Model

Still,more components could be derived to complement the SASI index.It is necessary for a more powerful index to include supplementary physiological parameters of seaweeds such as biochemical composition (tissue phosphate content,C : N and N : P ratios),photosynthesis,growth rate,and phosphate and nitrite/nitrate uptakes.Vergaraet al.(1993) demonstrated that the tissue C : N ratio is a powerful index for the physiological status of seaweed.Photosynthetic characteristics such as chlorophyll fluorescence are used as potential indicators of seaweed health status in the integrated cultivation of macroalgae (Figueroaet al.,2006).Seaweed growth rate will be measured for real nutrient removal rates by seaweeds in aquaculture systems during the culture period.As aquaculture effluents contain considerable inorganic phosphate and nitrate,the phosphate and nitrite/nitrate uptake rates of seaweed should be considered (Neoriet al.,1998).The co-occurrence of different N forms can affect N uptake rate (Abreuet al.,2011).The application of multiple seaweeds to IMTA can increase N removal efficiency because the uptake rate of each species is dependent on the form of different N sources and different species are complementary in using nitrogen source (Bracken and Stachowicz,2006; Kanget al.,2011).Furthermore,the effects of water temperature on the physiological characteristics of seaweeds should be considered due to a distinct seasonal trend in Korea.Wheeler and Srivastava(1984) reported that seaweeds have optimal temperature ranges over which nutrient uptake occurs.Kanget al.(2008) suggested thatCodium fragileis a suitable species for use in IMTA in summer.

4 Conclusions

In summary,this study developed a species-selection index applicable to seaweed-based IMTA systems for identification of seaweed species best suited for improvement of water quality and reduction of necessary cost and effort for sustainable,environmentally friendly aquaculture.The proposed index takes into account the physiological characteristics indicative of seaweed quality,such as biochemical composition and pigment,as well as the economic value and nutrient removal efficiency of the seaweed species.The seaweed species that have been used in IMTA receive higher scores by this index,identifying species suitable for seaweed-based IMTA system.The species-selection index requires additional parameters and further testing of its application.Although the proposed index cannot account for every eventuality and may incur some errors,it provides valuable information for IMTA seaweed species selection.

Acknowledgements

This research was supported by the ‘Greenhouse Gas Emissions Reduction Using Seaweeds’ program and the Jeju Sea Grant College Program funded by the Korean Ministry of Land,Transport and Maritime Affairs,Republic of Korea.

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