Feasibility of aquaculture cultivation of elkhorn sea moss (Kappaphycus alvarezii) in a horizontal long line in the Tropical Eastern Pacific

Seaweed aquaculture has become a profitable and an attractive alternative of cultivation thanks to its quick biomass production for food, feed, and other non-food applications. In addition, the ecosystem services generated by seaweed cultivation towards carbon fixation represents a more sustainable solution to the ocean’s acidification. The growth of elkhorn sea moss (Kappaphycus alvarezii) was evaluated in three plots with 200 propagules during a period of 70 days in a floating raft system covered by a fishing net underneath. Initial weight of propagules was 159.3 ± 12.74 g in wet biomass and 15.3 ± 1.43 g in dry biomass and were sampled up to 19 days (in the lag growth phase; period I), up to 33 days (in the exponential growth phase; period II) and up to 70 days (in the stationarity growth phase; period III). The variations of sea surface water temperature, salinity, turbidity (Secchi depth), total ammonium, nitrites, nitrates, and phosphate were determined. The growth increase was more evident in the exponential phase II when a dry biomass of 28.0 ± 2.48 (1153.3 ± 6.25 g in wet mass) was reached, more than 7 times the biomass of propagules with an average daily growth rate of 15.2% g.day–1. The carrying capacity of the zone was estimated at 86.2% in the area where 53 cultivation units would be projected. The economic analysis presented a financial feasibility with a net profit of 19% over the projected income and an IRR of 16.5%, recovering the investment in an estimated period of 4.3 years. We recommend to continue with larger-scale studies to optimize the cultivation of K. alvarezii in the study area.

Culture experiments.Within the floating rafts system, 3 non-contiguous experimental culture units (5 × 3 m) were chosen, each unit being considered as an experimental replica.In each experimental unit, 200 propagules were sown (manually fixed to the culture unit with a piece of polyester rope) with an initial weight of 159.3 ± 12.74 g of wet biomass and 15.3 ± 1.43 g of dry biomass (95% confidence interval as dispersion index in all measurements).Three sampling periods were conducted after sowing at up to 19 days (in the lag growth phase; period I), at up to 33 days (in the exponential growth phase; period II) and at up to 70 days (in the stationarity growth phase; period III).For the calculations of wet biomass growth, a sample of 15 tissue samples was taken at random in each period, which were weighed in situ with a digital portable scale (0.01 g precision).From this sample, 3 tissue samples were randomly taken for the evaluation of dry biomass, which were washed with fresh water and dried in an oven at 105 °C for 3 h following the recommendations of Ohno et al. 7 .The rest of the tissue samples were returned to the respective experimental replica.
For the growth curves we used the absolute dry weight end in each of the cultivation periods.For comparative use of other K. alvarezii cultures we use the equation of the daily growth rate in percentage (% DGR) in wet mass, proposed by Yong 27 with the Eq.(1) as follows: where Wo is the initial weight (g), Wt is the final weight (g), and t is the number of days of culture.The data is presented as mean daily growth rates for periods I, II and III, respectively.

Environmental factors.
To estimate the possible influence of environmental factors on algal growth, sea surface water temperature and salinity were measured using a YSI Professional Plus (Pro Plus) multiparameter probe.Similarly, water samples were taken to determine the concentration of total ammonium (NH 4 + ), nitrites Carrying capacity of culture area.As a feasibility factor in the study area, the carrying capacity was defined as the cultivation area that can be used for the mariculture activity of the macroalgae in a continuous way, considering that there are no social and ecological conflicts in the coastal system 2 .The calculations were based on the methodology applied by Azis 28 taking into consideration the following Eqs.( 2), ( 3) and (4).In this way, the system presented an area of 225 m 2 (45 m length × 5 m width-crop unit) (L 1 P 1 ).Additionally, a space between cultivation units of 10 m on all sides was considered, so the total projected area per cultivation unit was established at 1625 m 2 (65 m length × 25 m width) (L 2 P 2 ).The capacity of the water body was calculated according to the relationship: where L 1 = Width of a culture unit, L 2 = Appropriate width of a culture unit, P 1 = Length of a culture unit, P 2 = Appropriate length of a culture unit.The method used to calculate the adequate area without exceeding the load capacity of a specific area was based on the following relationship formulas: (2) where Wa = area (ha), Wc = capacity of water body (%).The maximum number of culture units that the carrying capacity of the water body can support was calculated using the following formula:

Capacity of water body
where C = Carrying capacity of the water body (ha), A = maximum area of use.

Economic viability.
To determine the economic feasibility of cultivation of K. alvarezii, profitability was estimated with data on the maximum load capacity of the cultivation area, which was projected with a total of 53 floating rafts of 45 × 5 m of simple construction with floats of PVC pipes with a diameter of 110 mm and a length of 3 m.These rafts had a capacity of 150 lines or 2134 propagules.Seaweed production was estimated at the harvest time with the highest yield assuming 11 harvests/year (harvests of 30-35 days).The operational costs included an already operational infrastructure for maintaining the propagules as well as various activities (drying, packaging, salaries, mobility, etc.) necessary for the commercial production of macroalgae.Calc software functions were used to determine costs and financial indicators [29][30][31] .The financial analysis was projected over 10 years based on export prices for the weight of dehydrated seaweed as well as current local market prices for used inputs, expressed in US dollars.The projected revenue from the sale of the algae was based on a 4% annual increase in the sales price in the main international markets located in the Philippines, Indonesia, and Tanzania 32 .

Statistical analysis.
The growth rates of the dry biomass of K. alvarezii during the initial, middle, and final periods were compared using a one-way ANOVA, after verification of the normal distribution and homogeneity of variances in the treatments (Shapiro-Wilk and Levene's tests, respectively), followed by Tukey's post hoc tests, according to recommendations of Zar 33 .Data of the environmental factors were analysed using the nonparametric Kruskal-Wallis test, establishing differences between periods using paired comparisons of Dwass-Steel-Critchlow-Fligner, and following the recommendations of Hsu 34 .The significance level for all tests was set at P = 0.05.

Results
Growth.No mortality was observed, nor was there any evidence of damage or grazing on the K. alvarezii cultivation during the study period.The final wet biomass average was 1620.7 g ± 12.74 g (95% CI, confidence interval), representing more than 10 times the initial one (159.3± 12.74 g).The increase was more evident in the period II when a wet biomass of 1153.3 ± 6.25 g was recorded.The growth shown in absolute values of dry biomass was similar, reaching an average initial dry biomass of the propagules (15.3 ± 1.43 g) to 28.0 ± 2.48, 108.3 ± 5.17 and 144.0 ± 8.61 g, for the I period (up to 19 days), II period (up to 33 days), and III period (up to 70 days), respectively, with the proportion daily growth rates of 3.0 ± 0.30, 15.2 ± 0.62 and 6.5 ± 0.25% g.day -1 , respectively, being the values in the intermediate period significantly higher (Fig. 2).

Environmental parameters.
The sea surface temperature presented an average of 28.9 ± 0.64 °C, with little difference among time periods (< 2 °C); however, the maximum temperatures recorded during the intermediate period (29.7 ± 0.06 °C) was significantly higher than the temperature in the final (28.7 ± 0.09 °C) and initial (28.3 ± 0.06 °C) periods (Fig. 3).The average turbidity was 1.3 ± 0.52 m, with a maximum of 2.0 ± 0.00 m in the initial period, significantly higher than that recorded in the intermediate (0.91 ± 0.025 m) and final period (1 ± 0.00 m).Regarding the dissolved inorganic nitrogenous compounds in the cultivation area, the values of NH 4 + , NO 2 − and NO 3 -, were generally 0.13, 0.13 and 1.92 ppm, respectively, without showing significant differences among the study periods (Fig. 4).Phosphate showed greater variability among periods, with values between 0.16 and 0.18, being the values in the initial period significantly higher.
Carrying capacity.Since each cultivation unit requires an area of 0.16 ha, the concession area in Bahía Las Conchas has a carrying capacity of the water body equivalent to 86.2%, which represents a total of 53 cultivation units in the concession system with horizontal floating rafts (Table 1).

Economic viability.
The 10-year financial study revealed a payback time from the fourth year of project start-up, with a return of 19% on the expected total income, with an expected annual production of 121.3 T in dry weight of K. alvarezii.The financial viability of the cultivation of the macroalga in floating farming systems was determined by the net present value (NPV) of $58,326.63, the internal rate of return (IRR) of 16.46% and the benefit-cost ratio (B/C) (1.67) (Table 2).

Discussion
Our trial study provides relevant new data on the biological feasibility of elkhorn sea moss (K.alvarezii) cultivation on the coast of the province of Santa Elena, Ecuador.During cultivation, we did not observe notable losses or damage to algae culture due to rupture, nor damage by grazers, which would attribute large losses in biomass production 35 .The recorded biomass growth after 70 days of cultivation, was more than 9 times (wet and dry) the original biomass sown, obtaining an average weight of 1600 g (144 g dry biomass) per propagule.In this period the % DGR was 6.5 ± 0.25% g.day -1 .
The growth was more accelerated in the exponential phase (from day 20 to 33), reaching biomasses of about 1150 g (108 g dry biomass).During period II (initial to 33 days) the % DGR was 15.2 ± 0.62% g.day -1 , which was more than twice if the algae were harvested after 70 days and 5 times more if the seaweed was harvested after 20 days.Slow growth in the first 20 days and after day 33 is characteristic in the culture of this species 36 .This suggests that the cultured biomass should be harvested after 30-35 days from out planting, when the highest growth rates are found 37 .Harvest time depends on the product yield and quality, and certain authors report that at a time close to a month after sowing, when the extraction of algae products (e.g.carrageenan) is feasible and recommended 36,37 .While, it has been documented that K. alvarezii has a lower concentration of carrageenan when harvested in a shorter period, other studies [49][50][51] have recommended harvesting over longer periods of time, about 45 days after sowing.
When comparing the % DGR of the present study harvesting K. alvarezii at 33 days, with previous studies in other tropical and subtropical regions (Table 3), the daily growth rate obtained for K. alvarezii growth (15.2%g.day -1 ) exceed all previous reports, either from studies carried out in the Atlantic (in Brazil, with the highest rate of 8.9% g.day -1 ) or in Asia with highest reported in India of 14% g.day -1 , but in several other countries did not exceed 10.8% g.day -1 .The high growth rate detected in the current study, in combination with environmental and littoral geography conditions of an extensive marine coastline (670 km) allow us to elucidate the positive biomass conditions for macroalgae production in Ecuador.Moreover, the possibility of culturing K. alvarezii in the upper estuary zone of Ecuador, where there is already a high level of aquaculture activity by coastal communities, reinforce the relevance of introducing the production of new species to diversify the aquaculture of the entire tropical Pacific region, in particular in Ecuador, where more than 95% is directly linked to shrimp farming 1 .
As such, to support the background idea of farming K. alvarezii and other macroalgae species in the region, more studies generating further evidence data should be promoted on the topic of assessing the production of macroalgae as one of the strategies to reduce the effects of climate change, counteract eutrophication and the crisis of biodiversity lost 52 .Given that macroalgae aquaculture is nowadays widely recognised as a strategic pathway to achieve a blue economy to meet more sustainability objectives 53,54 , the cultivation of macroalgae across the tropics, should be considered as one of main focus of public policies where the government, academic and private sector sectors must interact.
In our experimental design, although environmental factors related to the growth of macroalgae were recorded, there were no correlations detected with growth rates, except for the previously underlined intermediate II period, for which a positive correlation between increased temperature and higher biomass was observed.While temperature is a factor that modulates growth in aquatic organisms, including macroalgae and particularly www.nature.com/scientificreports/photosynthesis 55 , the difference among the studied periods did not exceed 2 °C, which was possibly not decisive in causing notable physiological effects in the macroalgae.This suggests that differences in growth rate might be associated with endogenous factors of post-adaptability of the algae, after its initial growth phase 56 .However, an important feature of the higher % DGR found in K. alvarezii cultures in Vietnam, is that the temperature was notably higher (33 °C) than the one recorded in the present study, which reflects the higher metabolism at high temperatures of this species.Further studies are necessary to understand the effect of temperature in tropical ranges on this algae species.Though the species can grow at lower temperatures, such as those occurring in the subtropics (17-31 °C), its production is lower (e.g.South Japan 41 and Bahía de Ubatuba in Brazil 45,46 ) than those observed in tropical areas, where the temperature is much higher and less variable 4,14,26,36,57,58 .
Regarding salinity measurements in our study, the culture in the province of Santa Elena was developed in a range of 30-35 ‰, which is considered optimal for K. alvarezii 8,9 .The chemical nutrients dissolved in the water measured as NH 4 + and PO 4 3-, were higher than the minimum required values of 0.3 to 0.6 ppm, both in NH 4 + , NO 2 -and NO 3 -11-14 , and (PO 4 3-) 0.009-0.05ppm, previously reported for a good development of the macroalgae 59 , which contributed to the good performance of the algae under cultivation.Both, the sowing density, and the depth of the cultivation set up, are also determining factors in productivity, and must be considered as one of the main factors affecting farm productivity 49,50 .
Quantification of the carrying capacity of an aquaculture system is important because its scale will determine the impact on the hydrodynamics of the area, the risk of spread of pests and diseases, as well as the probability of eutrophication due to the decomposition of the biomass 60 .However, eutrophication is an unlikely factor from the cultivation of this species as the biomass that breaks off and washes up on the shore is usually harvested due to its high commercial value.The calculations of the carrying capacity was based on multiple models and was adjusted to specific characteristics of the species and site where the project was performed 61,62 , the little information concerning to the cultivation of seaweeds is a limiting factor when comparing our results.For example, a recent work by Gomes Da Silva et al. 63 showed that a plant cover area of 2 ha is considered by the Brazilian Government to have a low ecological impact due to the oceanographic characteristics in the Southeast region of that country.Being the area of this study less than the determined cultivation capacity (8.6 ha) for Santa Elena, and considering a maximum load capacity of 86.15%, would allow a production of 121.3 T in dry weight of algae for the total concession area of 10 ha.
Until now, the economic viability for this species has mainly been reported for "family scale" developments < 0.5 ha.For example, in Colombia where the internal rate of return (IRR) was 65% 22 , and in Brazil between 38.1 and 87.8% 64 .However, Nogueira and Henriques 64 , concluded that the financial unfeasibility for large-scale macroalgae production in Brazil, is because of the required plant cover area and current legislation.New economic models in multitrophic cultures of Kappaphycus algae with bivalves 63 suggest features like those here reported, showing financial feasibility with an annual production of 121.3 T in dry weight of K. alvarezii, a period of recovery of 4.3 years of the investment and a rate of return of 16.46%.This activity generates an important socio-economic contribution to the sector (mainly constituted of associations of artisanal fishermen) since it guarantees the use of 259,200 man-hours for its development.Unlike Nogueira & Henriques 64 , this study To our knowledge, this is the first report on the cultivation of K. alvarezii and its feasibility in the Tropical Eastern Pacific waters.It was observed that the algae increased its biomass by more than 7 times after 33 days of cultivation, with an average daily growth rate of 15.2% g.day -1 .These values are almost three times higher than those proposed as suitable for commercial cultivation of eucheumatoides seaweeds worldwide 16 .The productivity and growth rates show the biological feasibility of K. alvarezii cultivation in the province of Santa Elena under the previously described conditions of temperature, salinity, and nutrients.
The load capacity established in the study area was 53 floating rafts in total, with a profitability of 67%.Although this profitability seems to be low, it is higher than that established by Gomes Da Silva et al. 63 in multitrophic culture (K. alvarezii with Perna perna, and Nodipecten nodosus) carried out in the state of Santa Catarina, Brazil.The data supplied for the economic feasibility exercise is real and comes from government support for the "Mariculture Macroproject on the Ecuadorian Coast", which is subject to various administrative control procedures that normally slow down and increase the cost of the initial investment, so the projection of costs in the present study could be overestimated.
We recommend continuing with the evaluation and refinement of K. alvarezii cultivation practices in the same study region and similar areas along the coast of Ecuador.Studies should include optimization of mass, number and seeding distance of propagules, control of biofouling, improvement of the product, etc. together with more detailed studies on phytopathology, product quality and commercialization.Special emphasis should be given to social inclusion, particularly in the active participation of women in the cultivation.The effect of different environmental factors on the culture should be evaluated before managing a large-scale commercialization phase, based on a K. alvarezii mariculture establishment with an adequate social and environmental impact within the framework of productive sustainability.Finally, given the systemic services that macroalgae can generate, we encourage and recommend to focus efforts on carbon sink studies for K. alvarezii cultures, as well as evaluating the effect on eutrophication reduction from discharge water systems from the shrimp industry and other similar discharge systems.

Figure 1 .
Figure 1.Panels (a) and (b) indicate the location of the cultivation site in the waters of Santa Elena province, Ecuador in South America.Panel (c) the propagules attached to the rope.Panel (d) a series of floating raft units, and finally panel (e) a branch of Kappaphycus alvarezii (Map source NASA: https:// soto.podaac.earth datac loud.nasa.gov/).
The seedlings of K. alvarezii were imported from Punta Laurel sector, Bocas del Toro Archipelago, in the Republic of Panama, with its respective phytosanitary certificate of origin and invoice.The experimental farm in Panama has been certified by the Undersecretary of Aquaculture as an exporting establishment of Kappaphycus macroalgae, being certified free of any pathological agent under the National Government of the Republic of Panama through the National Directorate of Animal Health of the Ministry of Agricultural Development.

( 4 )Figure 2 .
Figure 2. Growth estimates presented in absolute values of dry biomass of Kappaphycus alvarezii under cultivation in a floating rafts system on the coast of the province of Santa Elena, Ecuador.Numbers presented above the figure indicate mean % daily growth rates for periods I (from 0 to 19 days), II (from 19 to up 33 days) and III (from 33 to up 70 days).Vertical lines indicate 95% CI, confidence intervals.The three colours highlight the corresponding three different sampled periods from July to September 2016.

Figure 3 .Figure 4 .
Figure 3. Variation of sea surface temperature, salinity, and turbidity (Secchi depth) in the cultivation zone of Kappaphycus alvarezii on the coast of the province of Santa Elena, Ecuador.The three colours highlight the corresponding three different sampled period months from July to September 2016.

Table 1 .
Carrying capacity of the concession area of Bahía Las Conchas in the Province of Santa Elena, Ecuador.

Table 2 .
Financial evaluation of the floating raft culture of Kappaphycus alvarezii in the Pacific coast of Ecuador.a A total of 259,200 man-hours are estimated per production cycle.b A discount rate of 9.96% is used.*Although no mortality was observed in the study, we conservatively estimate a mortality of 10%.

Table 3 .
Culture parameters with estimated daily growth rate in percentages of the main culture studies of Kappaphycus alvarezii.Wo initial weight (g), Wt final weight (g) t is the number of days of culture.