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Growth and product quality of the seaweedKappaphycus alvarezii from different farminglocations in IndonesiaSimatupang, Nova F; Pong-Masak, Petrus R; Ratnawati, Pustika; et.al.https://research.usc.edu.au/discovery/delivery/61USC_INST:ResearchRepository/12142228640002621?l#13146209370002621

Simatupang, Pong-Masak, P. R., Ratnawati, P., Agusman, Paul, N., & Rimmer, M. (2021). Growth andproduct quality of the seaweed Kappaphycus alvarezii from different farming locations in Indonesia.Aquaculture Reports, 20, 1–8. https://doi.org/10.1016/j.aqrep.2021.100685

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Aquaculture Reports 20 (2021) 100685

Available online 10 April 20212352-5134/© 2021 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

Growth and product quality of the seaweed Kappaphycus alvarezii from different farming locations in Indonesia

Nova Francisca Simatupang a, Petrus Rani Pong-Masak a, Pustika Ratnawati a, Agusman b, Nicholas A. Paul c, Michael A. Rimmer c,* a Research Institute for Seaweed Culture, Ministry of Marine Affairs and Fisheries, Gorontalo, Indonesia b Research Centre for Marine Fisheries Product Processing and Biotechnology, Ministry of Marine Affairs and Fisheries, Jakarta, Indonesia c School of Science, Technology and Engineering, University of the Sunshine Coast, Queensland, Australia

A R T I C L E I N F O

Keywords: K. alvarezii Carrageenan Production Indonesia

A B S T R A C T

Indonesia is now the largest producer of carrageenophyte seaweeds, particularly Kappaphycus alvarezii (‘cottoni’ in the industry). Although it is widely acknowledged that there is substantial seasonal and regional variation in production and product quality of farmed K. alvarezii, this variability has not previously been systematically assessed in terms of production (growth and survival) and product quality (carrageenan content, gel strength and viscosity) in Indonesia. This study evaluated K. alvarezii cultured using standard techniques at 10 different geographic locations in Indonesia. Production over the 45-day trial period was highly variable, ranging from a high of (mean ± SE) 2819 ± 124 g.m− 1 per rope (Bontang site) to (mean ± SE) 848 ± 66.7 g.m− 1 to 495 ± 25.2 g. m-1 for the poorest-performing sites (Mamuju, Gorontalo, Manado and Banten). Carrageenan content of cultured K. alvarezii also varied between sites, from 26.4 % (Mamuju site) to 8.6–11.7 % (Lombok and Bantaeng sites). Gel strength ranged from 735.2 g.cm-2 (Manado) to 291.5 g.cm-2 (Bantaeng), and viscosity from 138.8 cP (Lombok) to 30.0 cP (Gorontalo). Overall, the sites with the ‘best’ product quality from culture K. alvarezii were Banten and Mamuju, and the ‘worst’ was Bantaeng. This variability in product quality data across growing regions is widely acknowledged by the industry but, to date, had not been evaluated using standardised approaches to farming. These results can be utilised to select and evaluate better-performing K. alvarezii cultivars, with a view to improving overall productivity of seaweed farming in Indonesia.

1. Introduction

Culture of the carrageenan-bearing seaweeds belonging to the genera Kappaphycus (colloquially known as ‘cottonii’) and Eucheuma (‘spino-sum’) is widespread in Indonesia which is now the dominant producer globally of carrageenophyte seaweeds (Kambey et al., 2020; Neish, 2008; Porse and Rudolph, 2017). The main products from these sea-weeds are carrageenans which are used in a variety of commercial ap-plications such as gelling, thickening, and stabilising agents, especially in food products and sauces (Necas and Bartosikova, 2013). Carra-geenans are also used in experimental medicine, pharmaceutical for-mulations, cosmetics, and industrial applications (Necas and Bartosikova, 2013).

Culture of Eucheuma denticulatum was initially trialled in 1967 in the Thousand Islands area near Jakarta and in the mid-1970s in Central Sulawesi (Hurtado et al., 2014; Neish, 2008; Porse and Rudolph, 2017).

In the 1980s Kappaphycus was introduced from the Philippines and farming commenced at Nusa Lembongan, Bali (Hurtado et al., 2014; Porse and Rudolph, 2017). From these beginnings seaweed culture expanded dramatically in Indonesia (Neish, 2013) and seaweed farming is now carried out in a range of locations throughout the archipelago, including Sumatra, Java, Bali, Sulawesi, East Kalimantan, West Nusa Tenggara (NTB), East Nusa Tenggara (NTT) and Maluku. Total Indo-nesian seaweed production in 2015 was estimated at around 11 million t wet weight (KKP and JICA, 2017) or 135,000 t dry weight. This repre-sents 65 % of the global total of ‘cottoni’ and 56 % of the global total of ‘spinosum’ (Porse and Rudolph, 2017). Farming of both Kappaphycus and Eucheuma in Indonesia is dominated by smallholder farmers (Larson et al., 2021; Neish, 2013). The uptake of seaweed farming amongst coastal communities in Indonesia is attributable to the ease of cultiva-tion, harvesting and drying techniques, the low level of initial capital investment required, and the short production cycle (typically about 6

* Corresponding author at: University of the Sunshine Coast, Locked Bag 4, Maroochydore DC, Qld, 4558, Australia. E-mail address: mrimmer@usc.edu.au (M.A. Rimmer).

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https://doi.org/10.1016/j.aqrep.2021.100685 Received 26 August 2020; Received in revised form 29 March 2021; Accepted 31 March 2021

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weeks) which provides a regular source of income to farming households (Neish, 2013). Neish (2013) notes that uptake of seaweed farming in Indonesia has been rapid and that by 2008 there were an estimated 20, 000 households in coastal areas involved in seaweed farming. The in-come earned from seaweed farming contributes to the well-being of households in seaweed farming communities, particularly transport, housing, basic needs, other (secondary) needs and education (Larson et al., 2021).

There is substantial seasonal and regional variation in production and product quality of farmed K. alvarezii with even farms a few kilo-metres apart reporting different seasonal growth trends (Neish, 2013). Based on published studies, this variation can be ascribed to:

(i) genetic variation amongst K. alvarezii ‘strains’ or cultivars (Lim et al., 2014; Zuccarello et al., 2006);

(ii) different environmental conditions, particularly temperature and nutrient levels (Kotiya et al., 2011; Munoz et al., 2004); and

(iii) culture methods, including planting density, depth and culture duration to harvest (Hurtado et al., 2008).

The objective of this study was to evaluate variability in production and product quality at 10 sites throughout Indonesia that broadly represent the locations where K. alvarezii is commercially cultured. This was done by undertaking in situ trials using a standardised methodology at each site to provide a level of resolution of culture performance at a much finer scale than that is reported at the farm or regional scale. This approach removes the third source of variability from the above list (i.e. culture methods) while retaining the inherent genetic variability of different culture ‘varieties or ‘strains’, as well as variability ascribed to local environmental variables. This approach is essential to evaluate genetic and environmental variability together, with a view to sepa-rating these factors in other experiments, to move towards evaluating inherent genetic variability for evaluation in transplant experiments or to develop genetic selection approaches (Rose and Garland, 2009).

2. Materials and methods

2.1. Research sites

Trials for seaweed growth and product quality were undertaken at 10 sites throughout Indonesia (Fig. 1, Table 1) concurrently during April – May 2017. The distribution of experimental sites broadly reflects the distribution of seaweed culture in Indonesia. All the sites chosen were in use for commercial cultivation of K. alvarezii. Vegetative propagules

Fig. 1. Inset: map of Southeast Asia showing location of Indonesia. Main: map of Indonesia showing the locations of the 10 sampling and trial sites for this study. Location details including administrative and geographical location for each site are listed in Table 1.

Table 1 Location details for the 10 sites used for growth and product quality trials for seaweed K. alvarezii in Indonesia. Numbers in the first column correspond to the numbered markers in Fig. 1.

No. (Fig. 1)

Province Regency Geographical location Site name

1 Lampung South Lampung

S 5 ◦ 43’84, E 105 ◦

47’8.98 Lampung

2 Banten Serang S 5 ◦ 57’49.09, E 106 ◦

17’48.678 Banten

3 West Nusa Tenggara

East Lombok

S 8 ◦ 54’1.75, E 116 ◦

30’41.04 Lombok

4 East Nusa Tenggara

Kupang S 10 ◦ 9’1.81, E 123 ◦

32’.31 Kupang

5 South Sulawesi Pangkep S 4 ◦ 35’45.42, E 119 ◦

24’55 Pangkep

6 South Sulawesi Bantaeng S 5 ◦ 29’32.82, E 119 ◦

54’12.36 Bantaeng

7 West Sulawesi Mamuju S 2 ◦ 37’55.43, E 118 ◦

57’38.86 Mamuju

8 Gorontalo North Gorontalo

N 0 ◦ 48’57, E 122 ◦

50’52 Gorontalo

9 North Sulawesi North Minahasa

N 1 ◦ 41’43.02, E 124 ◦ 57’19.98

Manado

10 East Kalimantan

Bontang N 0 ◦ 07’37.28, E 117 ◦ 31’35.35

Bontang

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were sourced from local commercial farmers and cultured in situ using standardised methodology and equipment, as described below.

2.2. Growth trials

Seaweed growth trials were undertaken in accordance with the ‘best practice’ long-line method recommended by Pong-Masak (2014). At each site, three ropes (4 mm diameter polyethylene) each 35 m in length and 1 m apart were established in an active seaweed farming area. The adjacent ropes were used for normal production by the seaweed farmer (Fig. 2). At each site, the K. alvarezii propagules used were sourced from local commercial farms. Individual thalli of K. alvarezii with initial weight 50 g were tied (using 1.5 mm polyethylene rope) at 15 cm in-tervals along each rope, giving 230 thalli of seaweed per rope. Mainte-nance (cleaning silt and epiphytes from the seaweed) was done once or twice per week by the local farmer. The seaweed arrays were sampled for production and product quality data after one 45-day production cycle.

Harvesting, weighing and drying of the cultured K. alvarezii was done in the field, using techniques similar to those used by commercial farms, in order to support the commercial application of our results by seaweed farmers. Each rope was weighed (drip weight) at the beginning of the trial, and again after 45 days to give a measure of production (final weight - initial weight). At the conclusion of the trial fresh samples of about 3 kg each were taken from each rope (three replicates), rinsed in seawater to remove any sediment, then sun-dried for 2–3 days to provide material for testing product quality (carrageenan content, viscosity and gel strength). Visual descriptions of each site cultivar were made of three representative thalli from each cultivar reporting colour and thallus morphology. At each site a representative seaweed thallus was photo-graphed and reported.

2.3. Water quality

Water quality parameters measured in situ were: temperature, pH, dissolved oxygen (DO), and salinity, measured using an Extech 700 meter. Because of logistical constraints, water quality was measured only once at each site except for the Pangkep site which was used for on- going seaweed growth trials (reported elsewhere) and where water quality could be measured regularly. A range for each variable is pro-vided for Pangkep. Consequently, water quality measurements are presented here for reference only, since a comparison between sites was not possible. Water samples for nitrate and phosphate were taken using 500 ml polyethylene sample bottles, and analysed at the Research Institute for Coastal Aquaculture and Fisheries Extension (RICAFE) Maros water quality laboratory in South Sulawesi. Nitrate was measured using the cadmium reduction method (APHA, 2005) and phosphate analysed using the ascorbic acid method (APHA, 2005).

2.4. Identification

Representative tissue samples were taken from each cultivar and preserved according to the methods of Zuccarello and Paul (2019). Sampling, analytical methods and results are reported in Ratnawati et al. (2020).

2.5. Proximate analysis

Proximate composition analyses were undertaken at RICAFE Maros, South Sulawesi, using Indonesian National Standards (SNI) 01-2354.2- 2006 for moisture, SNI 01-2354.1-2010 for ash, SNI 01-2354.4-2006 for protein, SNI 01-2354.3-2006 for fat, and SNI 01-2891-1992 for crude fibre. All these methods are aligned with those of AOAC Inter-national (1990). Briefly, moisture was analysed after drying the samples at 105 ◦C for 16 h using an oven (Memmert, Germany). Ash was ana-lysed using a muffle furnace at 550 ◦C (Barnstead, Thermolyne, CA, USA). Crude protein was determined according to micro-Kjeldahl pro-cedure (digestion, distillation, and titration). Lipid was extracted using chloroform and methanol. All analyses were done in triplicate.

2.6. Carrageenan content

Analysis of the carrageenan content of K. alvarezii samples from each site was done using potassium hydroxide (KOH) extraction (Per-anginangin et al., 2013) and precipitated used isopropyl alcohol (IPA) (Das et al., 2016; Husna et al., 2016) with some modification. A single 250 g sample of sun-dried seaweed was rinsed three times to remove any attached sediment. The sample was heated in 1.5 L of a 0.6 % KOH so-lution at 60–65 ◦C for 2 h. The sample was then steeped in freshwater until neutral pH (pH = 7) was obtained and then boiled in water at 90–95 ◦C for 2 h.

The extract was filtered using 100 μm mesh plankton net until gel formation occurred for 1–2 h at room temperature (25–27 ◦C). The extract was stored in a refrigerator at − 21 ◦C for 20 h. The filtrate ob-tained was further precipitated through the addition of 1.5 L of isopropyl alcohol for 30–60 min. The carrageenan fibre produced was dried in direct sunlight outdoors and then floured using a kitchen blender to obtain carrageenan powder. Carrageenan yield was calculated using the following formula (AOAC International, 1990):

Carrageenan content (%) = Weight of carrageenan fibre (g) × 100 ÷Dry weight of sample (g)

Carrageenan content, gel strength and viscosity were measured using duplicate samples with a composite average used for data analysis.

2.7. Gel strength

Gel strength was measured using the methods of Marine Colloid and FMC (1978); Basmal and Ikasari (2014) and Hayashi et al. (2017), from

Fig. 2. Diagram of seaweed culture array of the type used for this study. Note only three replicate 35 m ropes were used for the experimental cultures.

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the gel formed from 1.5 % solution of carrageenan in distilled water. Three grams of carrageenan powder and 0.6 g KCl were agitated in 197 mL of distilled water for 15 min, and then heated to 80 ◦C with constant agitation for 15 min. The hot solution was then transferred into two glass cylinders (height 4 cm, diameter 7 cm) and closed with glass plates. The gel was allowed to set overnight (12–15 h) at 10 ◦C. The glass plates were carefully removed before measurement. The gel strength of each sample (g.cm− 2) was measured using a Texture Analyser (TAXT Plus Stable Microsystem, UK) with a 1 cm Kobe stainless steel probe at test speed 1 mm.s-1 and the trigger force set at 3 G. Triplicate samples from each site were measured.

2.8. Viscosity

Viscosity was measured using a 1.5 % carrageenan solution at a temperature of 75 ◦C with a rotary viscometer (Brookfield RVT, USA). A 7.5 g sample of carrageenan powder was agitated in 500 mL of distilled water for 20 min. The solution was than heated on a hotplate for 20− 30 min to reach 80 ◦C with constant agitation. The viscosity (cP) was measured using a viscometer with spindle number 2 and 60 rpm test speed. Viscosity was measured in centipoise (cP) which are one milli-pascal.second (mPa⋅s).

2.9. Data analysis

Statistical analyses were carried out using Minitab v17. Production, carrageenan content, gel strength and viscosity data for each site were analysed using one-way analysis of variance (ANOVA) after checking homoscedacity using Levene’s test. Where ANOVA indicated significant differences between means (P < 0.05) individual means were compared using Fisher’s Least Significant Difference (LSD) test because of the high number of pairwise comparisons across the 10 sites.

3. Results

3.1. Seaweed morphology and colour and identification

As is usual with Kappaphycus species, there were considerable dif-ferences in morphology and colour observed across the various farming sites (Zuccarello et al., 2006). For the ten sites used in this study, sum-maries of morphology and colour are listed in Table 2. Cultured K. alvarezii from Mamuju, Lombok and Banten had a long thallus (20–30 cm) with numerous branches. In contrast, cultures from Gor-ontalo, Lampung and Bantaeng had a short thallus (Table 2). Colour varied from bright green to light brown and dark brown in seaweed from different sites (Table 2).

All samples from this study were genetically identified as K. alvarezii using the COI marker (Ratnawati et al., 2020). They were all of an identical COI genotype, corresponding to the main cultivated K. alvarezii genotype (Genbank Accession number: KT316556) (Dumilag et al., 2016; Ratnawati et al., 2020; Zuccarello et al., 2006).

3.2. Water quality

Water quality data from all 10 sites indicated conditions suitable for culture of K. alvarezii (Table 3). Water temperature ranged from 27.1–33.2 ◦C, salinity from 32.5–36.8, DO from 4.0–8.6 mg/L, and pH from 7.5–8.5 (Table 3). For most of the sites sampled, water quality parameters were close to the optimal range for K. alvarezii culture (Table 3). The salinity value for Banten was relatively high at 36.8. Nitrate values ranged from 0.04 to 1.66 mg/L, and phosphate ranged from not detectable to 0.54 mg/L. Although turbidity levels were not measured in this study, it is likely that turbidity may also have an impact on growth and product quality of K. alvarezii.

Table 2 Morphological and colour characteristics of seaweed K. alvarezii from 10 sites in Indonesia.

Site Photo Description

Lampung Smooth and shiny thallus with dark green colour, numerous but short branches.

Banten Moderate-sized, smooth and shiny thallus, brown in colour.

Lombok Large primary thallus, small secondary thallus, numerous branches and bright brown in colour.

Kupang Large thallus, dark brown in colour, with many new apical growing points in thallus.

Pangkep Moderate-sized thallus with adequate branches, and brownish-green in colour.

Bantaeng Small secondary thallus, light brown in colour.

Mamuju Extended and large thallus with numerous branches, yellowish-brown in colour.

Gorontalo Large primary thallus with many new buds, thallus is smooth and shiny and light green in colour.

Manado Long and lush thallus, brown in colour.

Bontang Large and extended thallus, few branches, green in colour.

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3.3. Growth and production

There were significant differences in biomass production (final weight of seaweed minus initial weight of seaweed) of K. alvarezii cultured in situ at 10 sites for a 45-day cycle (ANOVA, F = 25.5, P < 0.01). Seaweed cultured at the Bontang site showed the highest production, with (mean ± SE) 2.8 ± 0.124 kg.m− 1 (Fig. 3). The second- highest production was from Kupang and Pangkep sites (Fig. 3). In these in situ trials, Mamuju, Gorontalo, Manado and Banten sites showed the poorest production, with only (mean ± SE) 848 ± 66.7 g.m− 1 to 495 ± 25.2 g.m− 1 (Fig. 3).

3.4. Proximate composition

The proximate composition of representative samples of K. alvarezii from each site is listed in Table 4. Protein levels ranged from 3.1–4.9% (Table 4) which is higher than the 0.7 % reported for K. alvarezii from Seribu Islands (Santoso et al., 2006) and the 1.03 % for samples from Gorontalo (Adharini et al., 2019), but similar to the 3.0 % reported by Hong et al. (2007) from Vietnam. Lipid levels for all the samples in this study were below the limit of detection (Table 4). Other studies have also found low lipid levels in K. alvarezii: 0% (Adharini et al., 2019), 0.2 % (Santoso et al., 2006), 0.7 % (Hong et al., 2007), up to 1.5 % (Kumari et al., 2010). Fibre content in this study (4.3–6.6 %) is comparable with that reported by Hong et al. (2007) (6.3 %) but higher than that reported by Adharini et al. (2019) (0.87 %).

Moisture content from previously sun-dried samples in this study ranged from 10.5–15.1% (Table 4). There were three samples (Lombok, Banten and Gorontalo) that had moisture levels higher than the rec-ommended maximum of 12 % (FAO, 2007), but all samples were lower than the 30 % maximum value permitted by the Indonesian standard (BSN, 2015). The higher moisture content for these three samples may

be the result of less-than-optimal drying of the seaweed prior to analysis. Moisture content data for K. alvarezii measured by Adharini et al. (2019) are not comparable because they were calculated on a wet weight basis.

3.5. Carrageenan, gel strength and viscosity

Carrageenan content was significantly different between samples (ANOVA, F = 19.7, P < 0.01) (Table 5). Samples from Mamuju had the highest carrageenan content (mean ± SE: 26.4 ± 1.6 %) while samples from Lombok and Bantaeng had the lowest levels, from 8.6 ± 0.8%– 11.7 ± 1.3 % (Table 5). The values for carrageenan content obtained in this study were generally lower than reported in other studies (Table 6).

Gel strength also varied significantly between sites (ANOVA, F = 160.9, P < 0.01)5) (Table 5). The lowest values were for the samples from Bantaeng (mean ± SE: 291 ± 6.3 g.cm− 2) and Lampung (310 ± 13.1 g.cm− 2), and highest value (735 ± 14.8 g.cm− 2) was for the Manado sample (Table 5).

Viscosity also showed significant differences between sites (ANOVA, F = 26.1, P < 0.01) (Table 5). The sample with the highest viscosity was from Lombok (mean ± SE: 183.8 ± 16.3 cP) while the lowest value was for the Gorontalo sample (30.0 ± 0 cP) (Table 5). All our samples were within the range of viscosity values listed by Necas and Bartosikova (2013) i.e. 5–800 cP, and exceeded the minimum (5 cP) recommended by FAO (2007).

3.6. Product quality evaluation

There was no correlation between the three product quality variables measured in this study: carrageenan content, gel strength and viscosity. To give an overall picture of the seaweed from the different locations, mean values for carrageenan content, gel strength and viscosity were plotted on a three-dimensional graph (Fig. 4). The location with the overall poorest product quality (lower values for carrageenan, gel strength and viscosity) was Bantaeng (Fig. 4). Sites with overall higher values for these three variables were Banten and Mamuju (Fig. 4).

Table 3 Water quality for the 10 sites in Indonesia used for K. alvarezii growth trials. Measurements were taken regularly at Pangkep throughout the trial, whereas other sites were sampled only once. Optimal values taken from cited references. DO: dissolved oxygen.

Parameter Site Optimal

Lampung Banten Lombok Kupang Pangkep Bantaeng Mamuju Gorontalo Manado Bontang

Salinity 32.5 36.8 33.2 33.2 34–36 35 33.2 34.3 33.7 33.2 28–34 (Aslan, 1998) Temperature (◦C) 30.2 28.3 33.2 28.1 28.5–31.1 27.1 33.1 29.5 30.7 30.4 22.8–29.2 (Neish, 2008) DO

(mg/L) 4.1 8.1 8.6 5.4 5.2–8.6 4.0 7.6 7.5 7.4 7.4 >5 (Aslan, 1998)

pH 7.5 8.0 8.3 7.5 7.3–8.2 8.5 7.5 7.8 8.4 8.3 Nitrate (mg/L) 0.05 0.23 0.11 0.05 0.04–1.66 0.13 0.11 0.15 0.45 0.12 0.01–0.07 (Aslan, 1998) Phosphate (mg/L) 0.54 0.10 <0 0.09 0.01–0.13 <0 0.05 <0 0.01 0.11 0.1–0.2 (Aslan, 1998)

Fig. 3. Mean ± SE values for production of K. alvarezii from 10 sites in Indonesia, ordered from highest to lowest. Different letters denote significantly different means (Fisher LSD test, P < 0.05).

Table 4 The proximate composition of seaweed K. alvarezii cultured at different sites in Indonesia. Lipid in all samples was below the level of detection (<0.01).

Site Protein (%)

Lipid (%)

Crude fibre (%)

Ash (%)

Moisture (%)

Lampung 4.9 <0.01 5.0 46.4 12.0 Banten 3.1 <0.01 5.2 39.8 15.1 Lombok 3.8 <0.01 5.0 33.1 14.9 Kupang 4.5 <0.01 6.6 43.2 11.6 Pangkep 4.3 <0.01 6.1 45.2 10.5 Bantaeng 3.8 <0.01 5.1 42.6 10.6 Mamuju 4.1 <0.01 4.7 50.4 11.3 Gorontalo 4.8 <0.01 5.4 40.4 13.5 Manado 3.8 <0.01 5.1 42.2 11.7 Bontang 4.2 <0.01 4.3 44.5 11.1

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4. Discussion

This study highlighted key factors affecting production and product quality in commercial K. alvarezii culture across Indonesia. By using a standardised culture method this study allowed us to evaluate, for the first time, the variability in growth, morphology and quality of K. alvarezii produced under commercial conditions in Indonesia and ascribe this to inherent genetic variability and/or environmental con-ditions. The results showed substantial variation in production and product quality of K. alvarezii commercially cultured in Indonesia. Production from the Bontang site was about five-fold greater than the poorest-performing sites of Manado and Banten. Similarly, K. alvarezii from different sites showed substantial differences in carrageenan con-tent, gel strength and viscosity values, with samples from Banten and Mamuju scoring consistently higher for product quality parameters than samples from the other sites. The highest production site (Bontang) yielded an average product quality, whereas the highest product quality sites (Banten and Mamuju) unfortunately were also the lowest produc-tion sites.

This trial was carried out during the recognised production season for Kappaphycus growth, i.e. towards the end of the wet monsoon (rainy season) when rainfall was moderate. Light rainfall moderates surface seawater temperature and salinity and provides ideal conditions for the growth of K. alvarezii (Pong-Masak, 2014). It was noted that the site with the highest production, Bontang, had close to optimal environmental conditions (Table 3) and there was no sign of ice-ice disease, or signifi-cant biofouling in cultures at this site. In contrast, one of the poorer-performing sites, Banten, had a higher salinity (36.8) and the seaweed at that site was affected by sediment and biofouling which may have been a factor in the poor growth at that site. Culture environments

Table 5 Values (mean ± SE, n = 2) of carrageenan, gel strength and viscosity from K. alvarezii cultured at different locations in Indonesia. Different letters within rows denote significantly different means (Fisher LSD test, P < 0.05). cP: centipoise.

Location

Parameter Lampung Banten Lombok Kupang Bantaeng Mamuju Gorontalo Manado Bontang

Carrageenan (%) 18.6 ± 0.7b 19.4 ± 1.9b 11.7 ± 1.3ef 14.5 ± 1.0cde 8.6 ± 0.8f 26.4 ± 1.6a 15.8 ± 0.8bcd 14.0 ± 0.9de 18.1 ± 0.4bc

Gel strength (g. cm− 2) 310 ± 13.1e 604 ± 9.8b 540 ± 9.2c 412 ± 7.5d 291 ± 6.3e 510 ± 14.5c 598 ± 16.8b 735 ± 14.8a 430 ± 5.1d

Viscosity (cP) 153.8 ± 26.3ab 118.8 ± 3.8b 183.8 ± 16.3a 45.0 ± 5.0cd 35.0 ± 0d 73.8 ± 3.8c 30.0 ± 0d 60.0 ± 5.0cd 42.5 ± 7.5cd

Table 6 A selection of published values of carrageenan content, gel strength and vis-cosity for K. alvarezii from different locations and as reported by the cited studies. cP: centipoise.

Location Carrageenan (%)

Gel strength (g. cm− 2)

Viscosity (cP)

Reference

Gorontalo, Sulawesi, Indonesia

– 161.6 – Adharini et al. (2019)

North Gorontalo, Sulawesi, Indonesia

23.0–30.6 20.3–80.3 55–85 Harun et al. (2013)

Takalar, South Sulawesi, Indonesia

21.8–34.0 1493 – 2203 150 Arfini (2011)

Karimun Jawa, Java, Indonesia

19.5–43.9 94.5 – 152.3 6.1–8.2 Manuhara et al. (2016)

Brazil 20–32 % – – Hayashi et al. (2007)

Vietnam 18.8–26.4 503–1105 83–128 Hung et al. (2009)

China 56.9–58.0 25–45 – Rui et al. (1990)

Mexico 30.3–40.7 – 110.0–186.8 Munoz et al. (2004)

Japan 27.6–43.5 850–2000 890 Ohno et al. (1994)

Fig. 4. Three-dimensional plot of mean values for the three product quality variables (carrageenan content, gel strength and viscosity) for samples of K. alvarezii cultured at different locations in Indonesia.

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with sub-optimal conditions, such as low light intensity, low (< 20) salinity, and high temperature (up to 35 ◦C), may result in the devel-opment of ‘ice-ice’ disease, often associated with growth of epiphytes on the seaweed, resulting in low production or crop failure (Neish, 2013).

Growth and productivity data from other studies are difficult to compare objectively because of different cultivation methodologies employed by different authors (Hayashi et al., 2007). Kotiya et al. (2011) reported signficant differences in growth and production of K. alvarezii at nine different locations in Gujarat, India, as well as sea-sonal differences. Growth rate and carrageenan quality of K. striatum var. sacol are affected by stocking density, duration of culture, and culture depth (Hurtado et al., 2008). Munoz et al. (2004) found a sig-nificant negative correlation between growth rate and water tempera-ture for K. alvarezii cultured in Yucatan, Mexico, while Kotiya et al. (2011) found that growth of K. alvarezii at different sites in India was correlated with levels of NO3-N and Total P. In this study, there were no correlations between in situ production and any of the environmental factors measured, although this may simply reflect the limited number of data for these analyses (Table 3).

In this study, stocking density, culture duration and culture depth were constant across sites, and the trials were run concurrently, reducing the overall effect of seasonality. Thus the differences in pro-ductivity seen in this study (production ranging from 495 ± 25.2 g.m− 1

up to 2819 ± 123.8 g.m− 1) (Fig. 3) can be ascribed to genetic variation amongst the cultivars evaluated (which will require the use of additional genetic markers to comprehend: see Zuccarello and Paul (2019)) or can be ascribed to the local environmental conditions.

A wide range of factors associated with culture methods appear to affect the yield and quality of carrageenan in Kappaphycus species, including planting density, cultivation period (Hayashi et al., 2007) and culture depth (Hayashi et al., 2007; Hurtado et al., 2008; Iskandar et al., 2014). However, in this study all these factors were constant across sites, so the variation seen in carrageenan content, gel strength and viscosity are attributable to local environmental conditions and phenotypic variation amongst the cultivars, or a potential interaction between both factors.

Other studies on carrageenan content / yield, gel strength and vis-cosity, particularly for Indonesian samples, are summarized in Table 6. It is clear from Table 6 that there is substantial variation in product quality parameters reported for K. alvarezii, which in cases may be attributed to the different extraction methods used in different studies. Even seemingly minor changes in extraction method can impact the measured values for carrageenan content, gel strength and viscosity. For example, Manuhara et al. (2016) found substantial differences in all three parameters based on using different concentrations of KCl (ranging from 1.5 % to 3.5 %) in the extraction method.

Seaweeds in the genus Kappaphycus commonly demonstrate consid-erable morphological plasticity (Dumilag et al., 2016; Ratnawati et al., 2020; Zuccarello and Paul, 2019; Zuccarello et al., 2006) and this is commonly affected by environmental factors (Glenn and Doty, 1990). The morphological and colour variability seen in K. alvarezii sourced from 10 locations throughout Indonesia (Table 2) can at least partly be ascribed to the differences in a range of environmental variables at those sites (Table 3). Our ongoing work has shown that growing thalli sourced from different sites, at a common garden site, resulted in convergence of morphological and colour variation in K. alvarezii (Pong-Masak et al., unpublished data) again suggesting that much of the observed morphological variation is associated with local environmental conditions.

In the case of the locations evaluated in this study, details on the source of the cultivars and any subsquent introductions are not avail-able. While it is possible that different sites are now farming different ‘strains’ sourced from various and perhaps multiple sources, cultured K. alvarezii around the world shows a high level of genetic conformity. Zuccarello et al. (2006) evaluated samples of cultured K. alvarezii and concluded that all currently cultivated K. alvarezii from all over the

world have a similar mitochondrial haplotype, suggesting a common lineage. A recent genetic study of K. alvarezii sampled from throughout Indonesia (Ratnawati et al., 2020) further supported this finding. This study found that all the samples were of the E3 K. alvarezii haplotype and that genetic variation of cultured K. alvarezii in Indonesia, as measured in mitochondrial COI haplotypes, is limited (Ratnawati et al., 2020). Clearly, the relationship between the limited genetic diversity of cultured K. alvarezii and the highly variable levels of production and product quality measured in this study requires further research using traditional molecular tools (e.g. those used for population-level analysis) as well as new genomic tools such as transcriptomics that can capture multivariate responses in growth, morphology and product quality.

This particular study provides a ‘snapshot’ of the variation in pro-duction and product quality of K. alvarezii from across Indonesia. Such a study is essential to understand the overall variation to be found in cultured K. alvarezii in Indonesia, and to begin partitioning the factors that likely cause this variability: culture methods, environment and genetics. It is evident that a longer study, incorporating a temporal evaluation to cover the effects of seasonality, particularly in regard to water quality and its effects on ‘ice-ice’ disease and epiphytes, would likely demonstrate even greater levels of variability. However, our re-sults demonstrate that under standardised cultivation conditions there is still substantial variation in productivity and product quality of farmed K. alvarezii in Indonesia. Future research will further evaluate this variability in terms of isolating the genetic component of this variability, through ‘common garden’ trials comparing a variety of different culti-vars cultured under identical conditions at a single site. Overall, this research is intended to assess the potential for using better-performing ‘strains’ (rather than ‘best’ performing strains) in sites where local K. alvarezii strains appear to be under-performing. Following identifi-cation of better-performing K. alvarezii strains, these will be transferred to different locations to compare the results of ‘common garden’ trials with strain performance in diverse farming situations in various loca-tions in Indonesia, with a view to improving farm productivity and profitability for Indonesian small-holder seaweed farmers. However, any such transplant trials should be undertaken using the biosecurity framework proposed by Kambey et al. (2020) to limit any spread of pests and diseases that may impact on this nationally important aquaculture commodity.

CRediT authorship contribution statement

Nova Francisca Simatupang: Conceptualization, Methodology, Formal analysis, Investigation, Writing - original draft, Writing - review & editing. Petrus Rani Pong-Masak: Conceptualization, Methodology, Formal analysis, Investigation, Writing - review & editing, Supervision. Pustika Ratnawati: Conceptualization, Methodology, Formal analysis, Investigation, Writing - original draft, Writing - review & editing. Agusman: Formal analysis, Resources. Nicholas A. Paul: Conceptual-ization, Methodology, Project administration, Writing - review & edit-ing, Funding acquisition. Michael A. Rimmer: Formal analysis, Writing - original draft, Writing - review & editing.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

The authors thank the staff of the Research Institute for Coastal Aquaculture and Fisheries Extension Maros, South Sulawesi, as well as staff of the Research Institute for Product Processing and Biotechnology, Slipi, Jakarta, for their support for various analyses. The authors also thank the technical team from the Institute for Seaweed Research,

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Gorontalo, who participated in this research: Dwi Ayu Purwanti, Twi-nugroho, Handy Burase, and Elmiwia R. Baturante. We also thank the ACIAR Makassar office: Mrs Suliyanti Hakim, Mr Imran Lapong and Mr Nur Syamsul for administrative support to the project and assistance with revision of the maps, and Prof. Joe Zuccarello for assistance with the molecular identification of the seaweed samples. We particularly thank the farmers at each experimental site and local government offi-cers for their support of this study. This research was supported by the Australian Centre for International Agricultural Research (ACIAR) project ‘Improving seaweed production and processing opportunities in Indonesia’ (FIS/2015/038).

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