The growth feasibility of Lomentaria sp. in Laboratory conditions.

Ha Thi Thu Bui; Trong Quoc Luu; Ravi Fotedar

doi:10.4066/2529-8046.100016

Research Article - Environmental Risk Assessment and Remediation (2017) Volume 1, Issue 2

The growth feasibility of Lomentaria sp. in Laboratory conditions.

Ha Thi Thu Bui^*, Trong Quoc Luu, Ravi Fotedar

Department of Environment and Agriculture, Curtin University, Australia

*Corresponding Author:: Ha Thi Thu Bui
Department of Environment and Agriculture
Curtin University
Australia
Tel: +61 8 92667563
E-mail: ha.bui@postgrad.curtin.edu.au

Accepted date: May 03, 2017

DOI: 10.4066/2529-8046.100016

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Abstract

The growth feasibility of Lomentaria sp. in ocean water (OW) and inland saline water (ISW) at salinity 30‰ was tested in a series of four experiments. To grow Lomentaria sp., potassium chloride (KCl) was used to fortify ISW to approximately 100%, 66%, and 33% (ISW100, ISW66, and ISW33 respectively) of [K+] in OW and compared to two controls of OW and ISW. The results showed that the ISW66 medium resulted in the highest (P<0.05) Lomentaria sp biomass from day 14-56. The Lomentaria sp. was then cultured in OW, ISW and ISW66 enriched weekly with ammonium (NH4) 100 μmol by NH4Cl. A significantly slower reduction of specific growth rate (SGR) of Lomentaria sp. was recorded in the NH4 enriched waters than non-enriched waters. The effect of three temperature levels of 18-19°C, 21-22°C, and 25-26°C were also tested on the growth of Lomentaria sp. The 18-19°C resulted in highest biomass loss, whereas the higher temperatures resulted in similar SGRs of Lomentaria sp in both OW and ISW66. Four levels of NH4:PO4 including 0:0, 75:7.5, 150:15, and 300:30 μmol L-1 NH4:PO4 by NH4Cl and Na2HPO4, were weekly added to OW and ISW66, and these combined nutrient supplementation showed no effect on the Lomentaria sp. SGR. This study identified the suitable conditions for Lomentaria sp. growth in captivity as a temperature of 21-26°C, a supply of [NH4] no greater than 100 μmol L-1 in K+ fortification ISW 33-66% of [K+] in OW for higher biomass gain.

Keywords

Biomass, inland saline water, Lomentaria sp., potassium, temperatures.

Introduction

Of 5,000 red seaweed species, Rhodophyta, 1,300 species are found in Australian waters [1]. Rhodymeniales, which contains three families and 38 genera, 17 genera have been recorded in Australia, of which three species of Lomentaria genus have been identified in Southern Australia, including L. australis, L. pyramidalis, L. monochlamypdea [2]. The Lomentaria thallus “erect or forming entangled clumps, much branched, with or without percurrent axes, branches terete or compressed, hollow, basally constricted with solid septa; holdfast discoid or hapteroid. Structure multiaxial, with a cluster of apical cells developing an inner cortex 2-3 cells thick and an outer cortex of small cells sometimes forming rosettes” (p. 34) with a life cycle of isomorphic gametophytes and tetrasporophytes [2]. The red seaweed can be used as a source of food, to extract agar, and producing fertilizer [1]. However, little is known about the benefit of Lomentaria sp. yet, and there has been no record on growing Lomentaria sp either in ocean water (OW) and or in inland saline water (ISW).

In Australia, ISW is available in the form of large reserves of underground [3], which could provide a source of water for inland marine aquaculture [4]. About 2.2 and 5.7 million hectares of land was salt-affected in 1996 and 2000, respectively [3,5], which is expected to increase to 17 million hectares in 2050 [5]. Agricultural land, wildlife habitats and native vegetation are adversely affected due to ISW areas rising [6]. Inland marine culture can be a way to contribute to limit the impact of ISW expansion in Australia [6].

Potassium (K⁺) is crucial for algal growth [7], and it shares 1-2% of dry plant biomass [8]. K⁺ is an important internal cation in algae [9], and in the red algae Chondrus crispus and Porphyra tenera, it comprises 37 and 43%, respectively, of total internal cations [10]. K⁺ plays an important role in photosynthesis and respiration of the plant [11]. [K⁺] of 230-350 mg L^-1at 35‰ is suitable for the red seaweed Caloglossa leprieurii (Montagne) J. Agardh growth, but another red seaweed, Bostrychia radicans Montagne, prefers higher [K⁺] at 400-500 mg L^-1 [12]. K⁺ fortification for ISW to sustain the growth of marine species is needed [13-16] when K⁺-deficient ISW is common in Australia [17-19]. Studies on the K⁺ effect is important to determine the requirement of [K⁺] for seaweed growth.

Ammonium (NH₄), the most common type of ammonia (NH₃) in OW [20], and phosphate (PO₄) are the preferred source of nitrogen (N) and phosphorus (P) for seaweed growth [21-24] . However, N and P in water do not always meet the algal demand [25]. For higher seaweed growth, supplying NH₄ is more efficient than nitrate (NO3) [26]. In addition, the combination of NH₄ and PO₄ have a positive effect on the growth of Sargassum baccularia than either NH₄ or PO₄ alone [24]. As it is the first study on growing Lomentaria sp., it is necessary to identify the need of NH₄ and PO₄ for optimal Lomentaria sp. growth.

Temperature strongly affects the growth of algae [27]. The temperature of ISW in Western Australia (WA) is approximately 18°C, and varies around 6.3-28.1°C [28]. These temperatures are suitable for the growth of many red seaweeds. Hypnea cervicornis and Gracilaria tikvahiae prefer 20-25°C for optimal growth [29,30], when Hypnea musciformis and Gracilaria cornea grow well in the Florida Keys at 15-25°C [31,32]. At 15°C, Chondrus crispus and Furcellaria lumbricalis reach their maximum growths [29]. However, at temperatures exceeding 30°C, an inferior growth of Hypnea cervicornis and H. musciformis was recorded [30,32].

Studies on seaweed culture in ISW in Australian is limited to Gracilaria cliftonii Withell, Miller and Kraft, and Sargassum linearifolium [19] even though there are abundant studies about seaweed growth, chemical and nutrient uptakes worldwide [33- 38] . This study is the first attempt to grow Lomentaria sp. in the laboratory, testing the growth feasibility of Lomentaria sp. in OW and ISW, targeting on consuming the available ISW source to reduce adverse impacts of ISW on environment and agriculture [24].

Material and Methods

Seaweed collection

Lomentaria sp., was identified by WA Herbarium, was collected at Matilda Bay, Swan River, WA (latitude 31°97.9S, longitude 115°82.2E). This species currently is identifying by WA Herbarium and it maybe a new species. The Lomentaria sp. was transported in tanks holding ambient river salty water to Curtin Aquatic Research Laboratory (CARL) immediately after collection. The Lomentaria sp. were thoroughly cleaned in OW to remove all epibiotics.

Before using in experiments, the Lomentaria sp. was then acclimated for one day in aerated OW at 30‰ at 22°C in 114 L aquaria, under a downwelling photo-lux density of 120 μmol photon m^-2 s^-1 and a 14:10 h light: dark cycle [33].

Experimental setup

ISW had a salinity 45% was procured from a lake at Wannamal, WA (31°15″S, 116°05″E). OW had a salinity of 35‰ was procured at Hillary Habour (31°.83″ S, 115°.74″E). They were both brought to CARL, and were stored and aged in separate 10,000 L reservoirs. All waters were filtered through a 0.5 μm glass fibre membrane before using in the experiments. OW and ISW were then diluted with filtered fresh water to achieve needed salinity waters at 30‰.

A series of four experiments were conducted in order to determine (1) suitable [K⁺] levels for growing Lomentaria sp. in ISW, (2) the growth feasibility of Lomentaria sp. in NH₄ enriched water, (3) the effects of temperature and NH₄ on the growth of Lomentaria sp., and (4) the effects of NH₄ and PO₄ enrichment on the growth of Lomentaria sp.

Water salinity was maintained at 30-31‰, similar to the salinity of Swan River where the Lomentaria sp. was collected, by adding fresh water to compensate for evaporation. The tanks were exposed to light at 90 μmol photon m^-2 s^-1 on the surface and 22.5 μmol photon m^-2 s^-1 at the bottom.

Automatic heaters (Sonpar, HA-200, Zhongshan, Guangdong, China) were used to maintain temperatures at 25-26°C or 21-22°C.

Lomentaria sp. growth in K⁺-fortified ISW(K⁺ISW)

A total of 20 glass beakers, with a capacity of 1.5 L, holding 1 L culture medium were used for five fortnights from 19/6- 27/8/2013. The experiment determined the growth rate of Lomentaria sp. in four replicates at three levels of [K⁺] in ISW with two controls of OW and ISW at ambient room temperature. KCl was used to fortify ISW to approximately 100%, 66%, and 33% (ISW100, ISW66, and ISW33 respectively) of [K⁺] in OW at 30‰ salinity. [K⁺] at 30‰ in OW and ISW was 313 and 77 mg L^-1, respectively. Therefore, 451, 248 and 50 mg L^-1 of KCl were used to fortify ISW 30‰ to achieve ISW100, ISW66, ISW33, respectively.

The pH of cultured media was similar over the experimental period except at day 14, when ISW66 resulted in the highest pH among the five waters (P<0.05). The experiment was conducted in ambient room temperature, reflecting seasonal temperature changes during winter time. The temperature was significantly higher during the middle of the experiment, but the water temperature among the five treatments was similar as the experiment progressed (Table 1).

Time	OW	ISW	ISW33	ISW66	ISW100
pH
Day 1	17.92 ± 0.01	18.04 ± 0.03	17.95 ± 0.00	17.97 ± 0.00	18.06 ± 0.01
Day 14	28.46 ± 0.04ab	28.42 ± 0.01a	28.39 ± 0.01a	28.49 ± 0.04b	28.40 ± 0.02ab
Day 28	28.45 ± 0.03	28.39 ± 0.04	28.48 ± 0.04	28.41 ± 0.05	28.41 ± 0.03
Day 42	38.82 ± 0.07	38.71 ± 0.04	38.71 ± 0.06	38.72 ± 0.02	38.72 ± 0.05
Day 56	38.72 ± 0.08	48.85 ± 0.02	48.83 ± 0.03	38.79 ± 0.08	48.83 ± 0.06
Day 70	38.70 ± 0.02	8.92 ± 0.26	8.72 ± 0.02	8.79 ± 0.08	8.67 ± 0.04
Temperature (°C)
Day 1	118.95 ± 0.45	118.55 ± 0.35	118.50 ± 0.00	118.50 ± 0.00	118.55 ± 0.35
Day 14	2320.35 ± 0.09	2320.33 ± 0.06	2320.35 ± 0.10	2320.43 ± 0.09	2420.30 ± 0.06
Day 28	320.95 ± 0.59	2320.30 ± 0.31	2320.65 ± 0.59	320.88 ± 0.47	320.98 ± 0.21
Day 42	2320.60 ± 0.15	220.63 ± 0.11	320.85 ± 0.10	320.80 ± 0.15	2320.73 ± 0.14
Day 56	1219.88 ± 0.11	319.70 ± 0.04	2419.85 ± 0.14	219.88 ± 0.11	419.75 ± 0.03
Day 70	1219.68 ± 0.09	319.53 ± 0.06	419.65 ± 0.10	219.68 ± 0.17	419.53 ± 0.14

Table 1: The water pH and temperature in K⁺ISW for culturing Lomentaria sp.

Effect of ammonium enrichment in OW and ISW on the growth of Lomentaria sp.

Lomentaria sp. were cultured in 24 glass tanks 25/8-24/9/2013, receiving the results from previous experiment, when the ISW66 resulted in highest SGR of Lomentaria sp. Approximately 180 g of Lomentaria sp. was grown in each tank holding 45 L water in three replicates with aeration provided, at room temperature 17-19°C. The water included OW, ISW and ISW66 as control and the weekly enriched with NH₄ 100 μmol by NH₄Cl to give OW_NH₄, ISW_NH₄, ISW66_NH₄ (Table 2).

Waters	NH₄⁺ (µmol L^-1)	pH	Temp(°C)
OW	0	8.10 ± 0.02	19.00 ± 0.01
OW_NH₄	100	8.07 ± 0.02	19.10 ± 0.01
ISW	0	7.95 ± 0.03	18.99 ± 0.01
ISW_NH₄	100	7.97 ± 0.04	18.97 ± 0.01
ISW66	0	8.21 ± 0.02	18.93 ± 0.01
ISW66_NH₄	100	8.19 ± 0.02	18.95 ± 0.01

Table 2: pH and temperature in NH4 enriched water.

Effects of temperature on Lomentaria sp. cultured in OW and K⁺ISW

The effects of temperature on the growth of Lomentaria sp. were determined in two experiments.

The first experiment was conducted over four weeks, 25/8- 24/9/2013. Approximately 180 g tank-1 of 45 L cultured medium in four replicates, aeration provided were tested in three temperature conditions at 25-26°C, 21-22°C and 18-19°C. The water included OW, and OW enriched with 100 μmol NH₄ by NH₄Cl, OW_NH₄. An automatic heater (Sonpar, HA- 200, Zhongshan, Guangdong, China) was used in each tank to maintain the temperature. The pH and temperature of waters at the same temperature levels were similar over the experimental period (Table 3).

Waters	NH₄⁺(µmol L^-1)	pH	Temp(°C)
OW	0	8.14 ± 0.04	25.07 ± 0.01
OW_NH₄	100	8.16 ± 0.02	25.31 ± 0.00
OW	0	8.18 ± 0.01	21.75 ± 0.02
OW_NH₄	100	8.14 ± 0.02	21.63 ± 0.01
OW	0	8.10 ± 0.02	19.00 ± 0.01
OW_NH₄	100	8.07 ± 0.02	19.10 ± 0.01

Table 3: pH and temperature of the temperature-effect experiment in OW.

The second experiment was conducted 26/9-28/10/2013, at two temperature levels (25-26°C and 21-22°C) with three waters OW, OW_NH4 and ISW66_NH4 (the last two waters were enriched with 100 μmol L^-1 NH₄ by NH₄Cl), achieving the outcomes of NH₄ enrich for ISW66 and the two temperature levels in the first experiment of temperature effect. The Lomentaria sp. was selected by whole fond weight of approximately 3.5 g L^-1, grown in 1.5 L beakers holding 1 L of cultured medium and the beakers were placed in tank holding water. An automatic heater (Sonpar, HA- 200, Zhongshan, Guangdong, China) was used in each tank to maintain the temperature. The pH and temperature of the water were similar at the same temperature levels (Table 4).

Factors	21-22°C			25-26°C
	OW	OW_NH₄	ISW66_NH₄	OW	OW_NH₄	ISW66_NH₄
Temperature (°C)	21.64 ± 0.13	21.64 ± 0.06	21.69 ± 0.16	25.78 ± 0.22	25.67 ± 0.03	25.50 ± 0.06
pH	8.61 ± 0.03	8.74 ± 0.03	8.71 ± 0.03	8.47 ± 0.05	8.49 ± 0.05	8.45 ± 0.04

Table 4: pH and temperature in the temperature-effect (second experiment).

Effects of ammonium and phosphate enrichment on the growth of Lomentaria sp. in OW and K⁺ISW

A total of 24 1.5 L beakers were used for eight treatments in three replicates for the experiment 28/10-23/11/2013. Lomentaria sp. was cultured at a density of 3.5 g L^-1. The beakers were placed randomly into tanks filled with water. One automatic heater (Sonkar, HA-200, Zhongshan, Guangdong, China) and a pump (Grant Model GD 120, England) were used in each tank to maintain water temperature at 25-26oC, the optimal temperature for Lomentaria sp. growth (achieved from the temperature effect experiments). The water salinity was kept constant at 30-31‰ by adding filtered fresh water to compensate for evaporation.

Four levels of NH₄:PO₄ were provided weekly for OW and ISW66, by NH₄Cl and Na₂HPO₄: (1) T1 - no nutrients provided; (2) T2 - 75:7.5 μmol L^-1 NH₄:PO₄; (3) T3 - 150:15 μmol L^-1 NH₄:PO₄; and (4) T4 - 300:30 μmol L^-1 NH₄:PO₄.

Data collection

Water quality: The NH₄, NO₃, NO₂ and PO₄ concentrations in water were determined fortnightly applying the methods described by Bui et al. (in press).

The pH and salinity were recorded daily at 9-11AM using a pH meter (CyberScan pH 300, Eutech Instrument, Singapore), and a portable refractometer (RHS-10ATC, Xiamen Ming Xin Instrument, Xiamen, Fujian, China), respectively.

Temperature was recorded hourly by data loggers (HOBO Pendant temperature/light Data Logger UA-002-08, UA- 002-64).

Seaweed growth: The weight of seaweed was determined fortnightly, and at the termination of the experiment. All thalli were removed from the culture beakers/tanks by a small net and then dried using soft hand towels [33]. The thalli were immediately transferred to a weighing scale (AW220, d=0.1mg, Shimazu, Japan).

The cumulative specific growth rates (SGR) were calculated as: μ_a=(lnA_t - lnA_o) × 100/t. Where: μ_a is the SGR of seaweed (% d^-1); A_t and A₀ are the weight (mg) or length (mm) at the current time (t, day), and the commencement of the experiment (0, day); t is the current time of the trial (days).

Data analysis

All data were analysed using SPSS for Windows version 24.0. Data were tested for normality and homoscedasticity before applying parametric and non-parametric tests as appropriate. Analysis of variance (ANOVA), paired sample t-tests and Least Significant Difference (LSD) post hoc tests were used to determine significant differences at P<0.05 among the means of variables (Mean±SE). Correlations were used to find out the significant relationships among variables. Where the data did not have normal distribution and homogeneous variance, the Kruskal-Wallis (KW) test was used to test the overall difference in all treatments. In the case of significant treatment effects, a Mann-Whitney test was applied to analyse the significant differences among the means of all variables. Waters NH4

Results

Lomentaria sp. growth in K⁺ISW

Lomentaria sp. biomass remained unchanged in the first 56 days of the culture period, and a significant (P<0.05) reduction of the biomass was recorded in the last 14 days in OW, ISW and ISW100. Only ISW33 and ISW66 resulted in a significant increase of the biomass during the culture period (P<0.05), by day 42 and day 14-42, respectively. After that, the biomass reduced quickly (P<0.05). ISW66 also resulted in the highest (P<0.05) biomass at day 28 among the five treatments (Table 5). On average, ISW66 resulted in higher biomass growth than other waters in the first 56 days.

Time	OW	ISW	ISW33	ISW66	ISW100
Day 1	₁₂3.30 ± 0.62	₁₂3.32 ± 0.40	₁3.30 ± 0.47	₁3.31 ± 0.65	₁₂33.28 ± 0.58
Day 14	₁4.03 ± 0.41^ab	₁₂3.56 ± 0.15^a	₁3.70 ± 0.12^a	₂4.47 ± 1.88^b	₂3.97 ± 0.30^ab
Day 28	₁3.51 ± 0.23^a	₁₂3.63 ± 0.12^a	₁3.84 ± 0.25^ab	₂4.26 ± 0.17^b	₁₂₃3.29 ± 0.23^a
Day 42	₁3.83 ± 0.39	₁3.91 ± 0.28	₂4.51 ± 0.28	₂4.49 ± 0.35	₂3.75 ± 0.27
Day 56	₁3.47 ± 0.32^ab	₂3.01 ± 0.42^a	₁3.79 ± 0.24^ab	₁₂3.94 ± 0.20^b	₂3.58 ± 0.29^ab
Day 70	₂2.33 ± 0.61^ab	₃1.57 ± 0.36^a	₃2.51 ± 0.18^ab	₃2.53 ± 0.29^ab	₃2.86 ± 0.22^b

Table 5: The biomass (g) of Lomentaria sp. in K⁺ISW.

Values (mean±SE) within a row sharing a common superscript are not significantly different (LSD test; P>0.05; n=4). Values (mean±SE) within a column sharing a common subscript are not significantly different (LSD test; P>0.05; n=4).

Over time, the biomass and SGR were significantly (P<0.05) different among treatments. In the first two fortnights, the SGR of Lomentaria sp. was significantly higher than the rest of the experimental periods in all waters. ISW66 resulted in the highest SGR in the first fortnight, but ISW33 gave a higher SGR in the following fortnight. The Lomentaria sp. presented a similar fortnightly SGR over the last three fortnights of the experiment (Table 6). In the first 42 days of the culture period for growing Lomentaria sp., either ISW66 or ISW33 gave higher biomass gains than other water sources.

Time	OW	ISW	ISW33		ISW66		ISW100
Fortnightly
Day 1–14	₁1.44 ± 0.20^ab	₁0.49 ± 0.38^a		₁0.78 ± 0.29^a		₁2.08 ± 0.18^b		₁1.31 ± 0.54^ab
Day 15–28	₂-1.12 ± .57^ab	₁0.15 ± 0.41^ac		₁0.27 ± 0.33^c		₂₃-0.32 ± 0.30^abc		₂-1.45 ± 0.44^b
Day 29–42	₁20.60 ± 0.43	₁0.52 ± 0.62		₂1.24 ± 0.16		₂0.35 ± 0.32		₁1.02 ± 0.35
Day 43–56	₂-0.68 ± 1.11	₂2.03 ± 0.66		₃-1.26 ± 0.26		₃-0.90 ± 0.30		₂-0.35 ± 0.30
Day 57–70	₃-4.09 ± 2.34	₃-4.97 ± 1.88		₄-2.95 ± 0.39		₄-3.27 ± 0.53		₃-1.59 ± 0.38
Cumulative SGR
Day 1–14	₁1.44 ± 0.20^ab	₁0.49 ± 0.38^a		₁0.78 ± 0.29^a		₁2.08 ± 0.18^b		₁1.31 ± 0.54^ab
Day 1–28	₂0.20 ± 0.21^a	₁0.31 ± 0.13^ab		₁0.52 ± 0.26^ab		₂0.89 ± 0.13^b		₂-0.02 ± 0.22^b
Day 1–42	₂0.32 ± 0.25	₁0.37 ± 0.16		₁0.73 ± 0.16		₂₃0.71 ± 0.19		₂0.31 ± 0.17
Day 1–56	₂0.07 ± 0.16^ab	₁-0.23 ± 0.24^a		₁0.23 ± 0.12^ab		₃0.30 ± 0.07^b		₂0.14 ± 0.15^ab
Day 1–70	₃-1.05 ± 0.58	₂-1.28 ± 0.31		₂-0.56 ± 0.07		₄-0.83 ± 0.13		₂-0.47 ± 0.09

Table 6: The SGR (% d^-1) of Lomentaria sp. in K⁺ISW.

Values (mean±SE) within a row sharing a common superscript are not significantly different (LSD test; P>0.05; n=4). Values (mean±SE) within a column at one parameter sharing a common subscript are not significantly different (LSD test; P>0.05; n=4).

In the first two fortnights, the Lomentaria sp. showed promising signs of growth when new axial filament growth from different parts of the thallus, and the red colour of Lomentaria sp. remained. However, although the fresh biomass of the Lomentaria sp. increased until day 42, a sign of discolouration appeared, and defragmentation of the thallus began. By the end of the experiment, most of the red colour of the Lomentaria sp. disappeared and few tissues remained, providing small amounts of fresh biomass of the Lomentaria sp.

The [N] in water varied differently at different points of the culture period. NH₄ was negligible as the experiment progressed, whereas NO₂ decreased and NO₃ increased in all waters toward the end of the experiment. There was no significant difference of [NO₃] among water types during the first 42 days of the culture period, whereas, at day 56 and day 70, ISW66 and ISW33, respectively, resulted in higher [NO₃] than other waters (Table 7). However, NO₃ showed no significant correlation with the biomass of Lomentaria, but NO₂ did.

Time	OW	ISW	ISW33	ISW66	ISW100
NO₂
Day 1	₁₂0.021 ± 0.002	₁0.042 ± 0.017	₁₂0.022 ± 0.002	₁₃0.021 ± 0.001	₁0.021 ± 0.002
Day 14	₁0.063 ± 0.033	₁0.038 ± 0.005	₁0.038 ± 0.014	20.040 ± 0.008	₂0.040 ± 0.000
Day 28	₁₂0.034 ± 0.003^ab	₁0.041 ± 0.004^a	₁₂0.028 ± 0.007^b	120.028 ± 0.005^ab	₃0.045 ± 0.002^a
Day 42	₂0.005 ± 0.000^a	₂0.009 ± 0.001^b	₂0.006 ± 0.000^ac	30.005 ± 0.000^a	₄0.007 ± 0.001^c
Day 56	₂0.006 ± 0.000	₂0.006 ± 0.000	₂0.007 ± 0.001	30.007 ± 0.001	₄0.007 ± 0.001
Day 70	₁0.002 ± 0.000^a	₂0.004 ± 0.001^bd	₂0.007 ± 0.000^c	30.004 ± 0.001^b	₄0.006 ± 0.000^c
NH4
Day 1	₁0.825 ± 0.175^a	Neg.^b	Neg.^b	₁Neg.^b	Neg.^b
Day 14	₂Neg.	Neg.	Neg.	₁Neg.	Neg.
Day 28	₂0.003 ± 0.003	Neg.	Neg.	₁0.010 ± 0.004	Neg.
Day 42	₂Neg.^a	Neg.^a	Neg.^a	₂0.333 ± 0.236^b	Neg.^a
Day 56	₂Neg.	Neg.	Neg.	₁Neg.	Neg.
Day 70	₂Neg.	Neg.	Neg.	₁Neg.	Neg.
NO3
Day 1	₁1.23 ± 0.13^a	2.10 ± 0.22^b	2.05 ± 0.22^b	2.03 ± 0.15^bc	₁₃1.50 ± 0.15^ac
Day 14	₂₃2.28 ± 0.46	2.31 ± 0.44	2.02 ± 0.45	1.64 ± 0.35	₁1.87 ± 0.18
Day 28	₂2.69 ± 0.29	2.23 ± 0.09	2.03 ± 0.13	2.10 ± 0.43	₂2.37 ± 0.09
Day 42	₁₃1.67 ± 0.19	2.60 ± 0.58	1.70 ± 0.15	1.13 ± 0.06	₁1.73 ± 0.03
Day 56	₁1.18 ± 0.10^a	2.88 ± 0.80^ab	1.33 ± 0.32^ab	3.60 ± 1.08^b	₃1.13 ± 0.13^a
Day 70	₁1.53 ± 0.10^a	1.67 ± 0.16^a	3.03 ± 0.27^b	1.80 ± 0.11^c	₂2.30 ± 0.26^bc
PO4
Day 1	1.55 ± 0.12^a	₁1.68 ± 0.05^a	₁2.08 ± 0.11^bc	₁1.83 ± 0.08^ac	₁1.65 ± 0.10^a
Day 14	1.83 ± 0.20	₁1.78 ± 0.16	₁₃1.69 ± 0.11	₁1.83 ± 0.23	₁1.68 ± 0.06
Day 28	1.30 ± 0.06	₂1.17 ± 0.13	₂1.23 ± 0.20	₂1.23 ± 0.08	₁₂1.23 ± 0.10
Day 42	2.30 ± 0.85	₂1.03 ± 0.17	₂₃1.40 ± 0.12	₃1.40 ± 0.20	₂1.20 ± 0.29
Day 56	1.50 ± 0.15^a	₁1.73 ± 0.06^ab	₁1.80 ± 0.08^b	₂1.50 ± 0.04^a	₁₂1.60 ± 0.08^ab
Day 70	1.53 ± 0.14^a	₁1.47 ± 0.14^a	₄3.77 ± 0.20^b	₄3.97 ± 0.32^b	₃2.33 ± 0.10^c

Table 7: The water quality parameters of Lomentaria sp. cultured in K⁺ISW.

PO₄ was significantly reduced during the middle of the experiment; however, it increased towards the end of the experiment, and showed a significant correlation with the biomass of Lomentaria sp.

Values (mean±SE) within a row sharing a common superscript are not significantly different (LSD test; P>0.05; n=4). Values (mean±SE) within a column at one parameter sharing a common subscript are not significantly different (LSD test; P>0.05; n=4).

Effect of ammonium enrichment in OW and ISW on the growth of Lomentaria sp.

NH₄ did not affect the growth of Lomentaria sp. in OW, but it did show a significant effect on Lomentaria sp. growth in ISW. Both ISW_NH₄ and ISW66_NH₄ resulted in significantly higher biomass and SGR_w of Lomentaria sp. than ISW and ISW66, respectively (Table 8). NH₄ presented the highest effectiveness when used in ISW66_NH₄; this resulted in higher biomass and SGR_w of Lomentaria sp. by the end of the experiment than OW_ NH₄ and ISW_NH₄. In the waters not enriched with NH₄, the three water types gave similar biomass and SGR_w of Lomentaria sp. However, a significant reduction was found in the biomass of Lomentaria sp. over the experimental period in all waters.

Parameters	OW		ISW		ISW66
Parameters	OW	OW_NH₄	ISW	ISW_NH₄	ISW66	ISW66_NH₄
Biomass day 1	₁180.69 ± 0.09	₁180.45 ± 0.12	₁180.16 ± 0.13	₁180.37 ± 0.19	₁180.30 ± 0.15	₁180.50 ± 0.14
Biomass day 28	₂118.66 ± 11.77^a	₂131.22 ± 3.09^a	₂109.93 ± 10.78^a	2134.51 ± 5.13^b	₂126.22 ± 8.57^a	₂161.6 ± 4.08^b
SGRw	-1.48 ± 0.33^a	-1.10 ± 0.08^a	-1.74 ± 0.35^a	-1.02 ± 0.12^b	-1.24 ± 0.23^a	-0.38 ± 0.09^b

Table 8: Biomass (g) and SGR_w (% d^-1) of Lomentaria sp. in NH₄ enriched water.

Values (mean±SE) within a row at one water type sharing a common superscript are not significantly different (t-test; P>0.05; n=3). Values of biomass (mean±SE) within a column sharing a common subscript are not significantly different (t-test; P>0.05; n=3).

Effects of temperature on Lomentaria sp. cultured in OW and K⁺ISW

Temperature significantly (P<0.05) affected the biomass and growth rate of Lomentaria sp. during the four weeks growing in the tanks. The ambient temperature of 18-19°C resulted in the lowest Lomentaria sp. biomass and SGR_w. However in the OW_NH₄ water, 25-26°C gave a higher biomass and SGR than 21-22°C (Table 9).

Parameters	25–26°C		21–22°C		18-19°C
	OW	OW_NH₄	OW	OW_NH₄	OW	OW_NH₄
Biomass day 1	₁180.44 ± 0.23	₁180.15 ± 0.43	₁180.16± 0.13	₁180.50 ± 0.27	₁180.69 ±0.09	₁180.45 ± 0.12
Biomass day 28	₂152.73 ±1.36^a	₂150.99 ± 3.16^a	₂156.21± 2.36^a	₂113.97 ± 2.48^b	₂118.66 ± 11.77^a	₂131.22 ±3.09^a
SGR	-0.58± 0.03^a	-0.61± 0.07^a	-0.49 ± 0.05^a	-1.59± 0.07^b	-1.48 ±0.33^a	-1.10 ± 0.48^a

Table 9: Biomass and SGRw (% d-1) of Lomentaria sp. in three temperature levels.

Values (mean±SE) within a row at one temperature sharing a common superscript are not significantly different (test; P>0.05; n=4). Values (mean±SE) within a column sharing a common subscript are not significantly different (test; P>0.05; n=4).

In the second experiment, when only two levels of temperature and three water types were used, mortality of Lomentaria sp. started occurring on day 25. By day 45, there was no sign of living Lomentaria sp. in the beakers; therefore, the data of biomass and SGR_w were collected by day 25 of the experimental. At the 25- 26°C, both OW and OW_NH₄ resulted in a significant increase of biomass than at the beginning. However, these increases did not result in a significantly higher SGR_w of Lomentaria sp. than ISW66_NH₄. On the other hand, the temperature showed no effect on the growth of Lomentaria sp. in all waters, while at the same temperature levels, the three water sources resulted in a similar SGW_w. The length of the Lomentaria sp. showed no significant change over the culture period in all waters and temperatures (Table 10).

Criteria	21–22oC			25–26oC
	OW	OW_NH₄	ISW66_NH₄	OW	OW_NH₄	ISW66_NH₄
Biomass day 1	3.49± 0.07	3.49±0.26	3.53±0.07	₁3.20±0.13	₁3.23± 0.12	3.60± 0.14
Biomass day 25	4.53±0.50	4.89± 0.77	5.01±0.70	₂4.71±0.49	₂4.19±0.29	4.39± 0.43
SGR_W	1.01± 0.57	1.40 ± 0.25	1.32±0.56	1.59±0.16	1.08±0.08	0.80±0.17

Length day 1	10.88± 0.52	13.50 ± 1.10	13.60± 0.39	11.32± 0.66	12.43± 1.49	12.83± 0.60
Length day 25	11.98±0.30	13.13±1.20	14.28±0.47	11.67±0.67	13.00±1.53	13.00±0.64
SGR_L	0.41± 0.10^a	-0.12±0.22^b	0.20± 0.09^ab	0.13± 0.09	0.19±0.02	0.05±0.03

Table 10: Biomass (g), length (mm) and SGR (% d^-1) of Lomentaria sp. in two temperatures.

Values (mean±SE) within a row at one temperature sharing a common superscript are not significantly different (LSD test; P>0.05; n=4). Values (mean±SE) within a column of a parameter sharing a common subscript are not significantly different (test; P>0.05; n=4).

Effects of ammonium and phosphate enrichment on the growth of Lomentaria sp. in OW and K⁺ISW

Following the results from the previous experiment, this experiment lasted for only 25 days, to collect the dried biomass of the Lomentaria sp. By the end of the experiment, with no nutrient enrichment, ISW66 resulted in a significantly higher biomass and SGR_w of Lomentaria sp., and higher [NO₂] and [PO₄] content in water than in OW; however, these were similar at other nutrient levels (Tables 11 and 12).

Crite-ria	OW				ISW66
Crite-ria	T1	T2	T3	T4	T1	T2	T3	T4
Biomass
Day 1	3.37±0.01	₁3.38±0.02	3.40±0.01	₁3.40±0.02	₁3.35 ± 0.01	3.38±0.01	3.37±0.00	3.36 ± 0.01
Day 25	3.30 ±0.28^a	24.28 ±0.12^b	3.87±0.36^a	₂4.10±0.13^a	₂4.21±0.19	4.14±0.50	3.65±0.29	3.75 ± 0.17
SGR_W	-0.12±0.16^a	0.94± 0.14^b	0.49±0.40^a	0.75±0.12^a	0.91±0.16^a	0.71±0.50^ab	0.29±0.32^b	0.43±0.18^b
Dried content
Day 1	₁14.77± 0.11	14.77 ± 0.11	₁14.77 ± 0.11	₁14.77±0.11	14.77±0.11	14.77±0.11	14.77±0.11	14.77±0.11
Day 25	₂16.04± 0.64^a	14.51 ± 0.56^ab	₂16.45 ± 2.10^a	₂12.18±1.15^b	14.21±0.62	14.26±0.47	14.16±0.74	15.14±0.63

Table 11: Biomass (g), SGR_w (% d-1) and dried content (%) of Lomentaria sp. cultured in four nutrient levels.

Criteria	OW				ISW66
Criteria	T1	T2	T3	T4	T1	T2	T3	T4
NO₃
Day 1	₁0.97±0.03^a	1.47±0.03^ab	1.60±0.06^b	2.10±0.00^c	2.13±0.12^a	2.27±0.07^b	2.53±0.03^b	₁2.90±0.31^c
Day 25	₂1.17 ± 0.03^a	1.13±0.30^ab	1.33±0.27^b	1.43±0.27^b	1.50±0.21	1.87±0.32	2.53±0.62	₂1.00±0.06
NO₂
Day 1	Neg.^a	1.00±0.00^b	0.33 ± 0.00^ab	0.33±0.00^ab	Neg.	Neg.	0.33 ±0.00	0.33±0.00
Day 25	Neg.	0.09±0.08	0.01±0.01	0.12±0.05	0.42 ± 0.41	0.01±0.00	0.16±0.16	0.06±0.03
PO₄
Day 1	₁2.17±0.09^a	₁2.53±0.29^a	₁2.97 ± 0.09^a	₁3.93±0.20^b	2.23±0.09^a	2.73 ± 0.09^ab	₁3.17±0.43^b	₁4.47±0.52^c
Day 25	₂1.30±0.10	₂1.03±0.09	₂1.23±0.12	₂1.23±0.13	3.17±0.94^a	2.00 ± 0.40^b	₂1.73±0.03^b	₂1.73±0.28^b

Table 12: The water quality OW and ISW66 in which Lomentaria sp. was cultured at different nutrient enrichment levels.

Values (mean±SE) within a row in one water sharing a common superscript are not significantly different (LSD test; P>0.05; n=3). Values (mean±SE) within a column at one parameter sharing a common subscript are not significantly different (t-test; P>0.05; n=3).

Nutrient enrichment did not significantly affect the growth of Lomentaria sp. in ISW66. The biomass, SGR_w and dried content of Lomentaria sp. were similar after 25 days of culture in four NH₄:PO₄ levels. In OW, T2 resulted in the highest biomass and SGR, and the dried content of Lomentaria sp. cultured in T4 was lowest.

Although NH₄ was provided weekly, NH₄ in water was negligible during the experiment. By the beginning of the experiment, [NO₃] in ISW was higher than in OW, and in both waters, [NO₃] and [PO₄] were significantly increased in higher nutrient enrichment levels. However, by day 25, [NO₃] in ISW was only higher than OW at T3, and lower at T4. [NO₃] and [PO₄] were similar in OW. [NO₂] was negligible in the lower nutrient enrichment levels at the beginning, and showed no s