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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: [email protected]

Accepted date: May 03, 2017

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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: μa=(lnAt - lnAo) × 100/t. Where: μa is the SGR of seaweed (% d-1); At 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 ± 0.41ab 123.56 ± 0.15a 13.70 ± 0.12a 24.47 ± 1.88b 23.97 ± 0.30ab
Day 28 13.51 ± 0.23a 123.63 ± 0.12a 13.84 ± 0.25ab 24.26 ± 0.17b 1233.29 ± 0.23a
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 ± 0.32ab 23.01 ± 0.42a 13.79 ± 0.24ab 123.94 ± 0.20b 23.58 ± 0.29ab
Day 70 22.33 ± 0.61ab 31.57 ± 0.36a 32.51 ± 0.18ab 32.53 ± 0.29ab 32.86 ± 0.22b

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 ± 0.20ab 10.49 ± 0.38a 10.78 ± 0.29a 12.08 ± 0.18b 11.31 ± 0.54ab
Day 15–28 2-1.12 ± .57ab 10.15 ± 0.41ac 10.27 ± 0.33c  23-0.32 ± 0.30abc 2-1.45 ± 0.44b
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 ± 0.20ab 10.49 ± 0.38a 10.78 ± 0.29a 12.08 ± 0.18b 11.31 ± 0.54ab
Day 1–28 20.20 ± 0.21a 10.31 ± 0.13ab 10.52 ± 0.26ab 20.89 ± 0.13b 2-0.02 ± 0.22b
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 ± 0.16ab 1-0.23 ± 0.24a 10.23 ± 0.12ab 30.30 ± 0.07b 20.14 ± 0.15ab
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 ± 0.003ab 10.041 ± 0.004a 120.028 ± 0.007b 120.028 ± 0.005ab 30.045 ± 0.002a
Day 42 20.005 ± 0.000a 20.009 ± 0.001b 20.006 ± 0.000ac 30.005 ± 0.000a 40.007 ± 0.001c
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 ± 0.000a 20.004 ± 0.001bd 20.007 ± 0.000c 30.004 ± 0.001b 40.006 ± 0.000c
NH4          
Day 1 10.825 ± 0.175a 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 ± 0.236b Neg.a
Day 56 2Neg. Neg. Neg. 1Neg. Neg.
Day 70 2Neg. Neg. Neg. 1Neg. Neg.
NO3          
Day 1     11.23 ± 0.13a  2.10 ± 0.22b  2.05 ± 0.22b   2.03  ± 0.15bc  131.50 ± 0.15ac
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  ± 0.10a   2.88  ± 0.80ab    1.33 ± 0.32ab   3.60 ± 1.08b  31.13 ± 0.13a
Day 70 11.53 ± 0.10a 1.67  ± 0.16a 3.03 ± 0.27b 1.80 ± 0.11c 22.30 ± 0.26bc
PO4          
Day 1 1.55  ± 0.12a 11.68  ±  0.05a 12.08  ±  0.11bc 11.83  ±  0.08ac 11.65 ± 0.10a
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  ±  0.15a    11.73  ± 0.06ab 11.80  ±  0.08b 21.50 ± 0.04a 121.60 ± 0.08ab
Day 70 1.53  ±  0.14a 11.47  ±  0.14a 43.77  ±  0.20b 43.97 ± 0.32b 32.33 ± 0.10c

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 SGRw 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 SGRw 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 SGRw 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
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  ±  11.77a 2131.22  ±  3.09a 2109.93  ±  10.78a 2134.51  ±  5.13b 2126.22  ±  8.57a 2161.6  ±  4.08b       
SGRw -1.48  ± 0.33a -1.10  ±  0.08a -1.74  ±  0.35a -1.02  ±  0.12b -1.24  ±  0.23a -0.38  ±  0.09b

Table 8: Biomass (g) and SGRw (% 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 SGRw. 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 ±1.36a 2150.99 ± 3.16a 2156.21± 2.36a 2113.97 ± 2.48b 2118.66 ± 11.77a 2131.22 ±3.09a
SGR -0.58± 0.03a -0.61± 0.07a -0.49 ± 0.05a -1.59± 0.07b -1.48 ±0.33a -1.10 ± 0.48a

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 SGRw 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 SGRw 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 SGWw. 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
SGRW 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
SGRL 0.41± 0.10a -0.12±0.22b 0.20± 0.09ab 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 SGRw 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
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 ±0.28a 24.28 ±0.12b 3.87±0.36a 24.10±0.13a 24.21±0.19 4.14±0.50 3.65±0.29 3.75 ± 0.17
SGRW -0.12±0.16a 0.94± 0.14b 0.49±0.40a 0.75±0.12a 0.91±0.16a 0.71±0.50ab 0.29±0.32b 0.43±0.18b
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± 0.64a 14.51 ± 0.56ab 216.45 ± 2.10a 212.18±1.15b 14.21±0.62 14.26±0.47 14.16±0.74 15.14±0.63

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

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

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).

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, SGRw 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 significant difference among the nutrient levels as the experiment progressed. There was a significant reduction of [PO4] during the experiment, and [PO4] was significantly correlated with the biomass of the Lomentaria sp (Table 12).

Discussion

This is the first study on growing Lomentaria sp. in artificial conditions, particularly in ISW. Lomentaria sp. showed an ability to grow in ISW under special conditions of K+ISW and seasonal temperatures.

Potassium fortification was needed for ISW to sustain the growth of Lomentaria sp., when at the day 28, ISW66 resulted in higher Lomentaria sp biomass than in OW, and from the 28th day onward, the biomass of Lomentaria sp was similar in these two waters. The growth of seaweed is significantly affected by [K+], which plays an important role in photosynthesis and regulation of osmotic pressure of the seaweed cells [9,11]. The [K+] in the seaweed cells should be between 100-200 mM for proper protein synthesis [39]. Intracellular [K+] is regulated by internal and external [K+] exchange mechanisms, which are determined by external [K+] [39,40]. The osmotic gradient of aquatic plant cells is maintained by [K+], and is facilitated by a suitable ratio between Na+ and K+ internally [39,41]. Marine animals need the ISW to be fortified to 50-100% of [K+] in OW at the same salinity to obtain sufficient [K+] for a balanced osmo-regulation for a capacity to grow [14,17,18,19,42]. Similarly, Lomentaria sp. also needs higher [K+] than in ambient ISW for growing. In this study, the concentration of K+ of 103–206 mg L-1 (the Na:K ratio is 37:1-75:1) provided a higher biomass gain and SGRw of Lomentaria sp. than higher or lower [K+], and it is similar to the preferred Na:K for Ulva growth at 47:1 [43]. This [K+] range is lower than required by other rea seaweeds Caloglossa leprieurii and Bostrychia radicans [12]. If the culture period was less than one month, ISW66 would be a better choice than ISW33. However, Lomentaria sp. should not be cultured longer than 42 days for a higher biomass gain.

Ammonium is preferred source of N for seaweed growth over NO3 [44], which is why NH4 in water was negligible over the culture period, even in the waters supplied weekly with NH4. In previous work, the red seaweed Gelidium amansii grew faster at 80 μmol L-1 NH4 than at 200 μmol L-1 [44]. However, in this study, the Lomentaria sp. showed no response in 100 μmol L-1 NH4 in both OW and ISW in the tanks. This can be explained by the effect of the low temperature, since the ammonium-effect experiment was conducted at ambient room temperature in winter, when the temperature was approximately 19°C. This result was demonstrated in the temperature-effect experiment, where the reduction rate of Lomentaria sp. cultured in 18-19°C was higher than other two higher temperature levels. As the Lomentaria sp. cultured in tanks holding OW and OW_NH4 showed different responses to the 21-22°C and 25-26°C temperatures, the second experiment was conducted in beakers at these two temperature levels. In addition, ISW66_NH4 provided the lowest reduction SGR in the NH4-effect experiment, was also tested. A similar SGRw was found for Lomentaria sp. cultured in one water source at two temperature levels and cultured in four different water sources at one temperature level, and this revealed that the suitable temperature for Lomentaria sp. cultured in captivity was 21-26°C. This prefer temperature range was similar to the green seaweeds Ulva curvata [45], Ulva lactuca [46], and Ulva pertusa [47], and the red seaweed Hypnea cervicornis J Agardh [30], but was higher than the need of the red seaweeds Phycodrys rubens and Membranoptera alata [48] .

Contrary to the negative SGR found in Lomentaria sp. cultured in all temperature conditions in tanks, the Lomentaria sp. cultured in beakers at 21-26°C in the temperature-effect experiment and K+-fortification effect experiment at 18.5- 21°C resulted in a positive SGRw, revealing the scale of growing Lomentaria sp. This can only be explained by the different seasons of sampling. The Lomentaria sp. were collected from the field 2-3 days before the beginning of each experiment, reflecting the seasonal growth of Lomentaria sp. at different stages. The experiment conducted in the tanks were from the middle of winter to the end of autumn, whereas the beaker experiments were in early winter and late autumn to early summer. Observations in the field in early summer showed that the Lomentaria sp. grew quickly and the canopy was largest. Furthermore, the Lomentaria sp. standing crop decreased gradually by the end of summer, and reappeared in the spring.

At 21-22°C, the length of Lomentaria sp. cultured in OW_NH4 were reduced, resulting from apical cell breakage; however, the biomass gain was positive, indicating growth of the Lomentaria sp. The similarity of the SGRw and SGRL of the Lomentaria sp. cultured in ISW66_NH4 and the sources of OW showed the ability of Lomentaria sp. to grow in ISW66_NH4.

Although NH4 was necessary for Lomentaria sp. growth in ISW66, the combination of NH4 and PO4 did not show the good effect than single NH4. In addition to the weekly supplied NH4/ PO4, N and P in water were also produced by the decomposition of Lomentaria sp. NH4 combines with PO4 result in a higher growth rate of Sargassum baccularia than single nutrient sources [24]. Soluble N and P in water are quickly cycled by living microbes, so their concentrations are not stable, difficult to measure [49]. They are also consumed at different rates [50]. At the same concentrations, NH4 is uptaken faster than PO4 by seaweeds [51]. Consequently, NH4 was negligible in waters as the experiment progressed, NO3 was reduced over the culture period, and [PO4] was lower at the termination of the experiment than at the beginning in the last experiment, showing Lomentaria sp. growth.

In OW, the NH4:PO4 ratio at 75:7.5 μmol L-1 resulted in the highest SGR and a significant increase of biomass at the end of the experiment compared with the beginning. These nutrient concentrations were similar to those needed by the red seaweed Gelidium amansii [44]. However, in ISW, NH4:PO4 enrichment showed no effect on the growth of Lomentaria sp., since water not enriched with nutrients resulted in a significant gain of biomass over the culture period. This result verified those of the previous experiment, where ISW66_NH4 gained a similar SGR of Lomentaria sp. to OW and OW_NH4 at 21-26°C.

Conclusions

This study identified the suitable environmental parameters to grow Lomentaria sp. under laboratory conditions as a temperature of 21-26°C, a salinity of 30-31% and a supplied NH4 concentration of no greater than 100 μmol L-1. In ISW, K+ fortification is needed at 33-66% of [K+] in OW at 30‰ for higher biomass gain in the culture period of no longer than 42 days.

Acknowledgements

We thank Proof-reading-service.com for proofreading this paper. This work was funded by the Vietnamese Ministry of Education and Curtin International Postgraduate Research Scholarship, and conducted at Curtin University.

References