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Effects of Nitrogen Source and Concentration on the Growth and Biochemical Composition of the Red Seaweed Grateloupia turuturu (Halymeniaceae, Rhodophyta)
 
 
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Article

Physiological Impacts of Nitrogen Starvation and Subsequent Recovery on the Red Seaweed Grateloupia turuturu (Halymeniaceae, Rhodophyta)

by
Yining Chen
1,†,
Lan Lan
1,2,†,
Jing Zhang
1,
Qiaohan Wang
1,*,
Yan Liu
1,
Huiru Li
3,*,
Qingli Gong
1 and
Xu Gao
1
1
Key Laboratory of Mariculture (Ministry of Education), Fisheries College, Ocean University of China, Qingdao 266003, China
2
Marine Development Affairs Center of Wendeng District, Weihai 264400, China
3
Department of Aquaculture and Aquatic Science, Kunsan National University, Gunsan 54150, Republic of Korea
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Sustainability 2023, 15(9), 7032; https://doi.org/10.3390/su15097032
Submission received: 27 January 2023 / Revised: 14 March 2023 / Accepted: 18 April 2023 / Published: 22 April 2023
(This article belongs to the Special Issue Ecology, Diversity and Conservation of Seaweeds)
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Abstract

:
Grateloupia turuturu is a potential aquaculture species as it has a significant number of high-valued compounds. The purpose of this study was to evaluate the physiobiochemical performances of G. turuturu under nitrogen deficiency and resupply. In this study, G. turuturu was exposed to different lengths of nitrogen starvation (from 0 to 28 days) and subsequently subjected to a 21-day nitrogen-recovery period. The nitrate and ammonium uptake rates, growth rates, and nitrogenous compounds of G. turuturu were periodically measured. The results showed that the nitrogen-starved G. turururu absorbed ammonium much faster than nitrate after nitrogen recovery. Furthermore, an overcompensatory uptake of ammonium was induced via nitrogen deficiency in a short phase after nitrogen resupply. The time and rates of depletion of different compositions varied during nitrogen starvation. Specifically, pigment contents decreased faster than protein and total nitrogen contents, and the reduction rate of protein was the lowest. After nitrogen resupply, though G. turuturu gradually recovered, growth rates and pigments from long-term nitrogen starvations could not recover enough to reach their original values. Our study reveals the physiological changing processes of G. turuturu during nitrogen starvation and recovery and provides baseline information aiding in the development of strategies for G. turuturu cultivation.

1. Introduction

Nitrogen is a major and indispensable element for seaweeds as it is used to biosynthesize various structural and physiological compounds, such as proteins, nucleic acids, and pigments [1]. Nitrate and ammonium are the two main inorganic nitrogen forms utilized by seaweeds in seawater [2]. Their ocean concentrations experience dramatic spatial and temporal fluctuations that are due to physical and biological activities [3,4]. Hence, nitrogen is the nutrient most frequently observed to limit the growth and primary production of seaweeds [2,5,6]. Furthermore, the limitation and depletion of nitrogen not only repress natural seaweed populations but also impair the production and quality of commercial cultured and exploited seaweeds, such as Pyropia yezoensis [7] and Gracilaria gracilis [8], leading to extensive economic losses. Hence, given the fluctuations of nitrogen, it is crucial to investigate the dynamic physiological and biochemical acclimation process of seaweeds under nitrogen deficiency and subsequent resupply.
Previous studies have shown that nitrogen deficiency generally reduces algal growth rates and nitrogenous compounds, including chlorophyll a, phycobiliproteins, proteins, and amino acids [9,10,11,12]. In contrast, soluble polysaccharides and lipids were observed to increase in algae under the stress of nitrogen deficiency [11,13]. Additionally, accompanied by a decrease in pigments, photosynthesis II quantity and activity were also repressed during nitrogen starvation in diatom Thalassiosira pseudonana, whereas photoprotection was induced through increased nonphotochemical quenching [14]. Increased photoprotection is thought to reduce potentially photo-oxidative damage to cells because nitrogen deficiency can cause excess light absorption relative to its biosynthetic sinks [15]. With in inclusion of the physiological and biochemical responses of algae to nitrogen starvation, the transcriptomic and proteomic performances of algae, such as the downregulation of genes encoding photosynthesis and the upregulation of genes encoding ammonium transporters, have also been investigated [16,17,18,19,20]. The physiological, biochemical, and molecular responses of algae to nitrogen resupply were also investigated in previous studies, though most of them focused on microalgae [21,22,23,24]. For macroalgae, the overcompensatory uptakes of ammonium were observed after nitrogen starvation in Gracilariopsis lemaneiformis [25] and Sargassum horneri [26]. Liu et al. [11,12] observed that nitrogen resupply promoted the growth rates of nitrogen-starved G. lemaneiformis but failed to increase the phycoerythrin and soluble protein contents to their levels prior to nitrogen starvation.
The red macroalga Grateloupia turuturu is a Pacific species that was originally distributed in the low intertidal and shallow subtidal zones of China [27], Japan [28], Korea [29], and the far-eastern seas of Russia [30]. However, thanks to its high physiological tolerances and rapid reproduction rate, G. turuturu has successfully invaded seas across the world [31,32,33]. G. turuturu is a traditional sea vegetable consumed in Asia [34,35]. At present, it is regarded as a potential aquaculture species because it contains a significant number of high-value compounds, such as all the 18 basic amino acids, proteins, phycoerythrin, dietary fibers, minerals, and lipids, which are widely used in foods, cosmetics, and immunological analysis [36,37,38,39,40,41]. Additionally, G. turuturu is considered as an ideal seaweed for coculturing with other feeding species thanks to its nutrient uptake ability and negative impacts on pathogenic bacteria [42,43]. Seaweeds have generated growing interest nowadays thanks to their potential as a sustainable and valuable source for food and nonfood applications [44,45,46]. Accordingly, seaweed farming is also regarded as a sustainable aquaculture because seaweeds help to mitigate the impacts of climate change and human activities on seawater environments, such as ocean acidification, deoxygenation, and eutrophication [47,48,49]. As the limitation of dissolved nitrogen is one of the key challenges for seaweed aquaculture [50,51,52], the fundamental understanding of seaweed responses to nitrogen deficiency and recovery is critical for the sustainable and effective management of seaweed aquaculture. However, up to now, the physiological responses of G. turuturu under nitrogen deficiency and subsequent recovery conditions have remained unknown, though this provides an important reference for the sustainable maricultivation of G. turuturu.
In this study, to understand the dynamic physiological performances of G. turuturu under nitrogen starvation and subsequent recovery, we investigated the uptake rates of nitrate and ammonium, growth rates, and the nitrogenous compound contents of G. turuturu after different lengths of nitrogen deficiency (from 0 to 28 days) and 21 days of nitrogen resupply via indoor experiments. Our study helps to clarify the physiological adjustments and nitrogen utilization strategies of G. turuturu under conditions of nitrogen starvation and recovery. This study also provides important basic information for seaweed aquaculture.

2. Materials and Methods

2.1. Algal Collection and Preparation

The site at which Grateloupia turuturu tetrasporophytes were collected was the intertidal zone of Tuandao (36°02′ N, 122°17′ E), Qingdao, China. Collected samples were immediately transferred to our laboratory in cooler boxes filled with seawater. Tetrasporophytes in healthy conditions were chosen and rinsed well with sterilized seawater to remove diatoms and detritus. Afterward, samples were incubated in large conical flasks containing 3 L of enriched F/2 medium [53] with gentle aeration. These tetrasporophytes were precultured at 20 °C, with 86 μmol photons m−2 s−1 and a 12–12 h light–dark cycle until a large number of tetraspores could be detected by using an Olympus microscope. Next, one mature terasporopyte was placed in a Petri dish with a diameter of 20 cm and filled with 250 mL enriched F/2 medium. The bottom of the Petri dish was covered with 2 × 2 cm2 slides. After 24 h of dark cultivation at 15 °C, the slides were removed from aseptic conditions and placed in 20 Petri dishes with a diameter of 5 cm and filled with 15 mL of enriched F/2 medium. The cultural condition was the same as the precultivation. The culture medium was replaced every 3 days. After the young red thalli were visible to the naked eye, slides were cultured in 500 mL of aerated F/2 medium. The experiment was performed after the young thallus branches had grown up to 1–5 cm.

2.2. Experimental Setup

G. turuturu thalli were placed in 3 L of filtered, autoclaved, and nitrogen-free F/2 medium for 0, 3, 7, 14, 21, and 28 days for the nitrogen starvation treatments. During the period of nitrogen starvation, the culture media were not changed, though nitrogen-free F/2 stock solution was added every 3 days. After nitrogen starvation, samples were subsequently cultured in the F/2 medium with 200 µmol L−1 nitrate and 200 µmol L−1 ammonium for 21 days for the nitrogen-recovery treatments. During the period of nitrogen recovery, the culture media were changed every 3 days. The samples were aerated and cultured at 24 °C with a light intensity of 109–118 μmol photons m−2 s−1 and a photoperiod featuring a 12–12 h light–dark cycle. The salinity of the culture medium was 34 psu. Each treatment had four replicates. For analysis of growth and biochemical parameters, samples were measured with fresh weights and collected on days 3, 7, 14, 21, and 28 of nitrogen starvation for each treatment (hereafter referred as S3, S7, S14, S21, and S28), as well as on days 1, 3, 5, 7, 14, and 21 of nitrogen recovery for each treatment (hereafter referred as R1, R3, R5, R7, R14, and R21). Collected thalli samples were stored at −80 °C for further biochemical analysis. The RGRs and biochemical compositions on day 0 of nitrogen starvation (hereafter S0) were equivalent to those of G. turuturu without nitrogen starvation on R1. For analysis of the nitrate and ammonium uptake rates of nitrogen-starved G. turuturu after nitrogen resupply, the experimental media were collected after 1, 3, 6, 9, 12, and 24 h of nitrogen resupply. In addition, the medium without G. turuturu was also collected after 1, 3, 6, 9, 12, and 24 h as the control medium. Both experimental media and control media samples were stored at −80 °C for further measurements of their nitrate and ammonium concentrations.

2.3. Measurements of Nitrate and Ammonium Uptake Rates

The cadmium column reduction method [54] and the hypobromite oxidation method [55] were used to analyze the concentrations of nitrate and those of ammonium in culture media, respectively. The uptake rates of nitrate and ammonium were calculated according to Equation (1):
U (µmol g−1 h−1) = (C0 − CT) × V/(T × W)
where U is the uptake rate; C0 and CT are the concentrations of nitrate or ammonium (µmol L−1) in the control media and experimental media, respectively, at 1, 3, 6, 9, 12, and 24 h of nitrogen resupply; V is the medium volume (L); T is the experiment period (h); and W is the fresh weight (g) of the sample.

2.4. Measurements of Relative Growth Rates

The relative growth rates (RGRs) of fresh weights were calculated according to Equation (2):
RGR (% day−1) = 100 × (ln VN+T − ln VN)/T
where VN is the fresh weight (g) on the sampling day of N, VN+T is the fresh weight (g) on the sampling day of N + T, and T is the number (day) of experimental days.

2.5. Measurements of Total Nitrogen

For the measurement of total nitrogen, samples were dried at 80 °C for 48 h. After measuring their dry weight, samples were ground into a powder using a high-throughput tissue grinder. The total nitrogen content was analyzed by using an elemental analyzer (Vario EL III, Elementar, Langenselbold, Germany).

2.6. Measurement of Soluble Protein

Fresh samples (0.1 g) were ground with 0.9 mL of phosphate buffer solution (0.1 M, pH = 7.4) using a pestle and mortar. The extract was centrifuged at 12,000 rpm at 4 °C for 30 min. The absorption of the supernatant was measured at 595 nm by using an ultraviolet spectrophotometer. The soluble protein content was estimated by using Coomassie Brilliant Blue G-250 dye and bovine albumin (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the description of Kochert [56].

2.7. Measurements of Chlorophyll a (Chl a) and Phycoerythrin (PE)

For the measurements of PE, 0.1 g of fresh samples was homogenized with 6 mL of phosphate buffer solution (0.1 M, pH = 6.5) using a glass homogenizer at 0 °C. Next, the extract was centrifuged at 4000 rpm at 4 °C for 20 min. The absorption of the supernatant was measured at 565 nm. The content of PE was estimated according to Equation (3):
PE (mg g−1) = 12.4 × A565 × Ve/(I × W × 1000)
where A565 is the absorption rate of the supernatant at 565 nm, Ve is the volume (mL) of the extract solution, I is the optical path (cm) of the cuvette, and W is the fresh weight (g) of the sample.
For the measurements of Chl a, 6 mL of dimethylformamide (DMF) was added to the centrifuged deposit of samples for PE measurements and maintained at 4 °C in darkness for 1 day. Extracts were then centrifuged at 4000 rpm and 4 °C for 10 min. The absorption of the supernatant was measured at 750, 664, 647, and 625 nm. The content of Chl a was calculated according to Equation (4):
Chl a (mg g−1) = [12.65 × (A664 − A750) − 2.99 × (A647 − A750) − 0.04 × (A625 − A750)] × Ve/(I × W × 1000)
where A750–625 are the absorptions of extracts at 750, 664, 647, and 625 nm; Ve is the volume (mL) of DMF; I is the optical path (cm) of the cuvette; and W is the fresh weight (g) of the sample.

2.8. Data Analysis

All contents of the measured biochemical components were normalized to mg N g−1 DW (N, nitrogen; DW, dry weight). Two-way ANOVA was used to test the effects of nitrogen starvation time and recovery time on the uptake rates of nitrate and ammonium, RGRs, contents of total nitrogen, protein, Chl a, and PE during the nitrogen-recovery phase. One-way ANOVA was applied to test the effects of nitrogen starvation time on RGRs and the contents of total nitrogen, protein, Chl a, and PE during the nitrogen starvation phase. Prior to these analyses, the homogeneity of data variances was analyzed by using Levene’s test. Though the homogeneities of several parameters were violated, ANOVA was utilized as ANOVA is reasonably resistant to violations of the homogeneity of variance when the group size is reasonably similar [57]. When significant differences were detected (p < 0.05), pairwise comparisons were conducted with Bonferroni adjustment for multiple comparisons. Statistical analyses were carried out using SPSS 26.0 software. All data are given as mean ± standard deviation (SD).

3. Results

3.1. Uptake Rates of Nitrate and Ammonium

The uptake rates of nitrate were significantly affected by nitrogen starvation time (p < 0.001), recovery time (p < 0.001), and the interaction between starvation time and recovery time (p < 0.001; Table 1). The uptake rates of nitrate significantly increased in line with the time of nitrogen resupply and remained relatively stable after 12 and 24 h of nitrogen resupply (Figure 1A). After 1 and 3 h of nitrogen resupply, Grateloupia turuturu from all nitrogen starvation treatments had negative nitrate uptake rates. G. turuturu experiences the lowest nitrate uptake rates from 21 and 28 days of nitrogen starvation, followed by ones from 7 to 14 days of nitrogen starvation, while this rate remained relatively high in G. turuturu from 0 and 3 days of nitrogen starvation. After 6 and 9 h of nitrogen resupply, G. turuturu had positive values of nitrate uptake rates from 0 to 14 days of nitrogen starvation, but not from 21 and 28 days of nitrogen starvation. After 12 h of nitrogen resupply, the averaged uptake rates of G. turuturu from 21 and 28 days of nitrogen starvation increased to a positive value, though it was still the lowest among all treatments. Meanwhile, G. turuturu from 3 days of nitrogen starvation had the highest values of nitrate uptake rates. After 24 h of nitrogen resupply, no difference in nitrate uptake rates was detected between nitrogen starvation treatments.
The uptake rates of ammonium were also significantly affected by nitrogen starvation time and recovery time, individually and interactively (nitrogen starvation time: p < 0.001, recovery time: p < 0.001, interaction: p < 0.001; Table 1). The uptake rates of ammonium were highest after 1 h of nitrogen resupply and then significantly decreased in line with time (Figure 1B). After 1 h of nitrogen resupply, the uptake rates of ammonium in G. turuturu were highest from 7 and 14 days of nitrogen starvation and lowest from 0 and 28 days of nitrogen starvation. After 3 h of nitrogen resupply, the uptake rates of ammonium in G. turuturu from 7 days of nitrogen starvation were significantly higher than the values from 0, 3, and 14 days of nitrogen starvation, and the lowest values were found in G. turuturu from 21 and 28 days of nitrogen starvation. After 6 h of nitrogen resupply, G. turuturu from 0 to 14 days of nitrogen starvation absorbed ammonium significantly faster than G. turuturu from 21 and 28 days of nitrogen starvation did. Differences in ammonium uptake rates between treatments were not significant from 9 to 24 h of nitrogen resupply.

3.2. Relative Growth Rates (RGRs)

The RGRs of G. turuturu significantly decreased in line with the nitrogen starvation time (p = 0.008; Table 2). Specifically, the RGRs of G. turuturu after 21 and 28 days of nitrogen starvation were significantly lower than those of non-nitrogen-starved G. turuturu (Figure 2A). The RGRs decreased to as low as 0.38 ± 0.03% day−1 after 28 days of nitrogen starvation. After nitrogen recovery, the RGRs were significantly affected by the main effects of starvation time (p < 0.001) and recovery time (p < 0.001; Table 3). The RGRs were the lowest after 1 day of nitrogen recovery, and they then significantly increased from 5 to 21 days of nitrogen recovery (Figure 2B). The RGRs of the non-nitrogen-starved G. turuturu were generally higher than those of the nitrogen-starved G. turuturu, specifically so for the ones after 21 and 28 days of nitrogen starvation. After 21 days of nitrogen recovery, the RGRs of 21- and 28-day nitrogen-starved G. turuturu could not recover enough to reach their original values.

3.3. Total Nitrogen

The nitrogen starvation time significantly affected the total nitrogen content (p = 0.003; Table 2). Furthermore, the total nitrogen contents of G. turutru after 28 days of nitrogen starvation significantly decreased, by 48%, compared with the total nitrogen contents of the non-nitrogen-starved G. turuturu (Figure 3A). After nitrogen resupply, the total nitrogen contents were significantly affected by starvation time (p < 0.001), recovery time (p = 0.015), and their interaction (p < 0.001; Table 3). In general, the total nitrogen contents were the lowest after 1 day of nitrogen recovery, being significantly lower than the total nitrogen contents after 5 days of nitrogen recovery (Figure 3B). From 1 to 5 days of nitrogen recovery, the total nitrogen contents of G. turutru after 14 to 28 days of nitrogen starvation were significantly lower than those of G. turutru after 0 to 7 days of nitrogen starvation. The total nitrogen contents were restored to similar values in G. turutru from all starvation treatments after 7 days of nitrogen recovery.

3.4. Soluble Protein

Soluble protein contents also significantly decreased during nitrogen starvation (p < 0.001; Table 2). The rate of decrease in protein content from 0 days of nitrogen starvation to 28 days of nitrogen starvation was 12% (Figure 4A). In the nitrogen-recovery phase, there was an interaction effect between nitrogen starvation time and recovery time (p = 0.003; Table 3). A pairwise comparison showed that no difference in protein contents was detected among different nitrogen recovery days (Figure 4B). After 1 day of nitrogen recovery, protein contents from all treatments of nitrogen starvation were similar. However, after 3 days of nitrogen recovery, protein contents of G. turutru from 28 days of nitrogen starvation were generally lower than those of G. turutru from other nitrogen starvation treatments.

3.5. Chlorophyll a (Chl a) and Phycoerythrin (PE)

Both Chl a and PE contents significantly decreased with nitrogen starvation time (p < 0.001; Table 2). The rates of decrease in Chl a and PE from 0 days of nitrogen starvation to 28 days of nitrogen starvation were 77% and 63%, respectively (Figure 5A and Figure 6A). After nitrogen resupply, Chl a and PE contents were also significantly affected by the interaction between nitrogen starvation time and recovery time (Chl a: p < 0.001, PE: p = 0.027; Table 3). Chl a significantly increased from 1 to 5 days of nitrogen recovery and remained similar from 5 to 21 days of nitrogen recovery (Figure 5B). In general, the Chl a content was lower in G. turutru from 14 to 28 days of nitrogen starvation than from 0 to 7 days of nitrogen starvation. After 21 days of nitrogen resupply, the Chl a contents in G. turutru from 7 to 21 days of nitrogen starvation could not recover to their original values. The recovery patterns of PE were similar to those of Chl a. Though PE contents generally increased with nitrogen-recovery time (Figure 6B), the PE contents in G. turutru from 7 to 28 days of nitrogen starvation could not recover to their original levels after 21 days of nitrogen recovery.

4. Discussion

Our study showed that the nitrogen-starved Grateloupia turuturu had higher ammonium uptake rates than nitrate uptake rates after nitrogen resupply. This finding is consistent with that of Li et al. [26], who observed that the 10-day nutrient-starved Sargassum horneri absorbed ammonium much faster than nitrate after nitrogen resupply. Furthermore, several studies have illustrated that G. turuturu has higher uptake rates for ammonium than for nitrate in different macroalgal species, illustrated that a series of macroalgal species had higher uptake rates for ammonium than for nitrate, such as the green macroalga Ulva lactuca [58], the red macroalgae Porphyria perforata and Mastocarpus papillatus [59], and the brown macroalga Sargassum sp. [60]. The higher uptake rates of ammonium may be explained by the lower energy requirement of ammonium assimilation in comparison with nitrate assimilation [61]. Meanwhile, previous studies have shown that ammonium could inhibit the uptake of nitrate by 30% to 50% [62], which suggests that the nitrate transport systems is sensitive to ammonium inhibition and downregulated by ammonium. Additionally, the preferences of nitrate and ammonium for seaweeds are also related to the ratios of nitrate and ammonium in media [63]. Specifically, the uptake of ammonium was found to be surpassed by that of nitrate when the nitrate/ammonium ratio was over 2.2 [63].
G. turuturu from 3 to 21 days of nitrogen starvation exhibited an overcompensatory uptake of ammonium after 1 h of nitrogen resupply. This is in accordance with the study of Li and Lin [25], who observed that Gracilariopsis lemaneiformis from 10 days of nutrient starvation absorbed ammonium faster than the non-nitrogen-starved samples after nitrogen resupply. Likewise, the 10-day nutrient-starved S. horneri was also observed to have an overcompensatory absorption of ammonium after nitrogen resupply [26]. Furthermore, the overcompensatory uptake of nitrogen occurred in the first 3 h of nitrogen resupply and then decreased to values similar to the control ones. Such patterns also agree with previous observations on G. lemaneiformis [25] and S. horneri [26], illustrating that the rapid ammonium uptake after nitrogen starvation is a short phase, and therefore, its positive effect on seaweeds should be carefully evaluated.
During 28 days of nitrogen starvation, the decreased rate of total nitrogen (48%) was higher than that of soluble protein (12%), which suggests that protein is not the main nitrogen source that G. turuturu utilizes to maintain its basic metabolic activities during nitrogen starvation. Furthermore, though the rates of decrease in Chl a (77%) and PE (63%) were higher than those of total nitrogen, they were not the main nitrogen sources either, given their small proportion in the total nitrogen pool. As proteins and amino acids are the two main nitrogen pools in seaweeds [64,65], our results may indicate that G. turuturu utilizes mainly amino acids for metabolic activities during nitrogen starvation. Such an indication is supported by McGlathery et al. [4], who observed that the residual organic nitrogen (RON, comprising mainly amino acids) had a large decline during nitrogen starvation compared with proteins in the green macroalga Chaetomorpha linum and thereby concluded that RON can be incorporated into proteins to support macroalgal growth demands. Moreover, G. lemaneiformis was also observed to have sharply decreased amino acid contents after nitrogen deficiency compared with other nitrogenous compounds, illustrating that amino acids are first utilized during nitrogen depletion [66]. Additionally, the low rate of decrease in proteins during nitrogen starvation may be explained by the fact that proteins make up a fundamental part of seaweeds, and their consumption might thus impair seaweed structure. Hence, seaweeds are thought to utilize proteins after the depletion of amino acids, as reflected by the significant decrease in proteins occurring up to 28 days of nitrogen starvation. The above inference is supported by the investigation of Liu et al. [11], who found that G. lemaneiformis significantly reduced amino acid (i.e., asparagine, glutamine, and aspartic acid) contents after a mere 4 h of nitrogen deficiency; in contrast, the soluble protein content remained stable during the first 4 days of nitrogen deficiency and then significantly decreased the contents of proteins after 10 days of nitrogen deficiency. Additionally, the green macroalga Oedogonium [67] and microalga Scenedesmus acuminatus [13] have also been observed to have largely decreased contents of amino acids under nitrogen deficiency.
As nitrogen is a fundamental element for various macromolecules, such as protein, chlorophyll, and amino acids, and thereby essential for seaweed growth [1], it is not surprising that the RGRs and nitrogenous compounds measured in this study deceased during the nitrogen starvation. These results are in accordance with previous studies that also observed declines in growth and nitrogenous compounds under nitrogen-deficient conditions in different seaweeds, such as Pyropia yezoensis [16], G. lemaneiformis [11,12], and Ulva pertusa [68]. Noteworthily, several values of RGRs and pigments in our results had high standard deviations, suggesting that those samples’ readings are less reliable; we thus need to cautiously evaluate the patterns reflected by those data. Specifically, though RGR values continuously decreased in line with the nitrogen starvation time, the significant differences between the control and treatment groups were observed until after 21 days of nitrogen starvation thanks to the high standard deviations. In contrast, G. lemaneiformis and Ulva prolifera were observed to have significantly reduced RGRs after 4 h [12] and 4 days of nitrogen deficiency [69], respectively. In addition, Chl a and PE, which significantly decreased from 7 days of nitrogen starvation onward, declined faster than other parameters, suggesting that the regulation of pigments is more sensitive to nitrogen starvation. Comparatively, G. lemaneiformis also reduced the PE content after the first 2 days of nitrogen deficiency, whereas Chl a remained stable up to 12 days of deficiency, implying that PE and Chl a may have different functions and that PE plays a more important role in providing nitrogen for macroalgal survival during the long-term nitrogen deficiency [66]. Additionally, the relatively fast responses of pigments might be self-protection mechanisms of G. turuturu in response to nitrogen-deficient conditions, as a lack of nitrogen can lead to overaccumulations of electrons in photosynthesis and thereby induce photo-oxidative stress in cells [14,15]. Several studies have shown that the nitrogen limitation induces reactive oxygen species (ROS) scavenging antioxidants and enzymes [20,70]. Hence, the reduction in pigments, on the one hand, helps G. turuturu decrease the nitrogen demand and produce nitrogen for essential metabolic activities during nitrogen deficiency [71]; on the other hand, this reduces light reactions in order to prevent potential oxidative damages.
RGRs and biochemical compositions gradually increased upon the nitrogen resupply. Similar patterns have been shown in Galdieria sulphuraria [71] and S. acuminatus [13]. The recovery ability of G. turuturu and its rapid uptake of ammonium after nitrogen resupply suggest that G. turuturu is well adapted to the conditions of short or prolonged nitrogen deficiency, which is ecologically important for its survival in seawater with highly fluctuating nutrients or seasonally nitrogen deprivation [3,72]. Furthermore, the G. turuturu that underwent a longer period of nitrogen starvation took longer time to recover. Especially for the G. turuturu that underwent 21 and 28 days of nitrogen starvation, although their growth rates progressively increased, they could not recover enough to reach the original levels after 21 days of nitrogen resupply. In contrast, G. turuturu with 3 to 14 days of nitrogen starvation generally recovered enough to reach the controlled RGR levels after 3 days of nitrogen resupply. Furthermore, RGR values of G. turuturu with 3 and 7 days of nitrogen starvation even exceeded the control RGR values after 5 days of nitrogen resupply, even though the differences were not significant, thanks to the high standard deviations. A fast recovery time and overcompensatory growth were also observed in the 10-day nitrogen-starved G. lemaneiformis after nitrogen resupply [12]. Additionally, for G. turuturu with 21 and 28 days of nitrogen starvation, their RGRs after 7 to 21 days of nitrogen resupply were higher than the RGRs after the first 3 days of nitrogen resupply. Such increases in RGRs during the late phases of nitrogen resupply were also observed in P. yezoensis by Niwa et al. [51], who explained that the above pattern might be due to the increase in physiological activity following the recovery from intracellular damage induced by nitrogen deficiency. In turn, both the variation patterns of RGRs and the morphological observations in our study further support the above idea. During 28 days of starvation treatment, severe morphological changes in G. turuturu were observed, such as thalli ulceration and detaching. Similar visual changes were also observed in G. lemaneiformis thalli, which were fragmented after 12 days of nitrogen deprivation [66]. Hence, the above morphological traits indicate that macroalgae have to utilize the structural nitrogenous compounds to survive after they have depleted the storage pool of nitrogen during long-term nitrogen starvation, leading to inevitable intracellular damage.

5. Conclusions

This study set out to investigate the physiological and biochemical performances of G. turuturu during nitrogen starvation and subsequent recovery. The results identified that nitrogen-starved G. turuturu preferred to uptake ammonium rather than nitrate after nitrogen resupply. Furthermore, an overcompensatory uptake of ammonium was induced by nitrogen starvation in G. turuturu during the early stages of nitrogen resupply. The second major finding was that the time and rates of depletion of different compounds varied during nitrogen starvation, which provides clues as to the potential nitrogen utilization strategies of G. turuturu in surviving long-term nitrogen starvation. Furthermore, our research showed that the nitrogen-starved G. turuturu was able to gradually recover after nitrogen resupply, even for the sample starved for 28 days, implying a significant ability of G. turuturu to adapt to nitrogen fluctuations in nature. However, growth rates and pigments from long-term nitrogen starvation could not recover enough to reach their original levels after nitrogen resupply; nitrogen deficiency should thus be avoided in aquaculture conditions. Again, our findings on growth and pigments must be interpreted with caution owing to the relatively high standard deviation. Our study revealed the decline and recovery progresses of growth, nitrogenous compounds, and nitrogen uptake rates in G. turuturu under different lengths of nitrogen deficiency, which provide the basic information for the sustainable management of seaweed aquaculture. To further understand the responses of nitrogen pools in G. turuturu to nitrogen starvation and recovery, it is necessary to investigate the performances of major nitrogenous compounds, including free amino acids. Meanwhile, because the physiobiochemical responses were regulated through various metabolic pathways, further studies with measurements of enzymes and transcriptomes are desirable to better reveal the underlying acclimated mechanism of G. turuturu in response to nitrogen starvation and resupply.

Author Contributions

Investigation, data curation, writing—original draft, Y.C.; investigation, visualization, data curation, L.L.; methodology, data curation, J.Z.; conceptualization, formal analysis, writing—review and check, Q.W.; methodology, data curation, Y.L.; methodology, formal analysis, writing—original draft and review, H.L.; validation, resources, Q.G.; conceptualization, methodology, writing—review and check, project administration, funding acquisition, X.G. All authors have read and agreed to the published version of the manuscript.

Funding

This work was financially supported by the Taishan Scholar Foundation of Shandong Province (No. tsqn202211067), the Fundamental Research Fund for the Central Universities (No. 202262002), the Young Talent Program of Ocean University of China (No. 202212015), and the Key Laboratory of Mariculture, Ministry of Education of Opening Project (No. KLM202208).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The data in the present study are available from the corresponding author upon reasonable request.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. The nitrate (A) and ammonium (B) uptake rates of nitrogen-starved Grateloupia turuturu after 1, 3, 6, 9, 12, and 24 h of nitrogen recovery. Data represent mean ± SD. Different lowercase and capital letters indicate significant differences among nitrogen starvation times and recovery times, respectively (p < 0.05).
Figure 1. The nitrate (A) and ammonium (B) uptake rates of nitrogen-starved Grateloupia turuturu after 1, 3, 6, 9, 12, and 24 h of nitrogen recovery. Data represent mean ± SD. Different lowercase and capital letters indicate significant differences among nitrogen starvation times and recovery times, respectively (p < 0.05).
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Figure 2. Relative growth rates (RGRs) of Grateloupia turuturu during nitrogen starvation (A) and subsequent recovery (B). Data represent mean ± SD. Different lowercase and capital letters indicate significant differences among nitrogen starvation times and recovery times, respectively (p < 0.05).
Figure 2. Relative growth rates (RGRs) of Grateloupia turuturu during nitrogen starvation (A) and subsequent recovery (B). Data represent mean ± SD. Different lowercase and capital letters indicate significant differences among nitrogen starvation times and recovery times, respectively (p < 0.05).
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Figure 3. Total nitrogen contents of Grateloupia turuturu during nitrogen starvation (A) and subsequent recovery (B). Data represent mean ± SD. Different lowercase and capital letters indicate significant differences among nitrogen starvation times and recovery times, respectively (p < 0.05).
Figure 3. Total nitrogen contents of Grateloupia turuturu during nitrogen starvation (A) and subsequent recovery (B). Data represent mean ± SD. Different lowercase and capital letters indicate significant differences among nitrogen starvation times and recovery times, respectively (p < 0.05).
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Figure 4. Soluble protein contents of Grateloupia turuturu during nitrogen starvation (A) and subsequent recovery (B). Data represent mean ± SD. Different lowercase and capital letters indicate significant differences among nitrogen starvation times and recovery times, respectively (p < 0.05).
Figure 4. Soluble protein contents of Grateloupia turuturu during nitrogen starvation (A) and subsequent recovery (B). Data represent mean ± SD. Different lowercase and capital letters indicate significant differences among nitrogen starvation times and recovery times, respectively (p < 0.05).
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Figure 5. Chlorophyll a (Chl a) contents of Grateloupia turuturu during nitrogen starvation (A) and subsequent recovery (B). Data represent mean ± SD. Different lowercase and capital letters indicate significant differences among nitrogen starvation times and recovery times, respectively (p < 0.05).
Figure 5. Chlorophyll a (Chl a) contents of Grateloupia turuturu during nitrogen starvation (A) and subsequent recovery (B). Data represent mean ± SD. Different lowercase and capital letters indicate significant differences among nitrogen starvation times and recovery times, respectively (p < 0.05).
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Figure 6. Phycoerythrin (PE) contents of Grateloupia turuturu during nitrogen starvation (A) and subsequent recovery (B). Data represent mean ± SD. Different lowercase and capital letters indicate significant differences among nitrogen starvation times and recovery times, respectively (p < 0.05).
Figure 6. Phycoerythrin (PE) contents of Grateloupia turuturu during nitrogen starvation (A) and subsequent recovery (B). Data represent mean ± SD. Different lowercase and capital letters indicate significant differences among nitrogen starvation times and recovery times, respectively (p < 0.05).
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Table
Table 1. Results of two-way ANOVA examining the effects of nitrogen starvation time and recovery time on the nitrate and ammonium uptake rates of Grateloupia turuturu after 1, 3, 6, 9, 12, and 24 h of nitrogen recovery. Statistically significant values are indicated by asterisks (p < 0.05).
Table 1. Results of two-way ANOVA examining the effects of nitrogen starvation time and recovery time on the nitrate and ammonium uptake rates of Grateloupia turuturu after 1, 3, 6, 9, 12, and 24 h of nitrogen recovery. Statistically significant values are indicated by asterisks (p < 0.05).
FactorsdfFp
Nitrate uptake rates
Starvation time5696.190<0.001 *
Recovery time5273.300<0.001 *
Interaction2534.384<0.001 *
Ammonium uptake rates
Starvation time5973.315<0.001 *
Recovery time5153.071<0.001 *
Interaction2575.990<0.001 *
Table
Table 2. Results of one-way ANOVA examining the effects of nitrogen starvation time on relative growth rates (RGRs), total nitrogen, protein, chlorophyll a (Chl a), and phycoerythrin (PE) contents in Grateloupia turuturu after 0, 3, 7, 14, 21, and 28 days of nitrogen starvation. Statistically significant values are indicated by asterisks (p < 0.05).
Table 2. Results of one-way ANOVA examining the effects of nitrogen starvation time on relative growth rates (RGRs), total nitrogen, protein, chlorophyll a (Chl a), and phycoerythrin (PE) contents in Grateloupia turuturu after 0, 3, 7, 14, 21, and 28 days of nitrogen starvation. Statistically significant values are indicated by asterisks (p < 0.05).
ParameterFactordfFp
RGRsStarvation time59.7200.008 *
Total nitrogenStarvation time58.6590.003 *
ProteinStarvation time57.869<0.001 *
Chl aStarvation time552.869<0.001 *
PEStarvation time531.921<0.001 *
Table
Table 3. Results of two-way ANOVA examining the effects of nitrogen starvation time and recovery time on relative growth rates (RGRs), total nitrogen, protein, chlorophyll a (Chl a), and phycoerythrin (PE) contents in Grateloupia turuturu after 1, 3, 5, 7, 14, and 21 days of nitrogen recovery. Statistically significant values are indicated by asterisks (p < 0.05).
Table 3. Results of two-way ANOVA examining the effects of nitrogen starvation time and recovery time on relative growth rates (RGRs), total nitrogen, protein, chlorophyll a (Chl a), and phycoerythrin (PE) contents in Grateloupia turuturu after 1, 3, 5, 7, 14, and 21 days of nitrogen recovery. Statistically significant values are indicated by asterisks (p < 0.05).
FactorsdfFp
RGRs
Starvation time513.216<0.001 *
Recovery time56.880<0.001 *
Interaction251.2380.274
Total nitrogen
Starvation time514.325<0.001 *
Recovery time53.1490.015 *
Interaction253.309<0.001 *
Protein
Starvation time526.845<0.001 *
Recovery time52.4390.039 *
Interaction252.2020.003 *
Chl a
Starvation time582.955<0.001 *
Recovery time514.247<0.001 *
Interaction253.537<0.001 *
PE
Starvation time577.971<0.001 *
Recovery time55.655<0.001 *
Interaction251.7520.027 *
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Chen, Y.; Lan, L.; Zhang, J.; Wang, Q.; Liu, Y.; Li, H.; Gong, Q.; Gao, X. Physiological Impacts of Nitrogen Starvation and Subsequent Recovery on the Red Seaweed Grateloupia turuturu (Halymeniaceae, Rhodophyta). Sustainability 2023, 15, 7032. https://doi.org/10.3390/su15097032

AMA Style

Chen Y, Lan L, Zhang J, Wang Q, Liu Y, Li H, Gong Q, Gao X. Physiological Impacts of Nitrogen Starvation and Subsequent Recovery on the Red Seaweed Grateloupia turuturu (Halymeniaceae, Rhodophyta). Sustainability. 2023; 15(9):7032. https://doi.org/10.3390/su15097032

Chicago/Turabian Style

Chen, Yining, Lan Lan, Jing Zhang, Qiaohan Wang, Yan Liu, Huiru Li, Qingli Gong, and Xu Gao. 2023. "Physiological Impacts of Nitrogen Starvation and Subsequent Recovery on the Red Seaweed Grateloupia turuturu (Halymeniaceae, Rhodophyta)" Sustainability 15, no. 9: 7032. https://doi.org/10.3390/su15097032

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