Unlocking economic potential of the Ulva crop for low salinity environments: exploring the effect of salinity gradients on the performance and valuable compounds of Baltic Sea strains

2.1 Algal source material and taxonomic identification

Individuals of Ulva fenestrata originated from long-term indoor tank cultivation at Tjärnö Marine Laboratory, Sweden (TML, 58°52′33.7″ N 11°08′44.9″ E; Figure 1). Various populations of U. lacinulata, U. intestinalis, and U. linza were collected from different salinity conditions across the range to be found between the Atlantic and the inner parts of the Baltic Sea (Figure 1). Taxonomic identification of the Ulva strains was based on molecular identification of the tufA marker gene and followed the procedure as described by Toth et al. (2020). Collection data, including the salinity at the collection site, and information on molecular identity including GenBank reference numbers can be found in Table 1. During collection, populations were pre-identified using morphological characteristics defined in previous studies (Steinhagen et al. 2019a, 2023]). The collected samples were transferred in plastic bags with seawater from the collection site and transported to the lab within 24 h. In the lab, they were stored in a controlled temperature room (12 °C; 90–110 μmol m⁻² s⁻¹; light:dark regime of 16:8 h) in 14-L aquaria containing seawater (filtered [0.2 μm], deep-sea [40 m]) adjusted to the salinity of the sampling site using tap water, with permanent aeration, and supplemented with Provasoli Enriched Seawater (PES; Provasoli 1968). Acclimatization to these conditions was conducted for a minimum of one week prior to subsequent experiments. On this background, and based on previous experience, we assumed that the overall biological set up of the different strains was similar at the start of the experiment. The seaweeds were rinsed in 0.2-μm filtered seawater before the start of the experiments and samples were taken before the experiments (‘t0’) for subsequent biochemical analysis. Per population, three randomly selected individuals were subsequently identified by molecular techniques to assess their taxonomic species affiliation. Resulting sequences were uploaded to GenBank and are publicly available (see Table 1).

Figure 1:

Map of sampling sites (1–8, red circles) of Ulva strains used in the present study collected in 2023 across the Atlantic-Baltic sea gradient and used in the salinity experiments. Average sea surface salinity (black circles) is indicated.

2.2 Experimental setup and growth measurements

After a minimum of one week acclimation in the controlled temperature room (12 °C), the collected strains (one strain of U. fenestrata and U. lancinulata from salinity 30, four strains of U. intestinalis from salinities 30, 14, 9 and 7 and two strains of U. linza from salinities 14 and 9) were subjected to five salinity treatments of 5, 10, 15, 20 and 30 in biological replicates (n = 3) for a total of 8 weeks. The excess water of the biomass was removed by spinning the seaweeds in a salad spinner and 10 g fresh weight (FW) were submersed in 1-L aquaria supplied with sterile filtered seawater (0.2 µm + UV, 9 L h⁻¹) at salinities of 5, 10, 15, 20, and 30 (natural seawater [33] diluted with sterile distilled water; n = 3) and an average irradiance of 80–100 μmol m⁻² s⁻¹ under a 16:8 h L:D light regime (light source: OSRAM Lumilux Cool daylight L 58W/865) with permanent aeration. Growth medium PES was added once per week following the concentration specifications of Provasoli (1968) and was connected with a weekly performed water change. To prevent diatom growth, 1 mg L⁻¹ GeO₂ was added to all treatments. GeO₂ was found to negatively affect diatom growth whereas it was reported to not have any effect on Ulva growth (Rautenberger 2024). In combination with the weekly water change, each culture was weighed (Sartorius TE1502S, Göttingen, Germany), after removing the excess water with a salad spinner as described above, to determine growth rates. Afterwards, the biomass was re-adjusted to 10 g in any of the containers by removing excess biomass. Salinity and pH (WTW MultiLine 3420, Xylem Analytics, Weilheim, Germany) remained stable throughout the experiment in all treatments and no additional adjustments were necessary. Final measurements took place on the 8th week of the experiment. After the biomass had been weighed, samples were frozen and stored at −60 °C until lyophilized and homogenized into a fine powder by grinding the biomass with pestle and mortar for subsequent crude protein, pigment and phenolic content analysis.

2.3 Growth rate and crude protein content

To calculate average growth rate (AGR), the weekly recorded change in fresh weight was averaged over the eight measurement time points among the replicates (n = 3). Analyses of nitrogen content were performed according to the Dumas method on the biomass of the final sample point using an LECO Nitrogen Analyzer (TruMac N, LECO Corporation, USA). EDTA Calibration Sample (LECO Corporation, USA) was used as standard. Subsequently, the crude protein content was estimated based on the nitrogen-to-protein conversion factor of five for seaweeds (Angell et al. 2016).

2.4 Pigment (chlorophylls a and b, carotenoids) and phenolic content

Pigment and phenolic contents were determined at the end of the experiment on all treatments and replicates (n = 3) from lyophilized and homogenized tissue material. Extractions followed the detailed protocol available in Steinhagen et al. (2021).

Total contents of chlorophylls a and b and carotenoids in the 90 % aq. acetone extract were assessed spectrophotometrically (Lambda XLS+, Perkin Elmer, Waltham, MA, United States) using the formula and wavelength given in Jeffrey and Humphrey (1975) for chlorophyll and Parsons et al. (1984) for total carotenoids. Total phenolic content was spectrophotometrically determined at 765 nm (Lambda XLS+, Perkin Elmer, Waltham, MA, United States) using the colorimetrical Folin-Ciocalteu phenol assay and gallic acid (Sigma-Aldrich, St. Louis, MO, United States) as a standard. The total phenolic content was expressed as % of dw.

2.5 Statistical analyses

Data on growth, crude protein, pigment, and phenolic content of all samples were statistically analyzed in JMP (JMP^®, Version 15, SAS Institute Inc., Cary, NC, USA). The effect of salinity treatment on AGR, crude protein, Chla, Chlb, carotenoids and phenolic content was analyzed for each strain using one-way ANOVA with the different salinity treatments (5, 10, 15, 20, 30) as a five level, fixed factor. Significant differences among means were compared using Tukey’s HSD test. All data were visuallyed for homogeneity and normality with diagnostic plots (density-, normality- and QQ-plots).

3.1 Average growth rate (AGR) and crude protein content

After 8 weeks of exposure to different salinities, the AGR significantly (p < 0.05) differed among the salinity treatments in four (Fen30, Int30, Int9, Lin14) out of the eight tested Ulva strains (Table 2 and Figure 2) whereas no significant differences among salinities were observed in Lac30, Int14, Int7, Lin9. In Fen30, the AGR in the lowest salinity treatment, 5, was higher than in the other salinities (Figure 2A), and reached 17.7 %. In Int30, the highest salinity treatment (30) resulted in a lower AGR than the other salinity treatments (Table 2 and Figure 2C). Further, with decreasing salinity the AGR generally increased and algae grown at a salinity of five displayed an AGR of 15.7 % which was significantly higher than for algae grown at salinities of 15 and 20 (Table 2 and Figure 2C). Similarly, strain Int9 exposed to the lower salinity treatments (5 and 10) had higher AGRs compared to higher salinities (Table 2 and Figure 2E), and strain Lin14 also had a higher AGR (14.8 %) at five than at higher salinities (Table 2 and Figure 2G).

Figure 2:

Effect of salinity treatments on the average growth rate (AGR; % change in FW) of different Ulva strains: (A) Fen30, (B) Lac30, (C) Int30, (D) Int14, (E) Int9, (F) Int7, (G) Lin14, and (H) Lin9 (see Table 1 for strain abbreviations) after 8 weeks of culture at salinities of 5, 10, 15, 20, and 30 (n = 3). Error bars show standard deviations and means labelled with different lower-case letters are significantly different at p = 0.05, based on Tukey’s HSD test. Where no letters are shown, there were no significant differences for that strain.

There was a significant difference in crude protein content, explained by differences in salinity levels, in five (Fen30, Int30, Int7, Lin14, Lin9) out of the eight strains (Table 2 and Figure 3). Furthermore, the crude protein content in most of the strains and treatments was notably higher after 8 weeks than at the start (t0) of the experiments (Figure 3A, C–H) although a decrease of crude protein content during the experiments was observed for strain Lac30 (Figure 3B). In Fen30 we detected significantly higher crude protein content in salinities of 5 (27.97 %) and 10 (27.28 %) compared to the higher salinity treatments of 15, 20 and 30 (<25.7 %) (Table 2 and Figure 3A). In Int30, the lowest crude protein content (22.13 %) was observed at highest (30) salinity treatment. The highest crude protein content for Int30 (26.87 %) was found at salinity 10 (Table 2 and Figure 3C). With decreasing salinity, the crude protein content of strain Int7 increased and algae treated with a salinity of five displayed the highest tissue crude protein concentration of 33.67 %, which also was the highest crude protein value measured within this study (Table 2 and Figure 3F). Similar observations were made for strains Lin14 and Lin9 in which crude protein content increased with decreasing salinity and the highest values were recorded at salinity 5 (Table 2, Figure 3G and H).

Figure 3:

Effect of salinity treatments on the crude protein content (% DW; dashed line indicates protein content at t0) of different Ulva strains: (A) Fen30, (B) Lac30, (C) Int30, (D) Int14, (E) Int9, (F) Int7, (G) Lin14, and (H) Lin9 (see Table 1 for strain abbreviations) after 8 weeks of culture at salinities of 5, 10, 15, 20, and 30 (n = 3). Other details as Figure 2.

Overall, the trend of higher AGR and crude protein content in low salinity treatments was seen between species as well as at an intraspecific level and is widely displayed among the tested species and strains (Table 2, Figures 2 and 3). Significant results documented this phenomenon in at least half of the investigated strains, whereas a clear trend was observed in the full data set (Figures 2 and 3). Strain Lac30 was the most resilient to the salinity treatments and showed no significant differences in AGR or crude protein content (Table 2, Figure 2B, 3B).

3.2 Pigment (chlorophyll a,b, carotenoids) and phenolic content

There were no clear overall trends in pigment content nor in phenolic content in response to the salinity treatments (Table 2 and Figure 4).

Figure 4:

Effect of salinity treatments on the chlorophyll a (μg mg⁻¹), chlorophyll b (μg mg⁻¹), and carotenoid (μg mg⁻¹) content of different Ulva strains: (A) Fen30, (B) Lac30, (C) Int30, (D) Int14, (E) Int9, (F) Int7, (G) Lin14, and (H) Lin9 (see Table 1 for strain abbreviations) after 8 weeks of culture at salinities of 5, 10, 15, 20, and 30 (n = 3). Other details as Figure 2.

3.2.4 Total phenolic content

We detected significant differences in total phenolic content among the different salinity treatments in three (Int30, Int9, Lin14) out of the seven strains tested and analysed (Table 2 and Figure 5). Statistical analyses were not performed for strain Int7, because high mortality within the strain meant that there was insufficient sample replication.

Figure 5:

Effect of salinity treatments on the phenolic (μg mg⁻¹) content of different Ulva strains: (A) Fen30, (B) Lac30, (C) Int30, (D) Int14, (E) Int9, (F) Lin14, and (G) Lin9 (see Table 1 for strain abbreviations) after 8 weeks of culture at salinities of 5, 10, 15, 20, and 30 (n = 3). Other details as Figure 2.

In strain Int30, higher phenolic content was observed at salinities 10 and 15 than at other salinities, and the lowest phenolic content was at salinity 5 (Table 2 and Figure 5C). There were significant differences in the phenolic content of strain Int9 among the salinity treatments and the highest content was found at salinity 15 (Table 2 and Figure 4E), although the results for the highest salinity (30) treatment could not be analysed due to lack of replicates. For strain Lin14, the phenolic content at salinity 30 was lower than at all other salinities (Table 2 and Figure 5F).

4.1 Average growth rate

Generally, in most of the investigated species and strains we found a positive AGR in all tested salinity regimes. Notably, deleterious impacts of low salinity on growth in foliose Ulva species has been previously documented in different studies (Angell et al. 2016; Fort et al. 2024; Lu et al. 2006). Especially in mesohaline environments below salinities of 15, a significant decrease in growth rate was observed in various foliose Ulva species (U. australis, U. lacinulata, U. lactuca, U. ohnoi), suggesting a similar inhibitory mechanism in growth by low-salinity treatments in euhaline species (Angell et al. 2016; Fort et al. 2024; Lu et al. 2006). Our experiment, using long-term salinity exposure, however, documented that various genotypes, originating from an environments over a wide salinity range, such as the Baltic Sea (Reusch et al. 2018), performed significantly better in the lowest salinity treatment (5) than at higher salinity levels – independent of the strain’s original salinity (euhaline or mesohaline). It should be mentioned that detrimental effects of salinity treatments <10 were observed in only two (Int7; Lin9) out of the eight tested strains, such effects were not found to be species specific and the overall performance of strains was generally very low compared to the other strains. Nevertheless, such data underlines that genotype specific differences towards changing abiotic factors need to be distinctively monitored and that rather large screenings of many different strains are favorable to find best performing genotypes in dependence of the cultivation environment. As discussed in previous studies, varying growth rates likely have metabolic implications (Fort et al. 2024) and could be causal for variations in the nutritional content of Ulva biomass. Therefore, intricate connections between low salinity and its effects on growth necessitate further comprehensive investigation of the physiology and metabolic pathways to find causative mechanisms for the differences observed. It can however be summarized that genotypes originating from the wider Baltic Sea area seem to be capable to tolerate changing salinities whereas especially the low salinity treatments seem suitable for growth optimization and can contribute to the optimization of industrial cultivation protocols.

4.2 Crude protein

A notable shift in dietary patterns is observed, with a discernible move away from red meat consumption towards green and blue protein sources and this transition aligns with burgeoning concerns over environmental sustainability and the ecological footprint associated with livestock farming (Eckl et al. 2021; Wickramasinghe et al. 2021). Seaweeds, as marine macrophytes, present a possible compelling alternative as blue-green protein sources, offering an interesting nutrient profile while minimizing environmental impact (Juul et al. 2024; Samarathunga et al. 2023; Stedt et al. 2022a,b). Additionally, the increasing trend towards vegetarianism and veganism as well as the consumption trends of alternative protein sources predicted to increase by 9 % annually until 2054 (Probst et al. 2015), underscore the urgency of exploring novel vegan protein sources beyond over-exploited terrestrial options (Juul et al. 2024; Van den Boom et al. 2023; Wickramasinghe et al. 2021). Seaweed’s capacity to thrive in marine environments, coupled with their possibilities to attain protein contents similar to soy (Stedt et al. 2022a), positions them as promising candidates in a more diversified future food protein portfolio. Enhancing the downstream applications of seaweed proteins is paramount, and the refinement of novel protein extraction methodologies holds promise in achieving this objective (Juul et al. 2021, 2022]; Trigo et al. 2021, 2024]). By extracting the proteins, their purity, digestibility, taste and functionality can be enhanced (e.g. Trigo et al. 2021), expanding the potential applications of seaweed proteins in various products. However, it is imperative to underscore that the ultimate efficacy of downstream applications significantly hinges upon optimizing and increasing the intrinsic protein content within the seaweed biomass itself. Elevating protein content at the source ensures a more abundant and sustainable raw material for extraction, thus amplifying the overall efficiency and economic viability of seaweed protein utilization.

Our study has documented that salinity-induced alterations in crude protein content occur in Ulva spp., revealing a consistent trend of increased crude protein contents with decreasing salinity independent of species and strain origin. Across eight experiments, including different species and strains, five strains had their highest average crude protein content at the lowest salinity level of five.

Furthermore, it is noteworthy that the crude protein contents measured in the examined strains of this study were generally higher – especially in specific strains exposed to lower salinity treatments – when compared to previously reported protein values documented in both natural (4.8–8.2 %; Olsson et al. 2020a) and cultivated (4.67–20.79 %; Steinhagen et al. 2021, 2022a, b) Ulva biomass within the wider Baltic Sea region. Further, in seven out of eight experiments the crude protein content was higher in all salinity treatments than at the starting point. Our measured crude protein values, generally falling within the upper range reported for Ulva (Hofmann et al. 2024; Holdt and Kraan 2011), provide additional support for the impact of salinity treatments to be used in biochemical engineering endeavors for protein-enriched Ulva biomass. Moreover, our results suggest the viability of cultivating Ulva in low salinity environments, including fjords, estuaries, and the Baltic Sea. This expands the potential cultivation sites to brackish-water inland streams, and utilizing low-salinity environments in on-shore facilities may offer a cost-effective strategy for Ulva cultivation (Cardoso et al. 2023), particularly noteworthy as most current commercial Ulva farming is predominantly conducted in fully marine habitats (e.g. Bolton et al. 2009; Califano et al. 2020; Ghaderiardakani et al. 2019; Steinhagen et al. 2021). This approach furthermore has the potential to expand the cultivation of Ulva for in-situ bioremediation and nutrient capture in low salinity environments and further enables the reuse of nutrient-rich side streams from agriculture and industry (Sode et al. 2013; Stedt et al. 2022a,b).

A plausible rationale for the observed higher crude protein content at lower salinity levels may stem from the potentially increased nitrogen uptake rates exhibited by these species in environments with reduced salinity compared to higher salinity conditions. While scant research has delved into the impact of salinity on nutrient uptake in seaweeds (Roleda and Hurd 2019), conclusive attribution of our results to salinity-induced changes in uptake rates remains challenging. Nevertheless, Wu et al. (2018) noted a significant increase in nitrate uptake rates in U. prolifera at 5–15 compared to 20–50, concomitant with a decrease in tissue nitrogen content as salinity rose. In another study, Cohen and Fong (2004) hypothesized that the salinity tolerance of U. intestinalis could be linked to its adept nitrate uptake for osmoregulation. Substantiating this as an explanation for the observed crude protein content pattern would however necessitate further causal driven investigations.

Unexpectedly, no discernible parallels are evident between the patterns observed for crude protein and pigment content. While prior research has noted color variations in Ulva thalli in response to changes in biomass nitrogen content (Robertson-Andersson et al. 2009), a recent study by Stedt et al. (2022c) demonstrated the accurate estimation of nitrogen content in euhaline U. fenestrata through color image analysis. The absence of a clear relationship between protein content and pigment content in the strains tested here was unexpected.

4.3 Pigments and total phenolic compounds

Pigments and phenolics play crucial roles in the biology of seaweeds, contributing to various physiological functions and ecological adaptations. Pigments, such as chlorophylls, carotenoids, and phycobilins, are essential for photosynthesis, and hence for converting light energy into chemical energy (Harrison and Hurd 2001; Ramus et al. 1976) – therefore directly affecting the biochemical set-up of a seaweed. Carotenoids, on the other hand, aid in photoprotection by dissipating excess light energy and scavenging reactive oxygen species (Eismann et al. 2020). Phenolics, a group of secondary metabolites, serve multiple functions in seaweeds, including defense against herbivores, protection against UV radiation, and modulation of cell wall properties (Cotas et al. 2020; Farvin and Jacobsen 2013). These compounds possess antioxidant properties, helping to mitigate oxidative stress induced by environmental factors (Cotas et al. 2020). The intricate interplay of photosynthetic pigments and phenolics in seaweeds underscores their adaptive strategies towards changing biotic and abiotic factors and ecological significance. Increasing interest from economic sectors has arisen for seaweed derived pigments and phenolics as they find application in e.g. the pharmaceutical, cosmetic, and functional food industries.

Our study documented that, in terms of pigment and phenolic content, no discernible trend analogous to that observed for crude protein content or growth is evident. There was an absence of a definitive correlation between pigments and salinity that reflects across the investigated species and strains, with no consistent pattern of increase or decrease noted.

In the salinity regimes of 5–10, Chla content ranged from 0.94 to 1.75 μg mg⁻¹ and Chlb content varied between 0.60 and 1.55 μg mg⁻¹, falling within the anticipated range based on previous findings (Holdt and Kraan 2011; Steinhagen et al. 2021, 2022a, b). The carotenoid content ranged from 0.51 to 0.99 μg mg⁻¹ and was therefore within the expected range of carotenoids in Ulva spp. (Steinhagen et al. 2021, 2022a, b).

Although, significant differences were observed between different salinity treatments, the overall effect was rather small and values observed for pigments and phenolics at both inter- and intraspecific levels resemble previous findings and no distinct detrimental effects of varying salinity towards the pigment and phenolic composition of tested genotypes were observed. It requires further investigation if the photosynthetic apparatus and associated pigments of Baltic Sea strains are generally better adapted to salinity fluctuations, as previous studies have reported that e.g. in Ulva prolifera, photosynthetic rates decreased at low salinity, together with increased signs of oxidative stress (Luo and Liu 2011; Xiao et al. 2016). Further, U. australis exposed to short-term low salinity treatments accumulated higher amounts of photosynthetic pigments (Kakinuma et al. 2006). It should however be mentioned that our experiments were performed over a course of 8 weeks, whereas most previous work has investigated short-term effects of low-salinity treatments in Ulva (Fort et al. 2024; Kakinuma et al. 2006; Luo and Liu 2011; Xiao et al. 2016). It remains to be determined what effect the time of exposure has and whether euhaline Ulva strains might adjust as well over a longer course of treatment exposure.

Regarding the phenolic content, no discernible salinity-dependent trend was evident, indicating variability in phenolic acid content responses across Ulva species and strains. The phenolic content in the lower salinity range falls between 0.13 and 0.33 % of dry weight, aligning with previously reported values (Steinhagen et al. 2021, 2022a, b; Toth et al. 2020). Although this study did not observe values surpassing the expected range, it does not preclude the potential extraction of phenolics from Ulva biomass cultivated in low-salinity environments, hinting at potential economic value. It should however be mentioned that phenolic compounds are often regarded as anti-nutritional factors due to their ability to interfere with nutrient absorption and digestion (Bora 2014). These compounds can form complexes with proteins, carbohydrates, and minerals, reducing their bioavailability (Bora 2014). Further emphasis needs to be put into the downstream application of Ulva biomass used in food and feed stuffs. However, overall, our observed pigment and phenolic values in Baltic Sea strains of Ulva suggest commercial viability, indicating that the lower salinity environment prevalent in the wider Baltic Sea does not impede the exploitation of such compounds for potential commercial applications.

It is noteworthy to emphasize that, apart from specific photosynthetic pigments, the salinity treatments did not have a significant effect on the investigated variables in U. lacinulata (Figure 2B, 3B) suggesting that this is a resilient species with relative stability in response to changing abiotic factors such as salinity. Previous studies have underscored this phenomenon of stable performance in U. lacinulata, indicating similar reactions among different genotypes originating from various regions across Europe (see also Cardoso et al. 2023; Fort et al. 2024). Despite being frequently employed as a commercial crop species (Bachoo et al. 2023; Califano et al. 2020), U. lacinulata exhibits a proclivity for sudden biomass disintegration events, necessitating meticulous monitoring for its cultivation in aquaculture settings.