Introduction

Over the last several years, there has been a global increase in the demand for red algae in the carrageenan and agar industries (Bixler 1996; Zemke-White and Ohno 1999; Buschmann et al. 2001; McNeill et al. 2003; Buschmann et al. 2004). About 20% of the world demand for carrageenan has been supplied by extractive industries in Chile (Avila et al. 2001a, b). However, these local carrageenan industries depend entirely on the harvesting of natural beds of Gigartina skottsbergii (Buschmann et al. 2001), and other carrageenan producing red algae such as Sarcothalia crispata and Mazzaella laminarioides; all are harvested as a single group, known as “lugas” (Marin et al. 2002; Avila et al. 2001a, b). Since, at the global level, the cultivation of commercially important algae represents nearly 22% of all red algae used (Zemke-White and Ohno 1999; McNeill et al. 2003), culture techniques and repopulation methods are an important alternative for the recolonization of natural algae beds to ensure the supply of primary material for the hydrocolloid industry.

Due to its high level of carrageenan content (>70% of dry weight) (Piriz and Cerezo 1991), Gigartina skottsbergii is considered to be the most important carrageenan producing red alga commercially in Chile. Export volumes of carrageenan have increased from 26 tons in 1989 to 3,772 ton in 2005, totally US$ 29.5 million in value. The Magellan Region contributes approximately 9% of the total biomass produced in Chile and has over the last five years become the third most important port of landing for the resource (Zamorano, Pers. comm.). There exists a great internal demand and significantly increasing extraction of the species, which constitutes an alternative resource for artisenal fisheries, especially during the closed seasons for sea urchin and king crab and during outbreaks of red tide, a characteristic phenomenon of the region (Avila et al. 2001b). This situation has provoked an over-exploitation and in some cases the collapse of natural populations (Avila et al. 1999; Buschmann et al. 2004; Romo et al. 2006). The growing demand for this resource and the apparent deterioration of natural populations makes evident the importance of developing a production technology to allow the acceleration of some aspects of the developmental process, such as growth (Avila et al. 1999; Romo et al. 2006). In addition, the recently declared Cape Horn Biosphere Reserve has as a primary objective the establishment of research and demonstration projects that focus on the sustainable use of biodiversity for the betterment of social wellbeing in the archipelago (Rozzi et al. 2006).

Several endeavours on the part of researchers from diverse institutions, financed through a variety of sources, have allowed the generation of background information on the ecology, biology, physiology, and cultivation of G. skottsbergii in its distribution south of 40°S (Westermeier et al. 1999; Buschmann et al. 1999, 2001; Avila et al. 1999, 2003; Romo et al. 2001, 2006; Buschmann et al. 2004; Faugeron et al. 2004; Martínez et al. 2005). However, no prior studies exist on the effects of agricultural fertilizers on the development of G. skottsbergii in controlled systems. The objective of this study, therefore, was to determine the effect of traditional agricultural fertilizers on the growth and survival rates of the initial developmental stages of G. skottsbergii under laboratory conditions, which renders production more efficient, lowers costs associated with repopulation, and develops techniques valuable for the sustainable use of this resource.

Material and methods

Mature tetrasporic fronds of G. skottsbergii (Fig. 1) were collected by scuba diving from natural populations at Santa Ana Point (53°18’11.4’’S; 70°23’30, 5’’W), 70 km southwest from the city of Punta Arenas on the Strait of Magellan (Fig. 2). The material was transported in seawater inside hermetic containers to the laboratory at the Center for Aquaculture and Marine Resources, Department of Natural Sciences and Resources, University of Magallanes (UMAG). Once in the laboratory the material was washed with filtered, sterilized seawater to eliminate epiphytes and epibionts that could affect the subsequent cultures.

Fig. 1
figure 1

The life history of Gigartina skottsbergii. (Mod. Romo et al. 2004)

Full size image
Fig. 2
figure 2

Geographic location of collection site of Gigartina skottsbergii at Santa Ana Point, Strait of Magellan, Chile

Full size image

Tetraspore release

Liberation of tetraspores was induced by stress through dehydration of the fronds at ambient temperature for approximately 45 min, after which the fronds were rehydrated in sterilized and filtered (0.2 µm) seawater. Once the tetraspores were released, the fronds were removed from the water and the resulting suspension was filtered through a sieve in order to catch the remaining tissues and impurities, obtaining a homogenous density of approximately 2,000 tetraspores mL−1 to use as an inoculation solution on glass microscope slides.

Cultivation methods used

Five treatments were performed: three commercial fertilizers (Bayfoland 250SL, Superphosphate and Urea, Table 1), a laboratory medium (Provasoli) and a control (seawater without additives). Superphosphate and Urea were used as phosphate and nitrogen sources, respectively. Both are sold in their solid state, and for this reason it was necessary to prepare stock solutions. The concentrations of each nutrient were adjusted to the corresponding values of nitrogen and phosphorus as has been used in Gracilaria chilensis (Alveal et al. 1991): 0.06 g L−1of sodium nitrate (10 mg L−1 of nitrogen) and 0.01 g L−1 of sodium phosphate (3 mg L−1 of phosphorus). Bayfoland is a liquid commercial foliar fertilizer and was used at a concentration of 0.1 ml L−1, corresponding to 1/3 of the required minimum concentration for the cultivation of initial growth stages. The quantities of different agricultural fertilizers required to achieve the indicated concentrations of nitrogen and phosphorus are shown in Table 2.

Table 1 Commercial entities, chemical composition and components of the nutrient supply used in experimental treatments
Full size table
Table 2 Concentration of fertilizer additives in culture media used in experimental treatments
Full size table

The cultures were maintained inside a temperature-controlled room at 8 ± 1°C and 45 μmol photons m−2 s−1 PAR (provided by Philips TLT 20W/54 daylight fluorescent tubes) at 12:12h light-dark cycle. The culture media for each treatment was renewed weekly.

Experimental design

Slides inoculated with tetraspores were placed in 250 mL capacity Petri dishes. Fifteen experimental units (dishes) were prepared, distributed in the 5 treatments of 3 replicates each. The effects of the distinct nutrient solutions on the development of G. skottsbergii tetraspores were evaluated through the mortality and growth of 45 individuals of each experimental unit. Weekly and during the 81 days of cultivation, 45 individuals were photographed using a microscope integrated imaging system connected to an image analyzing software (Imagen Pro-Plus version 4.1), providing data on the increase in diameter of discs of G. skottsbergii.

Growth rate and data analyses

Increase of basal diameter was used as the parameter for estimating growth rates. The relative growth rate of G. skottsbergii discs was estimated using the formula proposed by Hansen (1980):

$$RGR{\text{ }}\left( \% \right){\text{ }} = {\text{ }}{{\left( {100{\text{ }}x{\text{ }}\ln {\text{ }}\left( {{{tf} \mathord{\left/ {\vphantom {{tf} {t0}}} \right. \kern-\nulldelimiterspace} {t0}}} \right)} \right)} \mathord{\left/ {\vphantom {{\left( {100{\text{ }}x{\text{ }}\ln {\text{ }}\left( {{{tf} \mathord{\left/ {\vphantom {{tf} {t0}}} \right. \kern-\nulldelimiterspace} {t0}}} \right)} \right)} T}} \right. \kern-\nulldelimiterspace} T}{\text{ }}$$

Where:

RGR (%):

relative growth rate expressed in daily percentage

t0 :

initial diameter of disc (µm)

tf :

final diameter of disc (µm)

T:

time in days.

Subsequently, the values for the growth rates and mortality percentages were transformed by applying arc-sine (Zar 1999) to achieve normal distributions and then subjected to an analysis of variance analysis (ANOVA), using the software program Statistica 5.0. When significant differences were found, a Newman-Keul test was applied a posteriori (Zar 1999). All conclusions were based on a 95% confidence level (p < 0.05).

Results

Growth rates

Gametophytes of G. skottsbergii cultivated with different nutrient solutions showed different responses in growth during their initial developmental stages. At the end of day 81, the greatest increase in disc diameter was obtained with Bayfoland (381.87 ± 54.99 µm) and Provasoli (377.88 ± 49.92 µm). Growth was less in the treatments with Superphosphate (88.36 ± 16.27 µm), in the control solution (77.65 ± 9.93 µm) and with Urea (70.06 ± 10.88 µm) (Fig. 3).

Fig. 3
figure 3

Diameter increase (µm ± SD) of G. skottsbergii discs during 81 days of culture using different nutrient solutions

Full size image

Therefore the highest growth rates were observed in the gametophytes cultivated with Bayfoland (6.19 ± 0.38% day−1) and Provasoli (5.70 ± 0.035% day−1), whereas the lowest growth rates were observed in the control and Urea treatments (3.76 ± 0.07% day−1 and 3.27 ± 0.23% day−1, respectively) (Fig. 4). The differences between all the treatments utilized were statistically significant (p = 0.000) (Table 3).

Fig. 4
figure 4

Daily growth rate (% day−1 ± SD) of G. skottsbergii discs during the first 35 days of the study in different treatments. Different letters refer to significant differences between mean values with Newman-Keuls post-hoc test (p < 0.05)

Full size image
Table 3 Analysis of variance (ANOVA) for the weekly growth rate, mortality and different enrichment solutions of G. skottsbergii gametophytes, cultured with different treatments, during the first 35 days. Treatments: Provasoli (Provasoli), Bayfoland (Bayfolan). (*) Indicates significant differences (p < 0.05)
Full size table

The a posteriori test applied to the interaction between the different treatments used and the time of cultivation, considering weekly periods, showed that during the first week, the gametophytes cultured with Provasoli had a higher growth rate than those under the other nutrient regimes (Fig. 5). During the second week, significant differences were observed in growth under Provasoli and Superphosphate treatments (p = 0.021) and Urea (p = 0.026) (Fig. 5, Table 4).

Fig. 5
figure 5

Growth rates (% day−1 ± SD) of G. skottsbergii disks during the first five weeks of culture (n = 3). (*) Indicates significant differences (p < 0.05)

Full size image
Table 4 A Newman- Keuls a posteriori test of the weekly growth rates of G. skottsbergii gametophytes, cultured in different enrichment solutions, during the first 81 days. Treatments: Seawater (S), Provasoli (P), Bayfoland (B), Superphosphate (S), Urea (U). (*) Indicates significant differences (p < 0.05)
Full size table

In the third week, the differences observed between the treatments were only in Bayfoland v/s the other cultures (control, p = 0.043; Superphosphate, p = 0.028; Urea, p = 0.000) (Fig. 5, Table 4). During the fourth week, the Provasoli and Bayfoland treatments showed significant differences in growth rate related to the other treatments (Fig. 5, Table 4).

Finally, beginning in the fifth week of the study, differences in growth rates became evident, mainly between the treatments Provasoli and Bayfoland compared to control, Superphosphate and Urea treatments (Fig. 5, Table 4). The a posteriori test indicated that the differences derived mostly from the control (least growth) versus Bayfoland (p = 0.033) and Provasoli (p = 0.021), Urea versus Provasoli (p = 0.011) and Bayfoland (p = 0.016), which showed the greatest growth at the end of the study (Table 4). The variance test used on the weekly growth rates showed that the nutrient solutions used, the time under cultivation, and the interaction between them, acted in a synergistic manner on the growth of the gametophytes.

A more detailed analysis of the behavior of the growth rates of different treatments (control, Provasoli, Bayfoland, Superphosphate, and Urea) during the five weeks of the study indicated that, although the growth rates of gametophytes cultivated with Bayfoland and Provasoli did not show significantly greater growth during the first 7 days of cultivation, they began in the second and third weeks to show better developmental responses in relation to the other treatments, recording rates of greater than 8% daily growth as compared with growth rates that did not surpass 6% in the other treatments. ANOVA carried out on the growth results of the Provasoli and Bayfoland treatments, the solutions that noticeably promoted the growth of G. skottsbergii gametophytes, showed no statistically significant difference (p = 0.431) between the growth rates of these two treatments (Table 3).

Survival

Beginning with day 39 and day 46, respectively, mortality was observed in the Urea (45%) and control treatments (11%). At 67 days of cultivation, all of the gametophytes cultured under the control treatment, Superphosphate, and Urea had died. The treatments using Provasoli and Bayfoland had disc survival rates of 100% on the 81st (final) day of the experiment (p = 0.000) (Table 3). The a posteriori test indicated that the observed differences were between the treatments of Bayfoland and Provasoli (p = 1.00) and cultures enriched with Superphosphate (p = 0.000) and Urea (p = 0.000) (Table 5).

Table 5 Newman-Keuls a posteriori test for mortality (%) of G. skottsbergii gametophytes, cultured in different enrichment solutions, during the first 5 weeks. Treatment: Seawater (S), Bayfoland (B), Superphosphate (S), Urea (U). (*) Indicates significant differences (p < 0.05)
Full size table

Discussion

Physiological explanations for differential response to cultivation media

There is a tendency to think of all algae as having similar nutritional requirements, but the heterogeneity of seaweeds makes such generalization difficult (Lobban and Harrison 1994). A wide variety of physiological and biological factors, such as inter-seaweed variability, nutritional history, type of tissue, life history stage, age, the surface area to volume ratio of the thallus, and morphology (Harrison et al. 1986; Harrison and Hurd 2001), will influence growth and uptake of nutrients.

Nitrogen and phosphorus are two nutrients that limit seaweeds growth and yields in most natural environments (Harrison and Hurd 2001). Nitrate and ammonia are important environmentally available nitrogen sources for the growth of marine algae. After these ions are absorbed from the environment, they are generally incorporated into amino acids and proteins, or used in the synthesis of pigments such as phycoerithrin, which is considered to be a store of available nitrogen (Lapointe 1985; Ryther et al. 1981). However, in many marine algae, nitrogen in the form of ammonia is frequently absorbed and assimilated more rapidly than nitrate and Urea (De Boer 1981). At the same time, it has also been found that Urea is an excellent source of nitrogen for many marine algae (Nars et al. 1968; Probyn and Chapman 1982; Thomas et al. 1985). However, experiments carried out by Buschmann et al. (2004), showed that G. skottsbergii achieves most of its growth rate with the addition of ammonia, rather than nitrate.

In our experiments, G. skottsbergii germlings grew more rapidly when cultivated in a solution of Bayfoland 250 SL and Provasoli, while growth rates in Urea and Superphosphate were significantly lower. These differences may be due to the chemical composition of each of the nutrient solutions used. A comparison of the solutions of Urea and Superphosphate with those of Bayfoland and Provasoli reveals that the latter solutions contain a broad variety of macro and micronutrients, namely all those required for the development of plants. In fact, the Provasoli solution was formulated specifically for the enrichment of media for cultivation of seaweeds and likely for this reason produced favorable growth in comparison with Urea and Superphosphate. On the other hand, Bayfoland has been formulated for the growth and development of terrestrial plants, and contains macro (N, P and K) and micronutrients (Fe, Cu, Mo, Ni, Mn and Bo among others), thus preventing nutritional problems in plants and making it also an effective medium for the cultivation of G. skottsbergii.

For their part, Urea and Superphosphate provide N (46%) and P (48%). Urea is an excellent N source for some seaweeds (e.g., kelp), but other seaweeds show reduced growth on Urea (De Boer 1981; Navarro-Angulo and Robledo 1999). While it is true that P and N are considered essential limiting elements for growth of algae (Lobban and Harrison 1994), in this study, their addition via Urea and Superphosphate did not increase growth rates in the G. skottsbergii germlings, possibly due to the absence of other elements which are present in Bayfoland and Provasoli.

This decrease in growth in two treatments, even in the presence of limiting nutrient, could be due to saturation of the uptake capacity for N and P. Some seaweeds do not show such saturation at high environmental nutrient concentration (as described by the Michaelis-Menten Equation), and their uptake rate increases linearly with high nutrient additions (Harrison et al. 1986; Taylor et al. 1998). However, here G. skottsbergii may need other elements and nutrients or have difficulty absorbing and assimilating Urea and Superphosphate. Nitrate is not as easily assimilated as ammonia, and there is a waste of energy in its capture and assimilation (Hurd 2000). Also, since our experiments were done without aeration, it must be considered that water motion is extremely important for seaweeds in natural conditions, since it determines the thickness of velocity and diffusion boundary layers (DBL’s) around the thallus and hence the movement of ions and gases to and from the thallus surface (Hurd 2000). Nonetheless, an adequate nutrient supply is important in the early stages (germlings) of G. skottsbergii development, as it determines future growth and reproduction processes.