Deleterious effect of TRIS buffer on growth rates and pigment content of Gracilaria birdiae Plastino

(Deleterious effect of TRIS buffer on growth rates and pigment content of Gracilaria birdiae Plastino & E.C. Oliveira (Gracilariales, Rhodophyta)). This work evaluated the effects of Tris (hydroxymethyl)-aminomethane (TRIS) buffer and its interaction with nutrient concentration on the development of Gracilaria birdie, a common species on the Brazilian coast that has been exploited for agar production. Responses to different conditions were assessed through growth rates and pigment content (chlorophyll a, phycoerythrin, phycocyanin and allophycocyanin). Provasoli’s nutrient solution with and without TRIS addition was tested at concentrations of 12.5, 25 and 50%. The pH was also monitored. G. birdiae grew better in the absence of TRIS and at low nutrient concentrations, 12.5 and 25% (growth rates of 10.8-11.3%.day). Higher contents of phycoerythrin and chlorophyll a were observed without TRIS at 12.5 and 25% (Phycoerythrin, 649.6-698.0 μg g fresh biomass; Chlorophyll a, 156.0-168.6 μg g fresh biomass). These findings highlight the deleterious effect of TRIS on growth and phycoerythrin and chlorophyll a content. They also demonstrate the importance of appropriate nutrient concentration for laboratory cultures, depending on the intrinsic characteristics of each species.


Introduction
In vitro cultures have significantly contributed to phycology development in the last decades.Life histories of many species of seaweeds have been accessed through these techniques, as well as physiological and genetic processes.In vitro cultures have also been important tools for selection and domestication of species (Van der Meer 1982).Environmental factors, such as light, nutrients, salinity and temperature, can be manipulated in the laboratory, which allows researchers to evaluate their effects on vegetative and reproductive development of different species or strains (Oliveira et al. 1995;Plastino 2003).Although in vitro cultures have been commonly utilized, some basic points still need further investigation, such as the adequacy of culture media for a selected species.
Many seaweed culture media still have a number of potentially undesirable characteristics, when their chemical composition is examined.Firstly, concentrations of some ions, notably phosphate and some micronutrients are substantially greater in the media than in natural seawater (Woelkerling et al. 1983).This is the case in seawater enriched with Provasoli's solution (PES -McLachlan 1973) and with von Stosch's solution (VS -Edwards 1970), commonly applied in rhodophytes in vitro cultivation.Secondly, concentrations of some other ions, especially calcium, sulphate and borate, while generally similar to those in seawater, appear to be totally unnecessary for ensuring stability of the medium (Woelkerling et al. 1983).Thirdly, some media employ organic buffers which are metabolizable, such as glycylglycine (McLachlan 1973) and Tris (hydroxymethyl)-aminomethane, TRIS (Hanisak 1979), which seem to have negative effects on life material.Of these, TRIS is the most controversial, because its effectiveness of stabilized pH has not been clearly demonstrated (Woelkerling et al. 1983).In addition, the deleterious effect of TRIS has been observed for some phytoplankton species (Harrison et al. 1980) and freshwater algae (Smith & Foy 1974).Studies on seaweeds and seagrasses have proposed that TRIS has a deleterious effect on photosynthesis by inhibition of mechanisms such as the transport of HCO 3 -across the plasma membrane (Axelsson et al. 2000;Hellblom et al. 2001) or O 2 evolution in Photosystem II (Sofrová et al. 1978;Rickert et al. 1991;Jegerschold & Styring 1996).This buffer also stimulates bacterial growth, leading to a negative effect on cultures (Fábregas et al. 1993).
Although most reports indicate a negative effect of TRIS, some authors have raised some of its beneficial effects, by enhancing the availability of carbon, vitamins and hormones when promoting the increase of bacteria in the growth media (Ogata 1966;Provasoli & Pintner 1980;Woelkerling et al. 1983).The question is if the beneficial effects of TRIS would compensate the damage caused by its addition, especially in the photosynthetic apparatus.
TRIS is a component of the PES medium, which is probably the most utilized for laboratory seaweed culture (Oliveira et al. 1995).Similarly to the majority of culture media, it was originally developed for cold area species from nutrient-rich waters.However, it has been widely used, in spite of the geographic origin of the alga.If it were taken into account, it would be expected that species from oligotrophic tropical waters do not need such high nutrient concentrations for their laboratory cultures.Nevertheless, PES has been commonly utilized for species from the tropical Brazilian coast (e.g.Berchez & Oliveira 1990;Yokoya & Oliveira 1992).
This work was planned to analyze the effects of TRIS buffer and its interaction with nutrient concentration on growth and pigment content of a tropical species.The target organism was Gracilaria birdiae, one of the main species exploited for agar production in Brazil (Plastino & Oliveira 2002), which has been the subject of color strain investigations in our laboratory (e.g.Ursi et al. 2003;Plastino et al. 2004).G. birdiae occurs from the northeast to the southeast coast of Brazil (Plastino & Oliveira 2002), which is predominantly characterized by oligotrophic warm waters (Boltovskoy et al. 1999).

Material and methods
Apical segments of three infertile tetrasporophytes of Gracilaria birdiae Plastino & E.C. Oliveira obtained in the laboratory were utilized in the experiments.These tetrasporophytes were originated from cystocarpic specimens derived from a tetrasporophyte collected in a natural population from Anchieta Beach (20°80'S and 40°65'W), Espírito Santo state (Brazil) (Plastino et al. 2004).Unialgal cultures were established as described by Plastino & Oliveira (1990).
Three different concentrations of PES (McLachlan 1973) were employed: 12.5, 25, and 50%.These concentrations were also tested utilizing PES without TRIS addition (PES-TRIS).Three replicates each with three apical segments from three different individuals were cultivated separately in flasks with 900 ml of enriched seawater.
Growth rates were evaluated by weekly weighing for 28 days.Fresh biomass was utilized to calculate the growth rates (Lignell & Pedersén 1989).Initial biomass of each replicate was 10 mg.
The pH values were measured after PES dilution in seawater using a pH meter (Denver mod.10234).After one week of cultivation, the apical segments were removed from the flasks and the pH of the medium was measured again.
Pigments were quantified by spectrophotometry using an HP 8452A spectrophotometer.Pigment extractions were carried out at the end of the growth experiment at 4 °C, according to Kursar et al. (1983) with modifications (Plastino & Guimarães, 2001).Briefly, the samples were disrupted by grinding with liquid nitrogen and 50 mmol/L phosphate buffer, pH 5.5.Crude extracts were centrifuged at 36,000 g for 25 min to get the phycobiliproteins.Chlorophyll a was extracted after dissolving the pellet in 90% acetone, and centrifuging at 12,000 g for 15 min.Pigment concentrations were calculated according to Kursar et al. (1983) for phycobiliproteins (allophycocyanin, phycocyanin and phycoerythrin), and Jeffrey and Humphrey (1975) for chlorophyll a.
Growth rates and pigment concentrations were analyzed by two-factor ANOVA (independent variables: PES concentrations and presence or absence of TRIS).Newman-Keuls post hoc tests were applied when necessary.

Results
Growth rates -The growth rates of Gracilaria birdiae were affected by both PES concentration and presence or absence of TRIS (interaction between factors F = 4.352, P < 0.05).Branches showed higher growth rates when cultivated in PES without TRIS and low nutrient concentration, 12.5 and 25% (Fig. 1, Tab. 1).Similar growth rates were observed in PES, regardless of concentration (Fig. 1, Tab. 1), whereas in the absence of TRIS, G. birdiae grew better in 12.5 and 25% than in 50%.
values observed after one week of cultivation were similar between seawater enriched with PES and PES-TRIS, showing that the buffer effect of TRIS was not maintained through cultivation (Tab.2).During one week, pH values of seawater with PES-TRIS varied less than the pH values of seawater enriched with PES.The pH variation was sensitive to PES concentration as well as to the presence or absence of TRIS (interaction between factor F = 18.881,P > 0.05).No difference in pH variation was observed among the nutrient solution concentrations when branches were cultivated in PES-TRIS.However, this difference was verified in PES, in pH measurement -The buffer effect of TRIS was evident when culture medium was added to seawater.
Pigment content -Concentrations of allophycocyanin and phycocyanin did not vary in any PES concentration tested (allophycocyanin, F = 1.475,P > 0.05; phycocyanin F = 3.896, P > 0.05), nor in the presence or absence of TRIS (allophycocyanin, F = 2.142, P > 0.05; phycocyanin, F = 2.301, P > 0.05) (Fig. 2).Both chlorophyll a and phycoerythrin concentrations were affected by PES concentration and presence or absence of TRIS, as demonstrated by the significant interaction between these factors (chlorophyll a, F = 5.540, P < 0.05; phycoerythrin F = 6.973,P < 0.05).The highest concentrations of chlorophyll a were observed in branches cultivated in seawater enriched with 12.5 and 25% of PES-TRIS (Fig. 2).The chlorophyll a content of branches cultivated in all concentrations of complete PES was similar to those of branches cultivated in PES-TRIS at 25% and 50%.Higher concentrations of phycoerythrin were observed among branches cultivated in seawater enriched with 12.5 and 25% of PES-TRIS, and in branches cultivated in complete PES at 50% (Fig. 2).Although pigment content varied according to culture condition, there was no color variation among branches.

Discussion
The higher growth rates of Gracilaria birdiae observed in PES without TRIS when compared to PES with TRIS corroborates the idea that the eventual benefits of this buffer in minimizing pH fluctuation may be smaller than its deleterious effects.However, it is clear that a consensus about TRIS effects does not exist because they vary among different organisms.From seven species of seaweeds cultivated in artificial marine culture medium (MCM - Woelkerling et al. 1983) with and without TRIS addition, five grew better in medium without the buffer, one showed worse growth in this condition and the other one was not sensitive to TRIS.The negative effect of this buffer was also reported for Kappaphycus alvarezii, which has shown lower growth rates in PES than in PES-TRIS (Paula et al. 2001), as observed for G. birdiae.The growth of some phytoplancton species was also lowered in cultures containing TRIS (Harrison et al. 1980).
Color variation among branches of G. birdiae cultivated in different conditions has not been observed.By contrast, Kappaphycus alvarezii (Paula et al. 2001) and G. domingensis (personal observation) showed changes in color when cultivated in media with TRIS.Although quantitative analysis of pigment was not performed on these species, it is reasonable to suppose that TRIS interfered in their pigment content, as observed here for G. birdiae.
Pigment analysis of G. birdiae showed that neither allophycocyanin nor phycocyanin was affected by TRIS or PES concentration, in contrast with phycoerythrin and chlorophyll a.The inner position of allophycocyanin and phycocyanin in the phycobilisome structure (Talarico 1996) may protect them from TRIS action, while phycoerythrin and chlorophyll a are probably more assessable to this buffer.
The type of interaction between TRIS and pigments is unknown.Our assumption is that this buffer may be negatively acting on nitrogen acquisition/assimilation, since phycoerythrin and chlorophyll a are highly nitrogenated molecules.Previous studies showed that low nitrogen availability diminishes the contents of phycoerythrin and chlorophyll a (Lapointe 1981;Lapointe et al. 1984).
A positive correlation was observed between chlorophyll a concentration and growth rates of G. birdiae, which is probably explained by an increase in the photosynthetic efficiency as the concentration of this pigment increased.In contrast with chlorophyll a, the content of phycoerythrin did not seem to be strongly related to the growth of G. birdiae.These results are expected since chlorophyll a plays a fundamental function in photosynthesis, while phycoerythrin is not essential for light-harvesting proposes if the light level is sufficient for algal metabolism.The metabolic pathway that involves phycoerythrin synthesis is complex.Apart from a lightharvesting function in photosynthesis, this pigment has a function in nitrogen storage (Lapointe 1981).This secondary function can explain the high concentration of phycoerythrin in 50% of PES, despite TRIS presence.The high nitrogen content in the medium might have compensated the negative effect of TRIS, maintaining the stability of phycoerythrin concentration.
Besides the deleterious effects of TRIS on growth and phycoerythrin and chlorophyll a content of G. birdiae, our results suggested that TRIS is not essential for buffering purposes.Its buffer effect was evident when culture medium was added to seawater.However, final pH values were similar between seawater enriched with PES and PES-TRIS, which showed that this effect was not maintained during the cultivation period.Studies on Callithamnion byssoides (Woelkerling et al. 1983) and on a variety of phytoplankton species (Harrison et al. 1980) also concluded that TRIS was not efficient in stabilizing pH during cultivation.
An increase of seawater pH during one week of cultivation was observed when PES was utilized, but it was not verified in PES-TRIS.These results suggest that pH increase might be attributed to bacterial growth and not to ordinary algal metabolism.The stimulatory effect of TRIS on bacterial growth has already been verified in microalgal cultures (Fábregas et al. 1993).
Our results suggested that G. birdiae might be adapted to a low nutrient content, since the higher growth rates were verified in higher PES dilutions.It demonstrates the need for adequate nutrient concentration, depending on intrinsic characteristics of each species.Dilutions of the original culture media can be more appropriate for laboratory culture of several species, especially those from oligotrophic warm waters.Therefore, it is not recommended the use of TRIS in future cultures of G. birdiae.Furthermore, this buffer can potentially impair the development of other species, being necessary further studies to evaluate how general this negative effect can be.

Table 1 .
Newman-Keuls post hoc test of the two-factor ANOVA of growth rates of Gracilaria birdiae Plastino & E.C. Oliveira after 28 days of cultivation in seawater enriched with different concentrations (50, 25, and 12.5%) of PES with (PES) and without TRIS (PES-TRIS).Independent variables: PES concentration and presence or absence of TRIS.*, significant differences (P<0.05).