The aim of the present contribution

The aim of the present contribution is to relate the
yield, chemical composition and physical properties of
agar with environmental factors and reproductive stages
along the year in order to contribute to a sustainable
commercial exploitation of Bahía Bustamante algal
resource.
MATERIALS AND METHODS
Sample collection
Gracilaria graciliswas collected between March 2006
and February 2008 from the subtidal in Bahía Bustamante (45°08′S, 66°32′W), Chubut Province, Argentina. Specimens were collected by SCUBA diving. The
sampling units were placed on four transects parallel
to the shore laid 100 m from each other and beginning 100 m away from the shore. On each transect
five square sampling units (0.25 m
2
) were located
at 100 m intervals. Each sampling unit was relocated by GPS, being latitude and longitude for the
first square sampling unit on each transect the following: 45°08.815′S, 66°29.107′W; 45°08.768′S,
66°29.142′W; 45°08.695′S, 66°29.190′W and
45°08.630′S, 66°29.198′W. For seasonal variation in
agar physico-chemical properties, at least 30 random
specimens from May 2006 (autumn), August 2006
(winter), October 2006 (spring) and January 2007
(summer) were sampled from inside each of the
square sampling units. Specimens corresponding to
each season were pooled, air dried and milled for
subsequent polysaccharide extraction.
Environmental physico-chemical variables
For each sampling site,in situ data of temperature,
salinity and pH were taken from the subsurface water.
Water samples were also collected for nutrient analysis,
keeping them refrigerated until arrival at the laboratory.
Nutrient concentrations were analyzed by colorimetric
techniques on a Technicon II autoanalyzer (Emeryville,
CA, USA), at the Marine Chemistry Laboratory of the
Instituto Argentino de Oceanografía (IADO). Nitrates,
nitrites and phosphates were determined as indicated
in Martín et al. (2010).
Polysaccharide extraction procedure
Previously milled random selected plants (10 g dry
weight) were mechanically stirred in water (1 L) at room
temperature (¥3) for 24 h. Room temperature extracts
were pooled (extract RTW). Extraction continued in: (i)
water (1 L) at 70°C (¥3) for 4 h (fractions W70.1–3) and
(ii) water (1 L) at 90°C (¥3) for 4 h (fractions W90.1–3).
After each extraction step, the residue was removed by
centrifugation and the supernatant was concentrated
and freeze-dried. Extracts obtained at room temperature
were thoroughly dialyzed using Spectra/Por tubing
(Spectrum Laboratories, Rancho Domínguez, CA, USA)
with a molecular weight cutoff of 3500 Da before freezedrying. Dialyses were performed during 48 h in running
tap water followed by 24 h dialysis in a closed system
against distilled water.
Composition of the products
Carbohydrate content was analyzed by the phenolsulfuric acid method (Duboiset al., 1956) without previous hydrolysis of the polysaccharide, using galactose
as standard. Sulfate was measured with the turbidimetric method of Dodgson and Price (1962) after hydrolysis of the samples with 1 M HCl for 4–5 h at 105–
110°C. The content of protein was estimated by the
method of Lowryet al. (1951).
Monosaccharide composition
Reductive hydrolysis of the polysaccharide samples
and further acetylation of the sugar mixtures were
performed as described by Stevenson and Furneaux
(1991).
Gas Liquid Chromatography (GLC) of alditol acetates
was carried out on a Hewlett-Packard 5890A Gas Chromatograph (Hewlett Packard, Avondale, PA, USA)
equipped with a flame-ionization detector and fitted
with a fused-silica column (0.25 mm i.d.¥ 30 m)
WCOT-coated with 0.20mm film of SP-2330 (Supelco,
Bellefonte, PA, USA). Chromatography of the alditol
acetates was carried out isothermally at 220°C. Nitrogen was used as carrier at a flow rate of 1 mL min
-1
. The
split ratio was 80:1. The injector and detector temperature was 240°C. Conversion of GLC areas to molar
basis was calculated according to the effective carbon
response theory (Sweet et al., 1975).
All data are expressed as means of duplicates.
Alkaline treatment and determination of
cyclizablea-galactose 6-sulfate units
One-pot alkaline treatment was carried out as described
by Navarro and Stortz (2003). The polysaccharide
(10 mg) was dissolved in water (5 mL) and NaBH4
(2 mg) was added. After 1 h, 5 M NaOH (1.7 mL) was
added and the solution heated at 80°C in a water bath.
According to previous results (Rodríguezet al., 2009),
the cyclization reaction was stopped after 5 h by neutralization with 4 M trifluoroacetic acid. The solvent
was evaporated-off and the residue derivatized to
the acetylated alditols according to Stevenson and
Furneaux (1991)