Introduction

Marine-microalgae are normally termed as seaweed belonging to the primitive family of non-flowering plant known as thallophyta (Fig 1). The sea plant lacks the leaves, stem and roots of true plants. The body of the seaweed is called the thallus named after the thallophyta. The flattened leaf portion of the microalgae is called the blade and its function is to increase the photosynthesizing surface area.

Seaweeds are attached to the bottom by a root-like structure called a holdfast. Seaweeds are autotrophic which grows in the intertidal and sub-tidal region of the sea. Seaweeds grow abundantly where rocks and coral are present as substratum (Marine Fisheries information Services October-November 1998; Kaliaperumal et al., 2004).

Figure 1: Shows the general structure of seaweed ( Thallophyta)( Reproduced from http://dtc.pima.edu/blc/183/03_183/03_183answers.html).

Seaweed may be classified into two categories namely phaephyceae (Brown) and Rhodophyceae (Red). Seaweeds contain different types of phycolloids such as alginate, carrageenan, agarose and agar agar.

Seaweed is mainly used in Asia as food, fodders, fertilizer and also as drugs in the pharmaceutical industry for its therapeutics properties (Marine Fisheries information Services, October-November, 1998).

1.1 Brown seaweed (Phaephyceae)

Brown seaweed is found in the family of seaweed which is called the phaephyceae. The brown seaweed composition varies according to the species, throughout the year and between different habitats.

Brown seaweed is composed mainly of carbohydrate such as alginate, laminaran, mannitol, fucoidan and small traces of cellulose as shown in Table 1. (Fasahati et al., 2012)

Table1. Brown seaweed composition

1.2 Alginate

Alginate is a major structural biopolymer found in cell wall and intercellular matrix which provides mechanical strength in brown seaweed. Seaweeds containing alginate are called alginophyte (Yabur et al., 2006). In the natural environment, alginate exists as a mixture of potassium, calcium and sodium salt (Gomez et al., 2009).

1.2.1 Structure of alginate

The structure differs from species, age and from different part of seaweed. Alginate consists of two monomers and is linked to each other alternately in a linear form. The monomers are namely β-D-Mannuronate and α-L-Guluronate in Fig 2(B) and 2(D) respectively. The acid form of mannuronate and guluronate, is mannuronic acid and guluronic acid (Fig 2(C)) respectively. Figure 2(A) depicts the structure of sodium alginate in a linear form.

Alginate is the salt of alginic acid and its simplest formula is NaC₆H₇O₆ in Fig 2(A) (Mahmood et al., 2009).

.

2(A)

2(B) 2(C) 2(D)

Figure 2: Repeat unit of (A) Sodium alginate (B) guluronate, (C) guluronic acid and (D) mannuronate (Falkeborg et al., 2014)

The monomers are linked through carbon 1 and 4 and have sequences forming blocks of MM(Fig 3(A)), GG(Fig 3(A)) or GM blocks in Fig 3(A) (Arzate-Vázquez et al., 2012, Morais et al., 2013). Mannuronates and guluronates both have carboxylic group on the carbon 5 of their respective structures (Falkeborg et al., 2014).

(A)

G G M M

(B)

G G M M

GMMMMGGGGGGGMGMGMGMGMMMMG (C)

MM blocks GG blocks MG blocks MM blocks

Figure 3: (A) Alginate structure with G and M repeat units, (B) showing the repeat of G and M joined together (C) block fractions of alginate polymer (Yuemei Lin et al., 2010)

1.3 Species of brown seaweeds

Alginate are commercially manufactured mainly from Laminaria hyperborea, Macrocystis pyrifera, Laminaria digitata, Ascophyllumnodosum, Laminaria japonica, Eclonia maxima, Lessonia nigrescens, Durvillea Antarctica, Turbinaria conoides, Turbianria ornata and Turbinaria decurrens, Sargassum, Turbinaria ,Hormophysa, Cystoseira and Sargassum sp (Bertagnolli et al.,2014; Chennubhotla et al., 2013; Kaliaperumal et al., 1974).

1.4 Extraction of alginate

Gomez et al., (2009) described the pre-treatment and the extraction of alginate from brown seaweed. The seaweed was crushed and was added to water to moisten the latter. 0.1 N Hydrochloric acid was added to the sample and the solution was stirred in order to reach a pH value of 4. This process was continued for 15 min at room temperature and the supernatant was eliminated. The pre-treatment was carried out three times for each sample using 0.1 N Hydrochloric acid.

The extraction was followed by the addition of the pre-treated solution to a beaker with 1 N sodium carbonate (pH of solution 10). The mixture was stirred mechanically for a period of 2 hours at a temperature of 60 ^oC. To the extracted solution a silicon based rock was added and was stirred for 15 minutes. The mixture was then centrifuged to obtain the sodium alginate supernatant.

Sodium alginate can be purified using three different routes namely (i) Ethanol route, (ii) Hydrochloric acid route and (iii) Calcium chloride route as summarised in scheme 1.

(i) Ethanol pathway

The ethanol pathway was performed by the direct precipitation of sodium alginate using ethanol. The precipitate was washed then with ethanol under soxhlet condition for 100 hours to obtain the pure biopolymer which was dried under vacuum.

(ii) Alginic pathway

The extract alginate was mixed up with 1 N of HCl at room temperature under constant stirring for 1 hour at a pH of 1. To obtain the alginic acid, the mixture was separated by centrifugation by adding water and 1 N sodium carbonate. The mixture was left at room temperature under stirring for 1 hour in order to obtain the soluble form of sodium alginate. The polymer was precipitated by the addition of 1:1 volume ratio of ethanol by the method described in the ethanol pathway.

(iii) Calcium chloride pathway

To the extract of sodium alginate, 1 M calcium chloride was added and the precipitate was thoroughly washed with distilled water using a soxhlet for 64 hours. The mixture was agitated at room temperature and distilled water was then added followed by the addition of 1 N Hydrochloric acid until a pH 2 was reached. The alginic acid which is insoluble was separated from the supernatant by centrifugation and the insoluble alginic acid was washed with 0.05 N HCl and the solution were stirred at room temperature. The precipitation of alginate was obtained by the addition of Na₂CO₃ to obtain sodium alginate. The purified alginate was collected by the soxhlet as discussed in the ethanol pathway.

Scheme1. Extraction of alginate (Gomez et al., 2009)

1.5 Yield of alginate

The yield of alginate is being affected by different factors such as the species, age, period of harvest, temperature of extraction, extracted with either alkali or acid and on the content of alginate in brown seaweed also the size of alginate also affects the yield.(Fertah et al., 2014)

Seaweeds that are grown in summer show a lower content of alginate in the branches and stems (15.1 %) and no trace of alginate in the receptacles (Bertagnolli et al., 2014).

Sample SS size is 1mm and BS for 1mmo