**2.1. Isolation, purification and separation by ultra-filtration or by solvent precipitation**

346 The Complex World of Polysaccharides

depth captured by a scuba-diver [3].

conformation [19].

polysaccharides formed the bulk of oceanic DOM [8].

and high molecular weight, present mainly as dissolved or particulate matter. Microscopic (transmission electron and atomic force microscopy) and NMR studies reveal that fibrillar

**Figure 1.** Macroscopic phenomenon of extracellular polysaccharide gellation in the Northern Adriatic Sea: (**a**) remote sensing by satellite showing gel phase in red color (adopted from [2]); and (**b**) at 10 m

Many papers showed that the aldose signatures of marine DOM obtained from different seawater samples around the world is similar to that determined on cultured phytoplankton DOM [9,10] and that the carbohydrate production could be very different among the species selected, growth and environmental conditions [5,7,11-17]. These results are very important in order to understand the role of algal exudation in the aggregation processes observed in all of the seas and in general in carbon cycling in the euphotic zone. Many authors showed that cultured diatoms growth in P-limiting condition determines an increase of polysaccharides

Among the three major classes of biopolymers, the polysaccharides show the greatest chemical and structural variety. The nucleic acids are constructed from a handful of nucleotide bases so that the polymeric structure obtained is invariably linear. The number of amino acid building blocks used to construct the proteins is approximately twenty but, again, the proteins are always linear polymers. On the other hand, polysaccharides display a wide chemical and structural variability that is not found among the polypeptides and polynucleotides mainly due to the multiple hydroxyl functionality of the five- and sixcarbon sugars. The replacement of one or more of such sugar hydroxyl functionalities by amine, ester, carboxylate, phosphate or sulfonate groups, leads to the frequent occurrence of tree-like branching and to the huge number of possible polymeric conformations of different solution behavior. For these reasons carbohydrate analysis involves, after isolation and purification, many steps, i.e. the determination of individual monosaccharides, of anomeric linkages, of branching and sequence, of anomeric configuration and, finally, of the chain

exudated by different diatoms species [4,6,7,11,12,15,17,18] both pelagic and benthic.

**2. Techniques for primary structure characterization** 

In the last two decades the filter (ultra-)fractionation technology has highly improved the methodologies for isolation and purification of polydisperse biopolymers, consisting of macromolecules, like algal polysaccharides, that present very often a large number of size fractions, going from oligomers of few sugar residues up to several million dalton of molecular weight. The tangential-flow filtration (known also as crossflow filtration) is one of the most useful tools for biopolymer separation and purification, both from seawater and culture medium. The principal advantage of this technique is that the residue which can obstruct the filter is substantially washed away during the filtration process by a tangential flow along the surface of the membrane. Depending on the biopolymer to be retained, membrane cut-off ranges from 1 kD to 1000 kD are used.

For large volumes, as seawater samples, a polysulfone multi-fiber system (hollow fiber tangential flow columns) is useful technique for simultaneous dia-filtration and concentration of samples given the large surface area available (on m2 scale).

The addition of a non-solvent (or a bad-solvent for polysaccharides) to a given sample containing dissolved polysaccharides (algal cultures or seawater samples) allows the separation of the carbohydrate fraction by precipitation. This is a very common method for many advantages. It is non-destructive, inexpensive and relatively fast allowing also a fractionation in terms of polysaccharide molecular weight. Cold ethanol, isopranol or acetone are often used and added to the cell-free supernatant of cultures or filtered samples in an appropriate volume to volume ratio (about 4:1). The precipitate is usually re-dissolved in pure water and the solution dialysed exhaustively against EDTA (0.01-0.1 M) and Milli-Q water [6,7,20,21] The precipitation/re-dissolution treatment is commonly performed threefour times depending on the purity to be achieved.

#### **2.2. Gas-chromatography of alditol acetates of neutral monosaccharides**

Although the excretion of photosynthetic compounds is recognised as the major source of carbohydrates in seawater [10], so far there are not many papers reporting on the molecular composition of carbohydrates in the exudates from diatom cultures [see for examples 6- 7,10,22-28]. The molecular-level characterization of dissolved polysaccharides may provide basic information on the origin, the bioreactivity and the fate of these biopolymers. For example, after monosaccharide analysis Hama and Yanagi reported that the turnover rate of dissolved glucose was the highest among dissolved neutral aldoses, while turnover rates of galactose, mannose, xylose, rhamnose and fucose were similar to each other and markedly lower than glucose [29]. This was a significant finding suggesting that the degree of degradability of autotrophic DOM depends mainly on the relative percentage of glucose with respect to other monosaccharides.

Gas-chromatographic (GC) methodologies for neutral monosaccharide analysis used to characterize the primary structure of marine polysaccharides were reported in several papers [5-7,20,30-39]. In general, the methodology is based on the acid hydrolysis of the polysaccharides and the suitable derivatisation of the saccharidic matter in order to obtain volatile compounds [40]. Thus, hydroxyl groups are subjected to chemical modifications obtaining silylated, acetylated, trifluoroacetylated, methylated or ethylated derivatives. Neutral and amino sugars are commonly analyzed after exhaustive hydrolysis with trifluoroacetic acid and the Neeser acetylation method [41]. The Neeser method provides a simple, rapid, and sensitive analytical method, which has been successfully used on glycoproteins and on plant and microbial cell-wall polysaccharide fractions in order to ensure complete release of amino sugars from glycoproteins, together with minimum losses of neutral sugars with an improved derivatization procedure by treatment with CH3ONH2. HCl in pyridine. In addition the occurrence of uronic acids requires a preliminar reduction process with carbodiimide and NaBH4 [42]. The N-acetylated form of amino groups, that often occurs in marine polysaccharides, are removed by hydrolysis.

A very popular method of neutral carbohydrate analysis is the alditol acetate method originally described by Blakeney et al.[43], based on the four-step reaction described as:

**Scheme 1.** Scheme 1

The complete separation of the mixture of neutral and amino sugars is usually obtained on polar capillary column (fused-silica coated with methyl silicone fluid).

As an example, the composition of extracellular polysaccharides produced by marine diatom *Chaetoceros decipiens* at the later exponential growth phase is presented in Figure 2. The exopolysaccharide fraction was isolated and purified by precipitation from bulk solution with isopropanol. The hydrolysis and the gas-chromatographic analysis yielded a suite of six neutral monosaccharides: glucose (glc), galactose (gal), mannose (man), xylose (xyl), rhamnose (rha) and fucose (fuc), present in different amount.

The molar ratio of monosaccharide presented in Table 1 shows rhamnose and fucose as the major components followed by galactose residue. The comparison with the composition of the exopolysaccharides obtained by Myklestad from *Chaetoceros decipiens* culture [5] shows a good agreement even at different growth conditions.


**Table 1.** Relative molar composition of exopolysaccharides from *Chaetoceros decipiens* cultures.

**Figure 2.** Monosaccharide pattern of the exopolysaccharides from cultured diatom *Chaetoceros decipiens* [44].

## **2.3. Anionic chromatography for charged and neutral monosaccharides**

Carbohydrates in seawater include neutral sugars, aminosugars and acidic sugars, mainly uronic acids, phosphorylated and sulphated sugars [45-47]. By using high performance anion exchange chromatography (HPAEC) coupled with pulsed amperometric detector (PAD) [38] the simultaneous analysis of mixture of such substituted carbohydrates is possible after acid hydrolysis and neutralization. Chemical derivatization is not required so that time consuming gas-chromatographic methods for uronic residue analysis are avoided.

Monosaccharides are completely separated by an isocratic elution [48-50] or in addition with a gradient course of two mobile eluent phases [51]. Seawater samples and culture media require a desalting step preceding the acid hydrolysis and the use of membrane dialysis (of about 1kDa) instead of resins is strongly recommended [51,52] avoiding the losses in carbohydrate yield.

#### **2.4. Methylation analysis and mass spectroscopy**

348 The Complex World of Polysaccharides

CH3ONH2.

HO

**Scheme 1.** Scheme 1

OH OH

O CH2OH

(H)OH

papers [5-7,20,30-39]. In general, the methodology is based on the acid hydrolysis of the polysaccharides and the suitable derivatisation of the saccharidic matter in order to obtain volatile compounds [40]. Thus, hydroxyl groups are subjected to chemical modifications obtaining silylated, acetylated, trifluoroacetylated, methylated or ethylated derivatives. Neutral and amino sugars are commonly analyzed after exhaustive hydrolysis with trifluoroacetic acid and the Neeser acetylation method [41]. The Neeser method provides a simple, rapid, and sensitive analytical method, which has been successfully used on glycoproteins and on plant and microbial cell-wall polysaccharide fractions in order to ensure complete release of amino sugars from glycoproteins, together with minimum losses of neutral sugars with an improved derivatization procedure by treatment with

HCl in pyridine. In addition the occurrence of uronic acids requires a preliminar

reduction process with carbodiimide and NaBH4 [42]. The N-acetylated form of amino

A very popular method of neutral carbohydrate analysis is the alditol acetate method originally described by Blakeney et al.[43], based on the four-step reaction described as:

NBH4

H2C OH HC OH HC OH

HC OH

H2COH H2COAc

AcOAc

HC OAc

HC OAc HC OAc H2C OAc

AcO CH

HO CH

The complete separation of the mixture of neutral and amino sugars is usually obtained on

As an example, the composition of extracellular polysaccharides produced by marine diatom *Chaetoceros decipiens* at the later exponential growth phase is presented in Figure 2. The exopolysaccharide fraction was isolated and purified by precipitation from bulk solution with isopropanol. The hydrolysis and the gas-chromatographic analysis yielded a suite of six neutral monosaccharides: glucose (glc), galactose (gal), mannose (man), xylose

The molar ratio of monosaccharide presented in Table 1 shows rhamnose and fucose as the major components followed by galactose residue. The comparison with the composition of the exopolysaccharides obtained by Myklestad from *Chaetoceros decipiens* culture [5] shows a

**Exopolysaccharide molar composition Rha Fuc Xyl Man Gal Glc** 

**Table 1.** Relative molar composition of exopolysaccharides from *Chaetoceros decipiens* cultures.

*Chaetoceros decipiens* 7 7 1 4 5 1 *Chaetoceros decipiens* [5] 7 7 0.5 1 3 0.5

polar capillary column (fused-silica coated with methyl silicone fluid).

(xyl), rhamnose (rha) and fucose (fuc), present in different amount.

good agreement even at different growth conditions.

groups, that often occurs in marine polysaccharides, are removed by hydrolysis.

CHO HC OH

HC OH HC OH H2C OH

HO CH

The most widely used method for determining anomeric linkage structure of a polysaccharidic chain is the methylation analysis. The polysaccharide is partially methylated, then hydrolyzed and the resulted partially methylated monosaccharides are acetylated. These methylated alditol acetate sugars allow to establish which carbons are involved in the anomeric linkage. The advent of combined GC and mass spectrometry allows the identification of monosaccharides and provides linkage information on complex polysaccharides. The methylation reaction is commonly performed using Harris' method [53].

Unpublished data (P. Sist) on axenic culture of *Chaetoceros decipiens* are presented in Table 2. The result of the methylation analysis of the exopolysaccharide allowed to identify the linkages among monosaccharides along the polymeric chain. The results showed that fucose and rhamnose were present mainly as terminal residues (t-Rha and t-Fuc) but a lower percentage of rhamnose (8.3%) was linked in the chain backbone (2-Rha) and 5.4% of fucose



**Table 2.** Methylation derivatives of monosaccharidic units of exopolysaccharide from *Chaetoceros decipiens* culture.

represented branched residues (2,3,4-Fuc). Galactose residues which were linked at carbon 2 and 4 (23.9% of 2-Galp and 4-Galp) resided predominantly in the backbone, while mannose was both a branched residue (2.9% of 2,4-Man and 2,3-Man) and mono-substituted in a linear chain (5.6% of 2-Man and 6-Man).

The relatively high percentage of galactose which could be present as or as anomeric configuration in the chain backbone suggested a possible extended and rigid chain conformation of the *D. decipiens* polysaccharide as also found for model polysaccharides in solution [54,55].
