**3. Results and Discussion**

### *3.1. Block Composition of Alginate*

Extracted alginate from the seaweed was fractionated using partial acid hydrolysis. The results showed (Table 1) that 19.2% MG-blocks, 62.4% MM-blocks and 19.3% GG-blocks made up the alginate chains, suggesting that MM-block is the main component of the sodium alginate we extracted. The commercial alginate (Sigma, City of Saint Louis, MO, USA) contained 13.8% MG-blocks, 53.5% MM-blocks and 32.7% GG-blocks, which also indicated that the main component in alginate was MM-blocks. These results were consistent with the literature reports that for most alginates MM-blocks dominated molecular composition [15]. According to the above result, the M/G ratios in the extracted alginate could be calculated to be 2.51, which was consistent with the literature in which M/G ratio was within the range of 0.33–9 [15]. It should be noted that different seaweed species could yield different M/G ratios, even the same species from different seasonal and growth conditions. For example, the M/G values of alginates from *Lessonia vadosa* blades collected at the same locality in winter and spring varied between 0.79 and 0.33 [22]. Therefore, it is of primary importance to pay attention to the chemical composition of the polysaccharide that was subjected to filtration. The alginate used in this study was extracted from the natural seaweed. It provided a different and more natural source for alginate blocks compared to the commercial alginate. In natural water environments, most of the TEPs form from the precursor materials (most of them are polysaccharides) secreted by algae via an abiotic pathway. Therefore, the alginate directly extracted from seaweed would be a good model for the TEP formation and polysaccharide fouling study.

**Table 1.** Molecular composition of extracted and commercial alginate.


### *3.2. FTIR Spectrums of MG-, MM- and GG-Blocks*

Figure 3 shows the respective FTIR spectrums of the MG-, MM- and GG-blocks derived from the seaweed and the commercial alginate. It can be seen that the alginate blocks showed similar absorption peaks to those obtained from commercial alginate. This observation suggested that the alginate we extracted had the same material composition with the commercial one, although all the alginate blocks had more impure peaks than commercial ones. The broad bands centered at wave numbers of 3393 cm<sup>−</sup>1, 3401 cm−<sup>1</sup> and 3381 cm−<sup>1</sup> were assigned to hydrogen bonded O–H stretching vibrations. The weak signals at wavenumbers of 2923 cm<sup>−</sup>1, 2935 cm−<sup>1</sup> and 2939 cm−<sup>1</sup> were due to C–H stretching vibrations and the asymmetric stretching of carboxylate O–C–O vibration contributed to the strong absorptions at 1618 cm<sup>−</sup>1, 1610 cm−<sup>1</sup> and 1615 cm<sup>−</sup>1. According to literature [19,23–25], the band at 1420/1412/1419 cm−<sup>1</sup> was assigned to C–OH deformation vibration with contribution of O–C–O symmetric stretching vibration of carboxylate group. The medium absorption at 1305 cm<sup>−</sup>1, 1303 cm−<sup>1</sup> and 1320 cm−<sup>1</sup> in the three figures, respectively, may be assigned to O–C stretching vibration of carboxylic acid and derivatives [23]. In addition, the medium to strong IR absorption bands at 1200–970 cm−<sup>1</sup> are mainly due to C–C and C–O stretching in the pyranoid ring and C–O–C stretching of glycosidic bonds [25]. An intense absorption in this spectral region is common for all polysaccharides [26,27]. The fingerprint or anomeric region at 950–750 cm−<sup>1</sup> showed three characteristic absorption bands in all polysaccharide standards and alginate polysaccharides. The band at 957/938/949 cm−<sup>1</sup> was assigned to the C–O stretching vibration of uronic acid residues. The one at 886/889/904 cm−<sup>1</sup> was assigned to the C1–H deformation vibration of β-mannuronic acid residues. Finally, the band at 814/820/812 cm cm−<sup>1</sup> is characteristic of mannuronic acid residues [22]. Consequently, alginate was the main polysaccharide extracted from seaweed and the three blocks had the same functional groups as the commercial ones.

**Figure 3.** FTIR spectrum of alginate blocks derived from seaweed, and the commercial ones. (**a**) MG-blocks; (**b**) MM-blocks; (**c**) GG-blocks

### *3.3. The Micro-Structures of the Alginate, MG-, MM- and GG-Blocks*

The dry powders of the alginate extracted from seaweed and the MG-, MM- and GG-blocks fractionated from the alginate were observed under an FESEM. As can be seen in Figure 4, the micro-structures of alginate and alginate blocks shared some similarities and discrepancies. All of the samples showed particle like structures (Figure 4a–d) but the detailed structures were different from each other (Figure 4e–h). These observations indicated that MG-, MM- and GG-blocks would have different chemical and physical properties which should have significant effects on their filtration behaviors.

**Figure 4.** The micro-structures of the alginate, MG-, MM- and GG-blocks derived from the natural seaweed. (**a**) Alginate; (**b**) MG-blocks; (**c**) MM-blocks; (**d**) GG-blocks; (**e**)–(**h**) were the amplificatory observations of (**a**)–(**d**).

### *3.4. The TEP Formation from MG-, MM- and GG-Blocks*

The TEP level formed from the 50 mg/L alginate blocks was measured to quantitatively assess their aggregation ability. To look into the size distribution of the TEP formed from MG-, MM- and GG-blocks, two sizes of filters were employed to conduct the measurements. As observed in Figure 5a, the highest TEP level was recorded in the MM-blocks determined with a 0.05 μm filter, achieving 13.6 mg Xeq/L from the 50 mg/L alginate blocks. The MG- and GG-blocks produced 1.1 and 7.8 mg Xeq/L TEP respectively. This finding revealed that in the water environments, the polysaccharide chains would aggregate through molecule crosslinking instead of existing as single chains even at a low concentration. The TEP they formed via molecule crosslinking was much larger than their molecule size. From the TEP results, it could be found that the extent of molecule crosslinking decreased in the order of MM-blocks > GG-blocks > MG-blocks, which is consistent with the literature [28]. As shown in Figure 5, the highest TEP concentration was observed in the solution of MM-blocks, which was also the main component of alginate, as determined in the alginate fractionation. It revealed that although fractionated from the same alginate source, the MG-, MM- and GG-blocks possessed different abilities in molecular crosslinking due to their discrepancies in chain characteristics [28]. In addition, the TEP detected with a size larger than 0.2 μm was remarkably smaller than the TEP formed with size bigger than 0.05 μm, as shown in Figure 5b. That suggested that the TEPs formed from all alginate blocks were small in size and most of the TEPs had sizes ranging from 0.05 μm to 0.2 μm.

**Figure 5.** The TEP formation from MG-, MM- and GG-blocks. (**a**) TEP measured using 0.05 μm filter and (**b**) TEP measured with 0.2 μm filter.

Besides, the TEPs formed from alginate blocks were gently retained on the 0.05 μm filters to provide a direct observation. As shown in Figure 6, the TEPs seemed like the result of crosslinked alginate blocks and had dimensions larger than a single molecule. TEPs actually were the aggregations of alginate blocks. The formation of TEPs from these alginate blocks would definitely affect their filtration behaviors in membrane filtration process. The intermolecular interactions of foulants was essential in understanding fouling mechanism [29,30] and more efforts should be devoted into exploring the complex crosslinking of various foulants in water environments.

**Figure 6.** The observation of TEPs formed from (**a**) MG-, (**b**) MM- and (**c**) GG-blocks with FESEM (0.05 μm filters).
