**1. Introduction**

Water scarcity is one of the most serious crises in the world as a result of the uneven distribution of water resources, poor water management and climate change. To cope with this situation, scientists and engineers have been working hard to develop treatment methods of every sort [1–4]. Most of the efforts aim to remove the pollutants in water bodies and to increase water supplies via the reliable reuse of wastewater and efficient desalination of seawater and brackish water [5,6]. In the past few decades, membrane technology has been widely used around the world as a promising technique for water treatment [7–9]. However, membrane fouling still remains one of the obstacles to the successful operation of membrane systems, as it has since the day that membrane filtration was employed [10]. Among all fouling problems, organic fouling has often been reported to be a major type and polysaccharides play a key role in organic fouling. Compared to other organic foulants, such as proteins and humic acid, the chains of polysaccharides are usually much longer and prone to gelling to form gel layer on a membrane surface via interaction of the molecular chain. However, the exact fouling propensities of polysaccharides are not clear, especially the influence of molecular structure on fouling. As a consequence of this, more efforts should be devoted to the investigation of the filtration behaviors of polysaccharides, relating the information of molecular structure with fouling tendencies.

Recently, it has been found that polysaccharides can aggregate together to form three-dimensional networks; i.e., transparent exopolymer particles (TEPs). The role of TEPs in membrane fouling has attracted more and more attention since Berman and Holenberg first pointed out that they may participate in the fouling problems in membrane systems [11]. TEPs are planktonic hydrogels in water environments which are mainly formed by polysaccharides via an abiotic pathway [12]. TEPs possess a high viscosity; thus, they can attach to the membrane surface easily to develop a gel layer [13]. More importantly, it is difficult to remove TEPs from a membrane system. TEPs are deformable; thus, they also can penetrate into membrane pores whose sizes actually are smaller than the dimensions of TEP under pressure. In addition, some of TEPs will break into small pieces at the feed side and then reassemble into larger forms at the filtrate side of membrane at the aid of stream turbulence and divalent cations [14]. Thus, TEPs are dynamic in membrane systems, which poses difficulties in identifying the exact nature of TEP fouling. The effect of TEPs on membrane fouling needs to be further explored.

Alginate is a typical model polysaccharide that is commonly employed in studies of membrane fouling. Alginate is unbranched binary copolymer consisting of b-d-mannopyranuronic acid (M) and a-l-gulopyranuronic acid (G) which combines intoMG-, MM- and GG-blocks in varying proportions [15]. These blocks play different roles in alginate chains; i.e., GG-blocks are responsible for the gel-forming capacity, and MG- and MM-blocks provide flexibility to the chains [16]. Their proportions, distributions and lengths determine the chemical and physical properties of alginate molecules. So far, there are more than 200 kinds of alginates derived from different sources [17] which are different in MG-, MM- and GG-blocks contents [18]. It is reasonable to consider that alginates extracted from different algal sources may behave differently during membrane filtration processes. Since alginate has been commonly used as a model organic foulant in numerous studies of membrane fouling, a new challenge on the interpretation and reliability of filtration data would appear if alginate used in filtration experiments was not well characterized. Therefore, in this study, alginate was directly prepared from dry seaweed using alkaline extraction and then block fractionation was performed. Subsequently, three kinds of alginate blocks were systematically characterized with Fourier transform infrared spectroscopy (FTIR) and field emission scanning electron microscopy (FESEM). Dead-end filtration tests were employed to examine the fouling propensities of MG-, MM- and GG-blocks under the same experimental conditions. In addition, TEP measurement was also performed to evaluate the influence of TEP on membrane filtration.

### **2. Materials and Methods**

### *2.1. Alginate Extraction from Raw Seaweed*

Alginate used in this study was extracted from Fuerji dried seaweed according to the following steps. Seaweed was chopped into small pieces and 5 g of chopped dry seaweed was soaked in 300 mL of 1% formaldehyde (Sigma, City of Saint Louis, MO, USA) for 4 h. After that, seaweed pieces were washed by deionized water (Milli-Q, Burlington, MA, USA) to remove excess formaldehyde residue. Subsequently, rinsed seaweed was soaked in 300 mL of 3% Na2CO3 (Merck, Kenilworth, NJ, USA) solution for 3 h, and alginate was converted to a soluble form so that it could be separated from the

insoluble seaweed residue. After alkaline soaking, the solution became very sticky and a clean cloth was used first to remove the bulk seaweed residue, and that was followed by filtration using filter papers. After careful addition of 50 mL of 10% CaCl2 (Merck, Kenilworth, NJ, USA) into the above alginate solution, wooly cloudlike aggregates formed. The aggregates were quickly transferred to a clean beaker. Ion exchange was employed to convert calcium alginate to sodium alginate. A series of ion exchanges, 20 min each, were carried out to get rid of the Ca2<sup>+</sup> until no white precipitate formed when Na2CO3 was added into the exchanged solution. Finally, an equal volume of ethanol (Merck, Kenilworth, NJ, USA) was added to precipitate sodium alginate.

### *2.2. Block Fractionation of Alginate*

The extracted alginate was fractionated according to the method proposed by Leal et al. as presented in Figure 1 [19]. Firstly, 10 g/L alginate was prepared with Millipore water. The solution was stirred for 2 h for complete dissolution. Then, HCl (Merck, Kenilworth, NJ, USA) was slowly added into the sodium alginate solution with stirring, forming white cloudlike aggregations. The solid-liquid mixture was subsequently heated at 100 ◦C in an oil bath for 0.5 h. After heating, the cloudlike aggregations were partially dissolved and the mixture was centrifuged at 10,000 rpm for 30 min. The supernatant solution was neutralized with 1 M NaOH (Merck, Kenilworth, NJ, USA) and precipitated with an equal volume of ethanol. The white precipitation was separated by another 30 min of centrifugation at 10,000 rpm and freeze-dried (MG-blocks). The insoluble residue from the first centrifugation was re-dissolved in 1 M NaOH and the pH was readjusted to 2.85 by adding 1 M HCl. At pH 2.85, new precipitation developed and centrifugation was used for separation. The soluble fraction was again neutralized with 1 M NaOH, precipitated by ethanol and freeze-dried (MM-blocks). The solid fraction was re-dissolved in 1 M NaOH, neutralized by 1 M HCl, precipitated by ethanol and freeze-dried (GG-blocks).

**Figure 1.** The alginate fractionation procedure employed to obtain MG-, MM- and GG-blocks.

### *2.3. FTIR Spectroscopy of Alginate Blocks*

In order to characterize the alginate blocks, the FTIR spectra of the MG-, MM- and GG-blocks fractionated from the extracted alginate were measured. The alginate blocks obtained from commercial alginate sodium (Wako, Tokyo, Japan) via the same procedure were also measured by FTIR to provide a direct comparison. Sample preparation was done by the FTIR Start Kit and 300 mg of KBr (Merck, Kenilworth, NJ, USA) was used to create the baseline. An amount of 2–4 mg of sample was mixed with 295 mg KBr and ground for a short time in order to mix it thoroughly. Then, the pure KBr disc and alginate-KBr mixture pellets were transferred to the disc holder which was subsequently inserted into the spectrometer. The FTIR spectra were recorded using a PerkinElmer FTIR Spectrum GX 50905 (PerkinElmer, Waltham, MA, USA). A total of 60 scans were taken for both background establishment and sample measurements.

### *2.4. Field Emission Scanning Electron Microscope (FESEM) Observations*

In order to visualize the micro-structures of the alginate, MG-, MM- and GG-blocks we prepared, the powders of them were completely freeze-dried and they were examined by an FESEM (Jeol JSM-7600F, Tokyo, Japan). These powder samples were coated with Pt just before observation to increase electrical conductivity. Besides, the TEPs formed from alginate blocks were also observed by the FESEM. Solutions of MG-, MM- and GG-blocks were gently filtered by the 0.05 μm filters under a pressure below 0.2 bars and then freeze-dried to keep the morphology of TEP. Subsequently, these samples were observed and at least 10 pictures for each sample were recorded. In addition, the fouled membrane pieces were also examined by the FESEM to provide a direct observation of the fouling development of the membrane. Membrane samples were taken from the same location from the fouled membrane and representative images were taken.

### *2.5. TEP Determination*

TEP measurements were carried out for the MG-, MM- and GG-blocks samples. The determination procedure was the same as in our previous study [20,21]. Alcian blue was used to stain the TEP sample and preparation involved dissolving alcian blue 8 GX (0.02%) (Sigma, City of Saint Louis, MO, USA) into 0.06% acetic acid before the tests. To quantify the TEP formed from different alginate block samples, solutions of MG-, MM- and GG-blocks were prepared. The alginate blocks had smaller sizes than a whole molecule chain of alginate; thus, filters with smaller sizes were used to determine the TEP. To look into the detailed size distributions of TEPs formed from alginate blocks, 0.2 μm and 0.05 μm polycarbonate filters (Whatman, Maidstone, UK) were used to conduct the TEP tests. Alginate block solutions were filtered through 0.2 μm and 0.05 μm filters at a constant vacuum of 0.2 bars. The retained TEP on filter was stained by alcian blue solution and subsequently rinsed with 1 mL of Milli-Q water after ~5 s staining. The filter together with stained TEPs were immersed in a beaker with 5 mL of 80% H2SO4 solution for 2 h, and we made sure all alcian blue was dissolved into the sulfuric solution. Finally, the absorbance of sulfuric acid solution was measured with a UV–Vis spectrophotometer (Shimadzu, Kyoto, Japan) at 787 nm wavelength and TEP concentration was expressed as mg gum xanthan equivalent per liter of water (mg Xeq.L<sup>−</sup>1).

### *2.6. Dead-End Filtration Tests*

Standard dead-end filtration, as shown in Figure 2, using nylon membrane with a pore size of 0.2 μm, was chosen to examine the different fouling potentials of the MG-, MM- and GG-blocks. The effective membrane surface area was 11.94 cm2, and the filtration process was driven by compressed nitrogen gas at 1 bar. The mass of the filtrate was recorded at the time interval of 10 s by an electronic balance with data-logging system installed in the connected computer. Feed solution was prepared with 0.01 M NaCl and 0.05 g/L alginate samples of respective blocks. A 20 min long initializing period with 0.01 M NaCl was conducted to make sure that stable performance of the membrane was

achieved. Subsequently, the samples of alginate blocks were filtered and the mass change of the filtrate was recorded.

**Figure 2.** Schematic diagram of the dead-end filtration system used in this study.
