**3. Components and market of alginate**

The cell walls of brown seaweeds have alginates as Ca, Mg and Na salts of alginic acid and are partially responsible for their flexibility.

The long chain of alginic acid consists of two uronic acids, mannuronic acid(M) and its C-5 epimer α-(1 → 4)-L-guluronic acid(G). Most of the chains consists of blocks as (−M-)n, (−G-)n, and (−MG-)n. The strengths of alginates depend on Ca2+ binding, with guluronic acid having a much greater affinity for Ca2+ than mannuronic acid. This effect depends on the more zigzag conformation of polyguluronic acid, which allows Ca2+ to fit into the spaces like eggs in an egg carton. The hold fasts of kelp have stiff guluronate-rich alginate and elastic, mannuronate-rich alginates dominants in the blades [11].

The properties of alginates from various brown seaweeds vary considerably as the arrangement of these M and G units(blocks) in the chain and the overall ratio of M/G is species specific. For example, the low G/M ratio of the alginates obtained from *Saccharina japonica* yields weakly gelling alginates that are only useful in textile printing and paper coating [11].

Composition of the blocks depends on the species being used for extraction and the part of the thallus from which extraction is made. Extraction procedures probably also affect alginate quality. Alginates of one kind or another seem to be present in most species of brown algae but they occur in exploitable quantities (30–45% dry weight) only in the larger kelps and wracks (Laminariales and Fucales). Not all large brown algae have sufficiently large quantities of alginates to merit exploitation, for example, *Sargassum muticum*, an adventive species from Japan that has recently arrived in the Atlantic and Mediterranean, has, when dry, only 16–18% alginates [12].

The uses of alginates are based on several main properties: First, increasing the viscosity of solutions (i.e. thicken) when dissolved in water. Second, forming a gel when a Ca salt is added in order to displace the Na in the alginates. They do not melt by heating so many products can be produced. Third, having the ability to form films of Na or Ca alginates [11]. Forth, using Calcium in the manufacture of beads made by dropping a sodium alginate solution into a bath of calcium sulphate solution, forming spheres of water with a calcium alginate coating. These beads are used to immobilize cells or to deliver specific chemicals [12].

Alginates are generally of yellowish brown to white in color and available in powder, granular, and filamentous form. Areas with high seaweed reserves caused the creation of alginate plants with relatively low labor costs. Some companies produce high-quality alginate (e.g., Norway), some produce alginate for food, printing and textile (Scotland, France), and others have specialized in low-grade production (e.g., China, Chile), whilst yet other companies have specialized in buying low-grade alginates and then purify them. Over the years, this complex situation has led to establish of one factory in Scotland and harvesting of Macrocystis in California [12].

**Alginate market** size was over USD 610 million in 2020 and is likely to grow at a CAGR of over 3.3% between 2021 and 2027 [13]. The global alginate market size is projected to reach USD 1.07 billion by 2028 registering a CAGR of 5.0% [14].

High M alginate market is projected to register over 3% CAGR through 2027. Alginates with a higher ratio of ß-D-mannuronic acid as compared to α-l-guluronic acid are referred to as high M alginates. They offer various benefits, such as high biocompatibility, ability to enhance cytokine production, and immunogenicity, stimulating their incorporation in biomedical science & engineering. Recent research indicate that natural biomaterials offer various benefits over synthetic alternatives, such as remodeling, biodegradability, and biocompatibility, triggering the requirement for high M products [13].

## **4. Application of alginate using microalgae**

Pollutants can be removed via physical, chemical and biological methods. For a typical physical removal process, for example, Mechanical filtration and foam separation are difficult to achieve complete removal of pollutants.

Conventional chemical removal methods such as ultraviolet (UV) light, ozone, neutralization, and precipitation produce many byproducts during the process. Biological treatment is widely used due to its lower cost [15].

Application of microalgae for wastewater treatment and nutrients recovery has been studied for decades. Removal of pollutants from wastewater using conventional treatment involves high energy cost for mechanical aeration to provide oxygen for aerobic digestion system. During this process, the aerobic bacteria rapidly consume the organic matter and convert it into single cell proteins, water, and carbon dioxide. Alternatively, this biological treatment step can be accomplished by growing microalgae in the wastewater.

Microalgae have shown great potential to treat industrial wastes such as petroleum, heavy metals, dyes, and toxic gases [16].

Microalgae can remove color from different dyes by biosorption, bioagulation, and bioconversion mechanisms. The biosorption ability of these microorganisms can be related to the high surface area and high binding tendency during the treatment. Also, the functional groups of microalgal surface such as hydroxyl carboxylate and amino phosphate can accumulate ions or dyes on the surface of the microalgal cell biopolymer [17].

Algal cell walls contain a fibrillary skeleton (cellulose) and an amorphous embedding matrix. The algal cell wall is made up of polysaccharides (alginic acid, chitin, xylan), including functional groups as metal-binding sites. Sulfhydryl, hydroxyl, imidazole, amine, phosphate, sulfate, and carboxyl groups are metal ion binding sites of algal surfaces. The algae uptake metals by bonding metal ions with their surface followed by internalization. Two mechanisms in algal biosorption are: (1) ion exchange: ions present on algal surfaces, such as Ca, Mg, Na, and K are displaced by metal ions, (2) complex formation between metal ions and functional groups. Carboxyl and sulfate groups can link with metal ions by covalent bonding. Biosorption can occur using dead biomass (passive process) and bioaccumulation by live algae (active process) [18].

Microalgae can remove toxic, organic substances through cell uptake. Then, the organic contaminant can be biotransformed and mineraliszd within the cells and providing nutrients and energy for growth [19, 20].

#### *Alginate: Wastewater Treatment DOI: http://dx.doi.org/10.5772/intechopen.110148*

Studies have also shown that removing of heavy metals from certain algae is much higher than from zeolite, activated carbon, and synthetic ion exchange resin. Microalgae are important in the secondary/tertiary treatment of municipal wastewater. They can increase removing of heavy metals, nutrients, and pathogens [18].

The advantages of using microalgae to treat wastewater include: the prevention of sludge handling problems; microalgae used in treating wastewater can also be used as fertilizer [21], the photosynthetic activity of microalgae releases oxygen into the water bodies for use by aquatic organisms and it does not require any carbon source for it to remove nutrients from wastewater [22].

Microalgae can grow in autotrophic and heterotrophic conditions, and biofuels like biodiesel and bioethanol can use as a result of the obtained biomass from the wastewater treatment process [17].

The living cells move independently within the treatment bottles. To ensure uniform cell distribution the application of immobilized cells is a good approach [23].

Despite the low cost and shorter hydraulic retention time (HRT) compared to conventional wastewater treatment, this system has low cell loadings and biomass removal issues. The nature of the cells that are negatively charged and small in size (5–50 μm) prevents cell aggregation [24]. Therefore, the removal of the algal biomass from the treated water requires high energy intensive and costly operation.

Various methods have been used to address the biomass removals such as centrifugation, filtration, flotation, and flocculation. On the other hand, immobilization techniques can be used to solve the biomass harvesting issue and simultaneously retain the high-value biomass for further processing [25] lowers the greenhouse gas emission by sequestering carbon dioxide, requiring less space [17] and improving the quality of the treated wastewater [26]. Additionally, immobilized microalgae can grow in more stable and enhancing cell tolerance to unfavorable conditions including high salinity, extreme temperature, metal toxicity, and pH value [17, 27].

Microalgae immobilization using polymers like alginate, agar, and polyurethane was found to be effective in retaining high initial cell loading and recovering algal biomass without significant input of energy while protecting algal cells from toxic pollutants and predation [25].

In comparison to the other immobilization methods, such as adsorption on a surface, encapsulation, entrapment within a matrix,entrapment of microalgal cells in natural(agar, cellulose, alginate, pectin), and synthetic (polyacrylamide, polyurethane, polyvinyl, polypropylene) gel polymers carriers presents advantages of higher nutrients/products diffusion rates, non- toxic, hydrophilic, more environmentally friendly character, greater stability and produce less hazardous waste upon completion of the process [23] and retention time of cells in the reactor is prolonged, metabolic activity of the cells is maintained for long periods, protection of entrapped microorganism from toxic substances in the wastewater by the polymers, increased nutrient removal is achieved because the population of the microorganisms increase within the polymer, competition of the entrapped microorganism with other microorganism in the wastewater is avoided [22], inert aqueous matrix with high porosity that help preserve the physiological properties and functionality of the encapsulated cells [28], reduces the environmental impact. Besides, beads in nutrient-rich wastewater, can be recovered after cultivation, dry, and used as a soil fertilizer [27].

Since the immobilized beads have a larger size than suspended free cells, a simple method by sieving/netting can be employed to harvest the beads from water without requiring high energy input compared to suspended free cells. Solvents are used to dissolve the beads and then oven-dried (where this process is also done in suspended free cells). Also, the immobilized microalgal cells exhibited higher removal efficiency of pollutants from wastewater as compared to the suspended free cells of microalgal culture [18, 23].

Alginate has been regarded as an excellent polysaccharide for immobilization process due to its mild procedure during the process [29]. Studies have shown that in biosorption of pollutants such as nutrients algal beads with alginate are more absorbent than alginate alone [30].

Generally, sodium alginate is used as a matrix for entrapment of microalgae because it has the ability to organize gel beads in the presence of multivalent cations, like Ca+2. The specific and strong interactions occur between calcium ions and guluronate blocks. During this process, known as the "egg-box" model, the solvent is confined within the spaces of a three-dimensional network, which is connected by cross-linking regions that involve the cooperative association of extended segments of polymer chains [31].

The "diffusion method" is the crosslinking of alginate to form gel beads usually resulting from external gelation with Ca+2. According to this method, an alginate-cell solution is dripped into a solution including Ca+2, like calcium chloride. The Ca+2 ions diffuse from the continuous phase into the internal of the alginate drops and an alginate matrix gell is formed. Cations tend to cross-link the surface of the bead first, bringing the polymer chains closer together to create a less permeable surface.

This results in a highly cross-linked and internal matrix with fewer cross-links [27].

There are several palm-oil mills around the world. Most of them have generated around 800 tones/day of palm oil effluent (POME) resulting from palm oil processing. POME has a high concentration of organic matter identified as chemical oxygen demand (COD) and biochemical oxygen demand (BOD) which are up to 51,000 and 25,000 mg/L, respectively could result in eutrophication of water bodies. Conventional technologies for POME treatment are sometimes still not capable of reducing the level of pollutants. Pollutants present in the wastewater can be assimilated by microalgae that their cell wall are porous which allow free passage of molecules and ions in aqueous solution. These microalgae can be used to produce high-level products such as biodiesel.

In a study, immobilization of the microalgal cells of *Nannochloropsis* sp. using sodium alginate was carried out to enhance the harvesting and efficiency of POME treatment [23]. The microalgal cell culture was mixed with the sodium alginate solution at a ratio of 1:1 (v/v) and extruded using a syringe to form a spherical shape of the beads in CaCl2 solution. The microalgal beads were then left overnight for the hardening stage. The procedure was repeated without the addition of microalgal cells culture with blank sodium alginate bead samples. According to the color of the beads microalgal cells were successfully grown inside the beads (**Figure 1**). The immobilized cells used in the study were more robust against high concentrations of POME, as indicated by a higher microalgal growth cell than the aforementioned suspended free cells of *the alga.*

A noticeable reduction of COD in 9 days was observed, which was 55% for 10% POME (p < 0.05) and 50% for 25% POME (p > 0.05), respectively. The *Nannochloropsis* sp. cells were in the heterotrophic growth metabolism, which consumed the organic matter as a carbon source for growth resulting in the reduction of COD over the treatment period. Organic carbon is one of the important nutrients for building up microalgal cells. Most of the composition of microalgal cells was made up of carbon [32]. These findings indicated that the immobilized cells were very effective in removing COD at 10% POME with initial COD of

#### **Figure 1.**

*Image of blank sodium alginate beads (A), immobilized sodium alginate microalgal cell beads before treatment (B), and immobilized sodium alginate microalgal cell beads after 10% POME treatment [23].*

252.5 mg/L [23]. Suspended free cells of *Chlamydomonas* sp. UKM 6 reduced COD within the range of 8–29% from various concentrations of POME [33], which was lower than immobilized *Nannochloropsis* sp. cells reported in the study of Emparan et al.

According to Maria and Anggraini [34], the cellular respiration of microalgal cells results in organic carbon assimilation process in the growth media. In this process, the organic compound and oxygen were used as electron donor and final electron acceptors, respectively. Cellular respiration was a source of energy for treatment and biosynthesis in dark conditions. The microalgal cells will consume inorganic carbon sources such as CO2 and organic carbon derived from the media for their growth during the mixotrophic culture mode [35].

According to [36], microalga *Nannochloropsis* sp. was immobilized in alginate gel beads and cultivated under optimal conditions that their growth. The immobilized cells were used in phytoremediation of secondary effluent from a palm oil mill and easily recovered by simple sieving method. The immobilized cells contributed to the removal of nitrogen and phosphorus >90% and CO2 mitigation >99%.

In the study by [25], the combination of *Scenedesmus obliquus*, *Chlorella vulgaris,* and *Chlorella sorokiniana* microalgae were co-immobilized with sludge bacteria in alginate beads for treatment of real meat processing wastewater and an annular photobioreactor was designed to facilitate efficient light transmission at high beadsto-wastewater ratios. Results showed that increasing alginate mixture from 1.25% to 2.00% (w/v) extended beads stability in real wastewater from three to seven days. When beads were suspended in wastewater the overshading effect at beads-towastewater ratio of 20% (v/v) was significant and average algal growth (0.17 mg/ bead) was lower than 2% (0.46 mg/bead) and 5% (0.36 mg/bead). Nevertheless, the ratio of 20% (v/v) was preferred as the higher beads count may compensate for the disadvantage, however, the light transmission efficiency needed to be improved. In comparison with the suspended photobioreactor, in improved annular photobioreactor algal growth increased from 1412.6 to 3191.0 mg/L, and removal of chemical oxygen demand (COD) and total nitrogen (TN) was improved from 78.5 to 82.9% and from 68.5 to 84.4% respectively with 89.4% total phosphate (PO43−) being removed. By the end of treatment, 135.9 mg/L TN and 99.2 mg/L total PO43− were removed and it was speculated that most of the nitrogen and phosphorus were removed by algal assimilation rather than the physical adsorption of alginate gel.

Halim and Haron [16] used *C. vulgaris* immobilized in calcium alginate to study the removal efficiency of main nutrients in wastewater such as ammonium and phosphate.

They used synthetic wastewater and concluded that the immobilized cells showed a higher percentage of ammonium and phosphate removal of 83% and 79% respectively, compared to free-suspended cells (76% and 56%). COD removal recorded was 89% and 83% for immobilized cells and free-suspended cells, respectively. Immobilized cells showed better growth than free suspended cells as reported by other studies reported similar observations on cell growth where the cell number increased rapidly once the beads were added into the medium.

The enhanced removal performance can be described based on the chemical interactions between the alginate matrix and the nutrients. The presence of carboxyl group (COO-) attracts cations and the free binding site on the surface of the alginate matrix provides the medium for physical adsorption for both NH4+ and PO4 3− ions before assimilation process occur by the encapsulated algae [37]. The calcium ions associated with alginate gelling can precipitate the PO4 3−-P ions [38]. In this study, the removal of nutrients by the alginate bead alone was reported to be minimal compared to the alginate-immobilized microalgae cells [37].

Even though the contribution of alginate in removing nutrients is evident, the primary mechanism of nutrient removal through microalgae uptake is more significant compared to the chemical and physical processes. Moreover, the utilization of immobilized cells using alginate matrix provides a protective environment against mechanical shear stress and external microorganisms contamination which enhanced the overall nutrients removal performance [16].

Oluwole and colleagues, in 2019, used *C. vulgaris* entrapped in calcium alginate beads for removing ammonium nitrogen from synthetic wastewater for 43 hours. They found that there was a rapid reduction in the concentration of NH4 + in the first 19 hours of treatment using the three beads (20, 40, and 80 mg/L concentrations. There was 86%, 97%, and 93% reduction in ammonium nitrogen at the end of the 43 hours experiment using 20 g/l, 40 g/l, and 80 g/l bead concentrations respectively. Too high and too low concentrations of beads are not very effective. High bead concentration (80 g/l) prevents proper light penetration into the bioreactor due to selfshading of dense beads. The metabolic activity and growth of the cells are thus affected. However, too low bead concentration (20 g/l) implies a low concentration of cells available for the treatment of wastewater.

In the research of Tam and Wong [39], *C. vulgaris* entrapped in calcium alginate as algal beads were employed to remove nutrients (N and P) from simulated settled domestic wastewater. A significantly higher nutrient reduction was found in bioreactors containing algal beads (at concentrations ranging from 4 to 20 beads ml−1 wastewater) than the blank alginate beads (without algae). Complete removal of NH4 + -N and around 95% reduction of PO4 3−-P was achieved within 24 h of treatment in bioreactors having the optimal algal bead concentration (12 beads ml−1, equivalent to 1:3 algal beads: wastewater, v/v).

Chen-Lin et al. [40] also showed that the specific growth rate and maximumNH4 + uptake of the immobilized *Nannochloropsis* sp. cells in calcium alginate bead were markedly higher than free cells.

Some research showed that algal-bacterial symbiotic system (ABSS) can remove pollutants from wastewaters higher than single algal or bacterial cultures. The algae consume CO2, nitrogen and phosphate, and other nutrients in wastewater and release O2 required for aerobic bacteria to grow and reproduce [15].

There are some examples of the removal of pollutants by algal immobilization in alginate beads in **Table 1**.
