Entrapment is one of the most common immobilization methods which consists of capturing the cells in a three-dimensional gel lattice, made of either natural (agar, cellulose, alginate, carrageenan) or synthetic (polyacrylamide, polyurethane, polyvinyl, polypropylene) polymers (de-Bashan and Bashan 2010; Hameed and Ebrahim 2007; Liu et al. 2009). Synthetic polymers are reported to be more stable in wastewater samples than the natural polymers, whereas natural polymers have higher nutrient/product diffusion rates and are more environmentally friendly (de-Bashan and Bashan 2010; Leenen et al. 1996).

Polysaccharide gel-immobilized algal cells have often been used for the removal of nitrate, phosphate, and heavy metal ions from their aqueous environment, in providing an alternative to the current physicochemical wastewater treatment technologies (Bayramoglu et al. 2006). Microalgae cells entrapped within either alginate or carrageenan beads were shown to have sufficient immobilization and significant nutrient removal efficiencies from aqueous environments (Chevalier et al. 2000). Aguilar-May et al. (2007) reported that the immobilization of Syn — echococcus sp. cells in chitosan gels had a positive effect on protecting the cell walls from the toxic effect of high NaOH concentration, with immobilized cells displaying higher growth than their free-cell counterparts.

Alginate beads are one of the most common encapsulation matrices, being an anionic polysaccharide found mostly in the cell walls of brown algae (Andrade et al. 2004). Major advantages of alginate gel are it being nontoxic, easy to process, cost-effective, and transparent and permeable (de-Bashan and Bashan 2010). Despite these advantages, alginate beads have some drawbacks such as not retaining their polymeric structure in the presence of high phosphate concentrations or high content of some cations such as K+ or Mg2+ (Kuu and Polack 1983). Faafeng et al. (1994) observed the degradation of sodium alginate beads, used for the immobilization of Selenastrum capricornutum, after keeping them in polluted wastewater with high phosphorous (P) and nitrogen (N) content for longer than two weeks. This degradation problem can be minimized if the stability of the target gel is enhanced. In this context, Serp et al. (2000) found that the mechanical resistance of alginate beads was doubled after mixing them with chitosan. Japanese konjac flour was also used to increase the stability of chitosan gels during tertiary treatment of wastewaters with high phosphate concentrations (Kaya and Picard 1996). Kuu and Polack (1983) suggested that increasing the gel strength of carrageenan and agar gels by integrating them with polyacrylamide results in a more rigid support for microorganisms.

Most of the entrapment processes have a similar protocol, namely mixing the microalgal suspension with the monomers of the selected polymer, followed by solidification of the resulting algae/polymer mixture by some physical or chemical process such as cross-linking of the monomers of the polymer with di- or multi­valent cations (Cohen 2001; de-Bashan and Bashan 2010). As an illustration, a general procedure for the entrapment of microalgae within alginate beads includes the following steps: (1) mixing of algal suspension with sodium alginate solution, (2) placing the homogenously distributed algae/alginate mixture in a vessel with a small orifice, such as a syringe, (3) gently dripping the mixture from the syringe as small droplets/beads into a cross-linking solution such as calcium chloride, (4) optimizing the time for algae/alginate beads inside the cross-linking solution to form cross-linked/hardened beads, (5) collecting the final algae/alginate beads, and rinsing them with deionized water several times (Smidsrad and Skjak-Brsk 1990). Since a manual dripping process for bead production is not practical for larger scale processes, automated prototypes were also proposed for the mass production of gel beads (de-Bashan and Bashan 2010; Hunik and Tramper 1993).

There are some drawbacks of cellular entrapment due to limitations of the oxygen and/or carbon dioxide transfer from the liquid environment through the immobilization matrix, which would cause difficulties mainly for aerobic micro­organisms (Toda and Sato 1985). Co-immobilization of the target microorganism with microalgal cells has been proposed as an interesting alternative to overcome any oxygen transfer limitations. Since microalgae are capable of generating oxygen from the photolysis of water, they function as ideal oxygen generators for their surrounding microenvironments (Adlercreutz et al. 1982; Chevalier and de la Node 1988). Selected microalgae-bacteria pairs have already been shown to benefit from each other, with microalgal cells generating oxygen and some organic compounds that are assimilated by bacteria. On the other hand, bacteria release some vitamins and phytohormones or provide an additional CO2 source that can enhance the algal growth (de-Bashan et al. 2005; Gonzalez and Bashan 2000; Mouget et al. 1995). Mouget et al. (1995) also found that Pseudomonas diminuta and Pseudomonas vesicularis bacterial cells isolated from the algal cultures of Chlorella sp. and Scenedesmus bicellularis stimulate the growth of those microalgal cells.

Previous attempts to immobilize viable algal cells inside gels faced other limi­tations, as the volume-to-surface ratios of spherical encapsulating materials are usually orders of magnitude larger than that of thin films. As a consequence, algal viability is a concern since the nutrients or reactants have to diffuse far into these materials to reach the algal cells. In order to overcome these problems, several other immobilization matrices have been proposed in the recent literature. Three different

immobilization matrices with different geometries and chemical properties are given in Fig. 2.1.

Algal biofilms are one of the alternatives to overcome the harvesting problems of algae in larger scale processes, where microalgal cells stick to each other on external surfaces (Chevalier et al. 2000; Wuertz et al. 2003). Microorganisms form a biofilm as a response to several factors, such as the cellular recognition of the specific functional groups on the targeted surfaces (Karatan and Watnick 2009). Microorganisms forming a biofilm on a surface secrete extracellular polymeric substance, which is mainly composed of phospholipids, proteins, polysaccharides, and extracellular DNA (Hall-Stoodley et al. 2004; Qureshi et al. 2005). Polystyrene disks (Przytocka-Jusiak et al. 1984), textured steel surfaces (Cao et al. 2009), aluminum disks (Torpey et al. 1971), and polystyrene surfaces (Johnson and Wen

2010) are some examples of biofilm surfaces used for algal growth for the primary application of nutrient removal from wastewaters.

The shape of algal cell composite material has two components, a global geometrical form and the surface detail which determines the texture of the surface, with nanomaterial processing techniques being the useful approaches for creating different shapes, from fibers to spheres and flat membranes (Crandall 1996). Var­ious nanofabrication processes have featured in recent research from the authors’ laboratories, albeit in using more unconventional types of immobilization matrices for the immobilization of Chlorella vulgaris cells, such as electrospun nanofibers (Eroglu et al. 2012), laminar nanomaterials such as graphene and graphene oxide nanosheets (Wahid et al. 2013a, b), microfibers of ionic liquid-treated human hair (Boulos et al. 2013), and magnetic polymer matrix composed of magnetite nano­particles embedded in polyvinylpyrrolidone (Eroglu et al. 2013). Electrospinning processes can create nanofiber mats with high porosities and surface-to-volume ratios and are generated by forcing a charged polymer solution through a very

small-sized nozzle while applying an electrical field (Kelleher and Vacanti 2010). On the other hand, a recently developed vortex fluidic device has been successfully used for the exfoliation of laminar materials within the dynamic thin films formed on the walls of this microfluidic platform (Wahid et al. 2013a, b). Scanning electron microscopic images of different nanomaterial matrices, used for the immobilization of C. vulgaris microalgal cells, are given in Fig. 2.2.