Immobilization of Microalgae

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Nirupama Mallick

Summary

Several microalgae synthesize metabolites of great commercial interest. Microalgae also act as filters for wastewater N and P, heavy metals, and xenobiotic compounds. However, the cost-effective harvesting of microalgae is one of the major bottlenecks limiting the microalgal biomass applications. In this context, immobilization of algal cells has been proposed for circumventing the harvest problem as well as retaining the high-value algal biomass for further processing. In recent years, innovative approaches have been employed in the field of coimmobilization and microencapsulation, which have proved the superiority of immobilized cells over the free cells. Further, the development in the field of biosensor technology with immobilized microalgae presents an early warning device to monitor pollutants in natural waters. This chapter reviews the various applications of immobilized microalgae and addresses the specific methods concerning the production of coimmobilized beads and the protocol for construction of optical algal biosensors.

Key Words: Coimmobilization; biosensor; bioreactor; heavy metal; microalgae; N and P removal.

1. Introduction

The commercial use of algae has a long history and many species of algae are being used as food, feed, and as sources of valuable chemicals. For example, several species of the macroalgae Caulerpa, Porphyra, Iridaea, Gigartina, and Ulva are harvested for human food or hydrocolloids (1-3). Cultivation of Porphyra, known as "Nori" in Japan, is one of the largest aquaculture industries in Japan (4). Moreover, the production of agar, carrageenan, and alginic acid from macroalgae are well-established industries. A few microalgae, such as some species of Nostoc and Aphanizomenon, are also harvested from the wild for human consumption (5); however, most commercially used microalgae are cultured. These include Dunaliella for P-carotene and Chlorella and Spirulina for protein production (6,7).

Table 1

Basic Requirements of an Efficient Immobilized Algal System and Properties of an Ideal Matrix for Immobilization

Table 1

Basic Requirements of an Efficient Immobilized Algal System and Properties of an Ideal Matrix for Immobilization

Requirements of an efficient

Properties of an ideal matrix for algal

immobilized algal system

immobilization

Retention of viability

Nontoxicity

Ability to photosynthesize

Phototransparency

High density of cells

Stability in growth medium

Continued productivity

Retention of biomass

Low leakage of cells from matrix

Resistance to disruption by cell growth

As early as 1949, Spoehr and Milner (8) suggested that mass culture of microalgae would help to overcome the global protein shortages. The basis for their optimism was that algae had crude protein content in excess of 50%, and biomass productivity of the order of 25 tons/ha/yr. Moreover, N- and P-rich wastewaters are also viewed as a valuable substrate for cultivation of algae (9). The cultivation of algae in wastewaters offers the combined advantages of treating the wastewaters and simultaneously producing algal biomass, which can further be exploited for protein complements and food additives (for aquaculture, animal and human feed), energies such as biogas and fuels, agriculture (fertilizers and soil conditioners), pharmaceuticals, cosmetics, and other valuable chemicals (10).

One of the major bottlenecks in microalgal biomass applications is harvesting or separation of algal biomass from the treated water discharge. Numerous efforts have been devoted to developing a suitable technology for harvesting microalgae, ranging from simple sand filtration to energy-intensive centrifugation (11-13). Autoflocculation (i.e., self-aggregation by stopping aeration followed by decantation), particularly for cyanobacteria, has also been practiced. Harvesting microalgae with ultrasonic waves or ultrasound has also been experimented by Bosma et al. (14), where the separation process is based on gentle acoustically induced aggregation followed by enhanced sedimentation. In this context, immobilization of algal cells has been proposed for circumventing the harvest problem as well as retaining the high-value algal biomass for further processing. Table 1 summarizes the basic requirements of an efficient immobilized algal system and the properties of an ideal matrix for immobilization.

1.1. Immobilization Techniques

Although several methods are available for immobilization of cells and enzymes, entrapment is by far the most frequently used method for algal immobilization. Several natural (e.g., collagen, agar, agarose, cellulose, alginate, carrageenan) and synthetic (e.g., acrylamide, polyurethane, polyvinyl) polymers are used for this purpose. However, for algal immobilization the most frequently used natural gels are alginate and carrageenan. The gel is generally formed into useful biocatalyst beads by first adding the algal cells as a suspension to an aqueous solution of the

Adhesive Flow Polymerisation Vesel

Fig. 1. Schematic representation of the multinozzle system: (1) double piston pump; (2) sterile barrier; (3) damper; (4) vibrator; (5) membrane of pulsation chamber; (6) concentric split; (7) pulsation chamber; (8) nozzle plate; (9) bypass system; (10) reaction vessel; (11) stirrer, and (12) input hardening solution. (From ref. 16 with permission.)

Fig. 1. Schematic representation of the multinozzle system: (1) double piston pump; (2) sterile barrier; (3) damper; (4) vibrator; (5) membrane of pulsation chamber; (6) concentric split; (7) pulsation chamber; (8) nozzle plate; (9) bypass system; (10) reaction vessel; (11) stirrer, and (12) input hardening solution. (From ref. 16 with permission.)

gelling material. This material is then formed into droplets by forcing it dropwise through a nozzle or orifice to an interacting salt solution. The droplets are subsequently stabilized into biocatalyst beads with the entrapped organisms via polymerization or other types of cross-linking. For example, alginate droplets can be stabilized with divalent ions such as Ca2+, and carrageenan droplets are typically cross-linked with K+. A new multinozzle system with thirteen nozzles for the encapsulation of microorganisms, enzymes, or cells was developed by Brandenberger and Widmer (16) (see Fig. 1). Based on the laminar jet break-up, monodispersed beads of calcium alginate are produced under sterile and reproducible conditions in the range of 0.2 to 1.0 mm. An in situ cleaning of the nozzles is implemented in order to guarantee several batch process cycles and a productivity of up to 5 L/h. Beads were analyzed and showed that the relative difference of the mean diameter of different nozzles was less than 0.3%.

Most recently however, coimmobilization of microalgae with the bacterium Azospirillum brasilense was reported, which was found to improve growth, pigment and lipid contents, and cell and population size of the entrapped microalgae (17). This novel coimmobilized biocatalyst was also found superior for removal of

Flow Chart Enzyme Immobilization
Fig. 2. Flow chart showing methods used to coimmobilize microalgae and MGPB. (From ref. 17 with permission.)

N and P rather than by the immobilized-algae alone. The device for coimmobilization is discussed in Subheadings 2.-4., and the flow chart is presented in Fig. 2.

Since 1990, 50% of all reports dealing with immobilized microalgae have involved their use in wastewater N and P removal, and others on the use of immobilized algae for metal accumulation and removal. Most recently, however, novel conductometic biosensors are developed using immobilized microalgae (1821). These fiber optic biosensors are used for monitoring toxic compounds such as heavy metals, herbicides, etc. in the environment. Developmental procedure for this biosensor is described in Subheadings 2.-4., and the device is shown in Fig. 3.

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