Application of Immobilized Algae

1.2.1. Production of High-Value Products

Reports showed that immobilization has a significant impact on algal cell productivity. Hydrogen evolution from the filamentous alga Anabaena was reported to increase threefold following immobilization (22,23). Brouers and Hall (23) and Bailliez et al. (24) demonstrated an enhanced production of ammonia and hydrocarbon, respectively, by Mastigocladus laminosus and Botryococcus spp. following immobilization. Santos-Rosa et al. (25) also found that Chlamydomonas reinhardtii cells immobilized in Ba-alginate provide a stable and effective system for photoproduction of ammonia. Glycolate production in alginate-entrapped cells was shown to be double that in the free-living cells (26). Leon and Galvan (27) studied the production of glycerol in C. reinhardtii cells immobilized in Ca-algi-nate. The immobilized cells registered a production rate of 7 g/L in comparison to 4 g/L by their free-living counterparts.

Efficiency of immobilized cells and thylakoid vesicles of the microalga C. reinhardtii CW-15 were compared with its free-living counterparts for production of hydrogen peroxide (28). H2O2 is an efficient and clean fuel used for rocket propulsion, motors, and for heating. This compound is produced by the photosystems in a catalytic cycle in which a redox mediator (Me viologen) is reduced by the electrons obtained from water. Under optimum conditions, the photoproduction, a rate of 33 |mol H2O2/mg Chl./h was maintained for several hours by Ca-alginate entrapped cells with an energy conversion efficiency of 0.25%. As compared with this the immobilized cyanobacteria (Anabaena variabilis and Anacystis nidulans) with poor catalase activity depicted a productivity of 150 and 60 |mol H2O2/mg Chl./h, respectively (29,30).

Rossignol et al. (31) entrapped the benthic diatom, Haslea ostrearia in agar as well as alginate beads, and studied it for marennine production. With identical conditions, the specific productivity of marennine was higher using a photobioreactor with immobilized cells rather than free cells. Based on this study a new photobioreactor has been designed by Lebeau et al. (32) for marennine production (see Fig. 4). Most recently, whole-cell immobilization of the microalgae Botryococcus braunii and B. protuberans in alginate beads under airlift batch culture resulted in significant increase in hydrocarbon production at the resting phase of growth (33).

A large number of reports are published on the use of immobilized microalgae for wastewater N and P removal (i.e., for the tertiary treatment of wastewater). In general, immobilized cells are found more efficient in removing N and P as compared to their free-living counterparts, and removal of phosphate is a much slower process than that observed for nitrogen (15). Further, a gradual decline in efficiency from the first to subsequent cycles was also observed. However, in 1986, Jeanfils and Thomas (34) with alginate-immobilized Scenedesmus obliquus demonstrated that nitrite uptake efficiency was not affected by immobilization, except

Application Immobilized Algae
Fig. 3. Device for an algal optical biosensor. (From ref. 18 with permission.)
Air Lift Pump Photobioreactor
Fig. 4. Photobioreactor used for marennin production. (From ref. 32 with permission.)

that a longer lag phase was observed in immobilized cells than the free ones. Megharaj et al. (35) provided evidence for the insignificant impact of culture age on nitrogen removal. In contrast to this, batch culture studies of phosphate uptake by alginate-entrapped Chlorella emersonii have shown that the exponentially growing cells remove phosphate from the medium five times more rapidly than the cells in the late stationary phase. When cells of different ages are immobilized in Ca-alginate and packed in a small-scale packed-bed reactor the effects of culture age are sufficient to produced significant differences in reactor performance

(36). Nitrogen starvation however, greatly increased the N uptake rate of immobilized S. obliquus (37).

Rai and Mallick (38) demonstrated a higher uptake rate for both N and P by immobilized Chlorella and Anabaena than their free-living counterparts. In subsequent studies Mallick and Rai (39,40) also observed that immobilized algae with a cell density of 0.1 g dry wt/L was the most efficient for nutrient and metal removal in a pH range of 6.0 to 8.0, and chitosan could be a promising algal support for wastewater detoxification. Vilchez and Vega (41) found that alginate-entrapped C. reinhardtii cells provide a stable and functional system for removing nitrogenous contaminants from wastewater. A group of parameters such as matrix concentration, cell loading, temperature, and pH were also considered in order to determine the best working conditions for the immobilized cells. In case of C. reinhardtii cells an alginate concentration of 3% is adequate for minimizing substrate diffusion problems, thereby allowing the beads to attain a physical consistency.

Garbisu et al. (42) studied phosphate removal efficiency of foam-immobilized Phormidium laminosum both in batch and continuous flow fluidized-bed bioreactors. Fluidized-beds were designed as funnel-shaped, column-type beds and beds in Erlenmeyer flasks. The bioreactors were continuously illuminated with cool white fluorescent lamps at an irradiance of 100 ^mol photon m2/s and were operated at 45°C by submerging the lower part of the bioreactor in a thermostatically controlled water bath. Preboiled, washed, and dried 5-mm foam cubes were placed in algal suspension and immobilization by adsorption was continued over 2 mo. Once the foam cubes were fully colonized, they were introduced into the bioreactors. Although cyanobacteria immobilized on the polymer foams did remove N and P from the system efficiently, the fluidized-bed reactors resulted in a heterogeneous system, which is unsuitable for most laboratory standardization. Several workers however, studied removal of N and P in packed-bed reactors. Poor light penetration, nonmixing of cells, and gaseous fluxes are limitations in operation of this type of reactors.

Kaya and Picard (43) developed a novel immobilized algal system for wastewater biotreatment. In this new process the green microalga Scenedesmus bicellularis, isolated from a secondary decantation tank, was grown in a synthetic medium for 12 d, and the cells collected after centrifugation were immobilized on alginate screens. The screens were inserted into a photochamber saturated at 100% relative humidity and a photoperiod of 16 h with an illumination of 150 ^E m2/s (see Fig. 5). After 48 h of nutrient starvation, the immobilized cells were used for the removal of ammonium and orthophosphate from a synthetic secondary wastewater effluent in a flexiglass reactor. In a subsequent study Kaya et al. (44) also demonstrated that intermittent CO2 enrichment accelerates tertiary wastewater treatment. However, one major problem with the use of gel materials is maintaining the integrity of alginate gels beyond a few weeks. Although agarose gel-based reactors showed comparatively more stability than others, these polysaccharide-based matrices are very much prone to microbial attack when placed in natural environment. To overcome these difficulties, Robinson (45) proposed a new type hollow-fiber reactor. Hollow-fiber cartridges are commercially available in a variety of sizes. Robinson (45) prepared the hollow-fiber reactor with 50 cylindrical

Application Immobilisation Enzyme

Fig. 5. Schematic description of wastewater treatment by microalgae immobilized on screens and starved in air, saturated at 100% relative humidity: (1) centrifugation; (2) immobilization; (3) starvation in air saturated at 100% relative humidity; and (4) incubation procedure in wastewater. (From ref. 43 with permission.)

Fig. 5. Schematic description of wastewater treatment by microalgae immobilized on screens and starved in air, saturated at 100% relative humidity: (1) centrifugation; (2) immobilization; (3) starvation in air saturated at 100% relative humidity; and (4) incubation procedure in wastewater. (From ref. 43 with permission.)

tubes of polysulfone, bundled and sealed in a transparent cartridge of 20 cm in length. Each fiber was of 1.1-mm internal diameter (id) and the fiber wall was perforated by numerous pores. Such a cartridge could be operated with algal cells contained within the lumen of each fiber and the nutrients pumped in through the shell space or vice versa. Preliminary results showed that phosphate uptake rate declined within a matter of hours, not resulting from any lack of activity but as a result of biomass settling within the reactor. However, when cells were suspended in a solution of 1% Na-alginate, settlement rates were significantly slow.

Sawayama et al. (46) designed a tubular photobioreactor with the thermophilic cyanobacterium, Phormidium laminosum, immobilized on cellulose hollow fibers. The removal of nitrate and phosphate from wastewater was studied at 43°C by continuously supplying dilute growth medium for 7 d and then secondarily treated sewage for 12 d. The removal of nitrogenous and phosphate ions from secondary-treated sewage were 0.24 and 0.11 mmol/d/L, respectively, under the same conditions. A wastewater purification system using thermophilic cyanobacteria has special advantage since contamination can be avoided because of their ability to tolerate high temperature.

Most recently, de-Bashan et al. (17,47) developed a coimmobilized system (a combination of microalgae, Chlorella vulgaris or Chlorella sorokiniana and a microalga growth-promoting bacterium (MGPB, Azospirillum brasilense strain Cd) to remove nutrients (P and N) from municipal wastewater. A. brasilense significantly enhanced the growth, pigment and lipid content, and cell and population size of both the Chlorella species when coimmobilized in the small alginate beads (47). Coimmobilization of the two microorganisms was found superior for removal of N and P than by the immobilized-microalgae alone, reaching removal of up to 100% ammonium, 15% nitrate, and 36% phosphorus within 6 d (varied with the source of the wastewater), compared to 75% ammonium, 6% nitrate, and 19% phosphorus by the microalgae alone (17), thus showing the potential of coimmobilization of microorganisms in small beads to serve as a treatment system for wastewater.

1.2.3. Metal Removal

One of the main interests for microalgae in biotechnology is focused on their use for heavy metal and radionuclide removal from effluents and wastewaters. In parallel to detoxification, it is also possible to recover valuable elements such as gold, silver and uranium after appropriate treatment of the loaded algal biomass (48).

Several reports are available on the accumulation of metals by immobilized microalgae (Table 2). The most interesting of them is the preparation of "AlgaSORB" by Darnall (50) of New Mexico State University. In this process the algae are packed in a column-shaped matrix of solid silica gel. The process of packing the algae in the solid matrix kills the microorganisms. Nevertheless, their cell walls still provide a plentiful source of binding sites, which can hook heavy metal ions from the solution. Subsequently, the removal of mercury from aqueous solution by a packed-bed reactor (PBR) containing Chlorella emersonii entrapped in alginate and agarose gels was studied (54). Reactors were constructed from chromatography columns packed with 200 gel particles of 4- to 6-mm in diameter. The effects of variation in cell stocking density, influent mercury concentration, and flow rate on mercury removal were investigated. It was found that levels of mercury volatilization could be reduced by using agarose rather than alginate as the immobilizing matrix. Lau et al. (55) designed a laboratory scale algal column reactor with the green microalga Chlorella vulgaris with 75 mL alginate-algal beads and was used to treat 30 mg/L Cu and Ni with a hydraulic retention time (HRT) of 30 min. At the end of loading 4 L metal solution, over 97% of Cu and 91% of Ni were removed from the wastewater. Up-flow was preferred to down-flow in maintaining a constant flow rate.

Torresday-Gardea et al. (56) immobilized the biomass of Synechococcus sp. PCC7942 in silica-polymer and the metal binding ability was studied under continuous flow conditions. Results showed maximum adsorption for Pb followed by Cd, Cu=Ni. 0.2 M HCl was found effective for recovery of the adsorbed metals. Experiments were conducted to determine if many cycles of metal binding/stripping by the immobilized biomass were possible. Cd, Cu, and Ni were found to be sorbed and desorbed three times, whereas for Pb as many as six times was

Table 2

Summary of Literature on Metal Accumulation by Immobilized Algae

Table 2

Summary of Literature on Metal Accumulation by Immobilized Algae

Algal genera

Type of treatment

Immobilizing matrix

Name of the metal

Ref.

Chlorella emersonii

Batch

Alginate

Hg

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