PBR, packed-bed reactor; FBR.fluidized-bed reactor.

PBR, packed-bed reactor; FBR.fluidized-bed reactor.

reported. Travieso et al. (58) compared the metal removal efficiency of Scenedesmus acutus and Chlorella vulgaris immobilized in polyurethane foam and K-carrageenan gel in fluidized- and packed-bed reactors. Immobilized cells are found tolerant to Cd, Cr, and Zn than free cells, thus implying their great possibility for wastewater treatment processes.

In 2000, Singh and Prasad (59) prepared one "AlgaSORB" column taking silica, a polymer matrix (poly-A-xylene-A, A-dicyclohexylethylenediamine dibromide) and a green microalga (i.e., Spirogyra) which showed unique ion selectivity for Cu from a host of diverse metal ions of the waste samples. Effects of flow rate, pH, equilibrium time, and other variables are also standardized. It has favorable kinetics without showing any clumping/clogging/leaching complications of the stationary support. This system has attractive features for application in single-column ion-chromatography with high durability, simplicity, and economy.

A new sorption system of Chlorella sorokiniana immobilized on the biomatrix of a vegetable sponge (Luffa cylindrica) was also tried for removal of Cd and Ni from the contaminated aqueous medium in continuous liquid flow column (61,62). As usual the immobilized Chlorella sorokiniana showed significantly higher efficiency than its free-living counterparts. Metal desorprtion with 0.1 M HCl was 100% and the desorbed immobilized system was found reusable with similar efficiency for the second cycle. This microalga-luffasponge-immobilized-disks (MLIDs) retained about 93% of the initial binding capacity for Ni up to five cycles of reuse. Thus, because of their high efficiency, low cost, simplicity of preparation, and repeated use for several cycles the MLIDs could provide an attractive high-affinity biosorption system for heavy metal removal.

Volesky and Prasetyo (63), however, used a new biosorbent material derived from the marine brown alga, Ascophyllum nodosum, in a packed-bed flow-through column. Removal of Cd was found to reach 99.98% from an effluent containing 10 mg Cd/L. Rai and co-workers (Banaras Hindu University, Varanasi, India) have studied the metal removal efficiency of the bloom-forming cyanobacterium Microcystis. Microcystis is an abundantly occurring nuisance cyanobacterium in many eutrophic ponds and reservoirs of India and other tropical countries. This cyanobactrium occurs in a natural immobilized state due to presence of a capsule or slime layer around the cell. Biosorption was found to be influenced by pH and temperature (64). Interestingly however, heat-killed, formaldehyde-treated, and metabolically inactive (DCMU-treated) Microcystis had the same biosorption potential as the metabolically active cells (65). This suggests that even the dead biomass could be equally useful for metal removal. This group is currently working on the feasibility of using the dried biomass for large-scale application of Microcystis as an "AlgaSORB" for metal accumulation removal.

1.2.4. Biosensor Development

Recent interest in immobilized microalgae in biotechnology is focused on the development of biosensors for detection of hazardous chemicals such as heavy metals and pesticides in the environment. In 2000, Naessens and co-workers (18) developed a biosensor with the microalga Chlorella vulgaris immobilized on a removable membrane and placed in front of the tip of an optical fiber bundle inside a homemade microcell (the detailed procedure for construction of a biosensor is described in Subheadings 2.-4.). The detection of herbicides was based on the kinetic measurement of chlorophyll fluorescence. A continuous culture was set up to produce algal cells in reproducible conditions for measurement optimization. Effects of flow rate, algal density, temperature, and pH on biosensor response to various herbicides were studied. Detection of PSII herbicides was achieved at sub-ppb concentration level (20). However, for detection of heavy metals a biosensor is constructed based on the activity of alkaline phosphatase (AP), present on the external membrane of Chlorella vulgaris (19). It appeared that these conducto-metric biosensors were more sensitive than bioassays to detect low levels of cadmium ions (the detection limit for Cd was 1 ppb). The main advantage of these AP-based biosensors was their high stability, because contrary to enzymatic biosensors they use whole algal cells with APs on their cell walls (21). The enzyme remains in its natural environment which favors long-term stability and reflects the mechanism of toxic inhibition, being therefore of ecological interest.

1.3. Concluding Remarks

Biotechnology of immobilized microalgae, though not a subject of current augmentation, has gained importance in recent years as a result of the development of some new production systems and environmental technologies. It is quite clear that a combination of solar energy utilization and algal immobilization can be advantageously applied in the development of photo-activated systems for the treatment of wastewaters and production of valuable algal products. However, some preliminary reports have indicated that immobilization affects the cell's behavior and reduces productivity (66,67). But recent development in the field of "microencapsulation" has clearly proven the superiority of cell immobilization over the free cells. Under the batch culture of aerobic conditions, the thickness of the capsule membrane and CO2 supply did not affect the growth of microalgae. The cell concentrations of microencapsulated Dunaliella bardawil and Haematococcus pluvialis were found to be five times greater than that of the free cells (68). Based on these results, microencapsulation for the culture of microalgae was suggested as an effective method for the high-division cultivation, and thus higher production. Further, current approach on coimmobilization of microalgae with the bacterium Azospirillum brasilense strain Cd was found to enhance growth and increase synthesis of pigments and lipids in three test Chlorella species (47), thus clearly indicating a significant change in the metabolism of the microalgae in presence of this growth-promoting bacterium. However, this new coimmobilzation system is capable of reducing the nutrient load at a much higher rate than the immoblized algae alone from the regular municipal wastewater (17). Although the removal rate is still under 1 mg/L, this coimmobilization system provides a new approach to the biological removal of nitrogen and phosphorus from wastewater.

Recent developments in the field of biosensor technology with immobilized microalgae, either those detecting the presence of toxic metals by inhibition of alkaline phosphatase activity or those measuring chlorophyll fluorescence for detection of PSII-inhibiting herbicides, are found to be highly sensitive, as detections are observed at sub-ppb level. These chlorophyll fluorescence-/AP-based biosensors deliver fast and reproducible signals that detect some herbicides below regulation concentrations limits. The immobilization method provides inexpen sive, ready-made membranes that are easy to handle, and thus this optical algal biosensor could be used as an early-warning device to monitor the target pollutants in natural waters. For environmental management, these small and quick-answering biosensors can be considered as competitive in situ tools as soon as the effects of pollutants on the system can be reversed, which means the membranes can be reusable after reversion. Studies led on AP activity showed the efficiency of ethylene diamine tetraacetic acid (EDTA) for reversing the action of some heavy metal ions (69,70). However, examining to what extent this is also possible in the complex environmental samples is an important task for future investigation.

2. Materials

2.1. For Coimmobilized Bead Preparation

1. Azospirillum brasilense strain Cd DMS 1843.

2. Axenic Chlorella cultures.

3. Sterile mineral medium (C30; see Note 1).

4. Nutrient broth (see Note 2).

5. Sterile saline solution: 0.85% NaCl solution (w/v).

2.2. For Preparation of Algal Optical Biosensor

1. Chlorella vulgaris strain CCAP 211/12.

2. Glass microfiber filter GF/C, 45.7-mm filter diameter, 1.2 mm pore diameter (Whatman, England, [email protected]).

4. Fluorometer equipped with a microcomputer.

7. 0.1 mMMethylumbelliferoyl phosphate (MUP).

3. Methods

3.1. Procedure for Production of Coimmobilized Beads (17)

1. Cultivation of axenic microalgal cultures in sterile mineral medium.

2. Culture Azospirillum brasilense strain Cd in liquid nutrient broth.

3. Take 100 mL of the axenic microalgal culture containing approx 6 x 106 cells/ mL, wash twice in sterile saline solution (0.85% NaCl), and resuspend in 10 mL of same sterile saline solution.

4. Take 50 mL of A. brasilense culture, wash twice in sterile saline solution, and resuspend in 10 mL of saline solution (the optical density should be adjusted to 1 at 540 nm to have approx 109 CFU/mL).

5. To coimmobilize the two microorganisms in the same bead, mix both the cultures (20 mL volume).

6. Now mix the 20 mL of cultures with 80 mL of a sterile 2% alginate solution and stir slowly for 15 min.

7. Drop the solution from a syringe into a 2% CaCl2 solution and leave the beads for 1 h in the solution for stabilization (see Note 3).

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