Algae Biotechnology: Features, Techniques and Achievements

Read this article to learn about the features, techniques and some major achievements of algae with biotechnology.

Beneficial Features and Suitability of Algae:

Algae present a diversity of form, chemistry, genetic complexity and range of habitats that are exploitable and adaptable to commercial processes.

Distinct benefits are derived from joining Phycology with technology.

The Features of Algae that makes it a Suitable System for Biotechnology are as follows:

(i) For industrial biosynthesis of specific compounds, algae feature high rates of growth and natural products chemistry distinct from other organisms.

(ii) Many natural products, including vitamins, organic acids, polypeptides and polysaccharides are secreted by algae. Algal cells need not be free floating but may be immobilized on surfaces where they can serve as biocatalyst.

(iii) Algae rarely produce effective quantities of toxins and many species of algae are edible.

(iv) The life-cycle of many algal species can be controlled.

(v) Algal strains are genetically stable and are likely to perform more accurately than bacteria in many processes. For production of proteins, algae provide subunit assembly. Correct folding and clipping to form the furnished product.

(vi) Pigmentation can be controlled in some species of algae.

(vii) Harvest methods for large algal forms are often much simpler than those for bacteria or yeasts.

(viii) In general, the freshwater to highly saline culture media and phototrophic or heterotrophic bioprocessing methods used for algae are far less complex than those required for mammalian or vascular plant tissue culture. Microalgae cultured in fermentation vessels show doubling times as rapid as four hours and with dry weight densities up to 40 to 50 grams per liter.

Techniques Involved in Algae Biotechnology:

Techniques specific to plant biotechnology are applicable to algae. Most algal biotechnological research and applications will be involved in developing new products or algae that will synthesize commercial products competitively.

A wide Range Methods are Available for Algae System:

1. Production of protoplasts in algae is relatively simple in some forms and difficult in others. Protoplasts of Chlamydomonas are useful tools for investigation of vegetative plant cell fusions. Enzymes utilized for protoplast isolation in seaweeds are cellulase, macerozyme, agarase, ablone acetone powder and papain.

These enzymes are used in various combinations. Grateloupia sparsa is a carrageenan producing seaweed. The tissue was incubated with cellulase, agarase and papain. Purified protoplast pellets were cultured in Provasolis enriched seawater medium at proper temperature and light. Cell wall was resynthesised and discs were formed with marginal meristematic tissue one cell layer thick. Discs regenerated leafy thalli.

2. Flowcytometry can be utilized to isolate desirable cells for regeneration to whole thalli. Monoclonal antibodies to specific algal biochemicals may be used to enhance the sensitivity of cell sorting, especially where the detection of small quantities is necessary. Flowcytometry is also useful for isolating unialgal cultures, selecting high-quality wild strains, sorting organelles and selecting nuclear characteristics.

3. Propagation of commercially important mariculture crops will increase in efficiency through use of clonal regenerants of tissue culture. The isolates that are necessary for establishment of protoplast or callus cultures provide an excellent foundation for maintenance of pure stocks and gene banks. In addition to the commercial importance of the genebanks, the mass propagation potential of tissue cultures may be important for ecological reconstruction.

4. The bioprocessing methods for transformed algae are vast and may result in such systems as Spirulina or Chlamydomonas reactors producing enzymes or hormones or other chemicals of value.

5. Accumulation of elemental gold and uranium on lyophilized preparations of Chlorella vulgaris was demonstrated with building capacity exceeding that of other microorganisms.

6. DNA Transfer Techniques that have been used in Algae include:

(i) Bombardment of cells with DNA-coated gold particles fired by a biolistic device (gene gun),

(ii) Microinjection—transfer of DNA into cells via fine glass needles,

(iii) Electroporation—the use of electrical charge to temporarily open pores in the cell membrane and agitation of wall-less cells with DNA and glass beads or silicon carbide whiskers (Steven and Purton 1997),

(iv) Recombinant viruses (Henry and Meints, 1994) and plasmid vectors represent two additional means of transferring DNA. Either foreign or naturally occurring (endogenous) plasmids, isolated from cells of the organism to be transformed, can be modified for use in transforming algal DNA. Cyanobacterial plasmids have been used to construct plasmid vectors, which have been successfully employed to stably integrate prokaryotic and eukaryotic genes into cyanobacterial cells. Heterologous promoters are used in case of diatoms. Homologous promoters were effective in transformation of the carageenan-producing red alga Kappaphycus alvarezii, the red alga Porphyra miniata and the green seaweed Ulva lactuca (Graham and Wilcox, 2000).

A number of reporter genes have been used in algae, but those coding for antibiotic resistance, commonly used in transforming other organisms are avoided because several group of algae are naturally resistant to these compounds. The gene ARS, which encodes the enzyme arylsulfatase and is normally expressed only under sulfur starvation, causes transformed algal cells to develop a coloured product that is easily detected when it is used as a marker (Stevens and Purton, 1997).

7. Agrobacterium-mediated stable genetic transformation has been reported in marine red alga Porphyra yezoensis (Cheney et al. 2001). Amongst marine algae, stable genetic transformation has only been accomplished in a few unicellular species (a few diatom and dinoflagellate species). Porphyra, known as Nori, is one of the most widely eaten and cultivated seaweed in the world, as well as being one of the most ancient seaweeds and multicellular eukaryotes that exist today.

The method here is unusual in that it utilizes Agrobacterium tumefaciens that has not heretofore been used to transform any alga or any marine plant. Transformation has been confirmed using both GUS and GFP reporter genes coupled to either a heterologous cauliflower mosaic virus 35 S promoter gene or one of two homologous reporter genes that have been tested (RPBL & GAPDH). Transgene expression has been observed in primary transformants for more than eight months periods as well as in progeny through T1 and T2 generations. This gene transfer system permits for the first time the study of red algal promoter gene structure and function.

8. In the past 20 years, the use of immobilized enzymes or cell components for the production of a series of metabolites has become a branch of biotechnology of rapidly growing importance. In the initial stage most of the work on immobilization dealt with systems designed for the release of products, synthesized by enzymes or multi-enzyme complexes.

A more recent development focuses on the immobilization of complete cells or cell agglomerates. To a certain extent these systems resemble natural environmental conditions as many microorganisms grow in a biotope where they are also immobilized by encapsulation in slimes or as a partner of symbiotic systems.

A number of work on immobilization are being carried out with plant cells, algae, cyanobacteria and photosynthetic bacteria. The phototrophic microbes offer several prospects for use in immobilization techniques because they can use sunlight as their sole, or major, energy source to make products from the substrates of photosynthesis.

For the immobilization of algae in polyurethane foams, small cubes of foam are cut and washed three to five times in distilled water for a few days. About one cube per milliliter of algal growth medium is placed into the flask containing the sterilized culture medium, inoculated with algae. Cubes may be removed, rinsed and aseptically re-suspended with fresh medium.

For immobilization of microalgae in carrageenan, one part of algae suspension is mixed with three parts of carrageenan solutions at 38°C to obtain a final concentration of 2.5% carrageenan. The mixture is dropped into a gently stirred 2% KC1 solution at 20°C, where the beads are soaked for 30 minutes to increase their stability. Agar is another natural compound that has occasionally been used for algal immobilization, performed as follows: Equal amounts of Tris-HCl buffer containing 4% agar, and algae suspended in Tris-HCl buffer, are mixed and immediately cooled to room temperature. The solidified gel is cut into small pieces. These methods have been described by Becker (1994).

9. High efficiency transformation of C. reinhardtii by electroporation: A high efficiency method has been established for transforming C. reinhardtii by electroporation. Electroporation of strain CC3395 and CC 425, cell-wall less mutants devoid of arginine succinate lyase (encoded by ARG7), in the presence of the plasmid pJD 67 (which contain ARG 7) was used to optimize conditions for the introduction of exogenous DNA (Shimogawara et al. 1998).

The conditions that were varied included osmolarity, temperature, concentration of exogenous DNA, voltage and capacitance. Following optimization, the maximum transformation frequency obtained was 2 ∞ 10³ trans formants per µg of DNA. This frequency is two order of magnitude higher than obtained with the current standard method using glass beads to introduce exogenous DNA.

10. One of the latest techniques of transforming plants is via subcellular organelles such as chloroplast. The limitations of nuclear gene transformation methods necessitated deriving a technique for introduction of foreign genes at specific locations on a particular chromosome; also needed was a technique to introduce block of genes conditioning a character such as nitrogen or carbon-dioxide fixation. Plastids (compared to nuclear-cytoplasmic compartment) provide more favourable environments for certain biochemical reactions and for accumulating large amounts of some gene and enzyme products.

In addition, this organelle has the great advantage of having a smaller genomic size. Chloroplasts with 60 copies of a single circular chromosome have only 120-150 genes. Researchers have confirmed the expression of DNA after it was injected directly into individual chloroplasts in the photosynthetic tobacco leaf cells.

Since many chloroplast genes occur in operons it may be possible to introduce blocks of foreign genes into a single operon. Furthermore, since the same kind of promoter is involved in transcription, location of foreign gene in any position of the circular chloroplast chromosome will not make much difference in its expression.

Another advantage is that the chloroplasts are transmitted via ovules and there will be no pollen from transgenic to pass on the foreign gene(s) to nearby plants. It will now be possible to bioengineer chloroplasts for N₂-fixation. The present difficulty that prevents this is the sensitivity of nitrogenize to oxygen which is released as a result of photosynthesis.

This problem may be solved by deriving a method which cyanobacteria have adopted by keeping the two processes, photosynthesis and N₂-fixation separate temporally by means of heterocyst. Engineering of plants capable of fixing their own nitrogen is an extremely complex task requiring the coordinated and regulated expression of 16 nif-genes in an appropriate cellular location.

In plastids a favourable environment may be created provided the nitrogenize can be protected. Photosynthesis could be temporally separated from N₂-fixation in chloroplasts by restricting nitrogenase synthesis to dark period.

Some Major Achievements in the Field of Algal Biotechnology:

1. Biotechnology Involving Cyanobacteria:

Most achievements in algal genetic engineering have been achieved with cyanobacteria, because of their relatively simple genetic structure and economic importance.

There are Two Strategies for Gene Cloning in Cyanobacteria:

(a) Use of shuttle plasmid vector, that carry both cyanobacterial and E. coli replicon. The cloned gene can be maintained on the autonomous plasmid or integrated into the cyanobacterial chromosome.

(b) The other strategy exploits the efficient recombination system of Anacystis nidulans for a direct integration of the cloned gene into the chromosome.

To Mobilize Genetic Material into Cyanobacterium the Target Cyanobacterium should be:

(i) Transformable by exogenous DNA,

(ii) Have no host restriction,

(iii) Be Rec^– when stabilization of autonomous vector is required,

(iv) Allow the generation of meroploid,

(v) Express genetic markers,

(vi) Recognize regulatory elements required for transcriptional and translational activities (Cohen and Gurevitz, 1992).

A simple method to introduce foreign DNA into the chromosome of unicellular cyanobacteria was developed by Williams and Szalay (1983). They constructed chimeric DNA in an E. coli vector consisting of a DNA fragment derived from Synechococcus PCC 7942 that had been interrupted by the foreign (Donor) DNA fragment aimed for introduction into the cyanobacterium.

This construct could propagate in E. coli and transform cyanobacteria. The transformation was achieved via double recombination between homologous chromosome and plasmid DNA sequences. This allows site directed insertion of foreign DNA into the cyanobacterial chromosome.

(i) Genetic Improvement of N₂-Fixing Cyanobacteria:

In order to achieve enhanced grain yields the modern agricultural fields are chemicalized with agronomically recommended high doses of synthetic nitrogenous fertilizers and herbicides caring least for the adverse effect these may exert on the soil microflora of naturally—occurring nitrogen-fixing cyanobacterium. Presence of fixed nitrogen in the soil repress the nitrogenase synthesis by the cyanobacterium. Herbicides injure the cyanobacterial photosynthesis as these are designed to be either photosynthetic inhibitors or photosynthetic energy deviators.

Under the prevailing situation synthetic N-fertilizer is essentially required which, unfortunately, on the other hand represses the N₂-fixing activity of the surviving cyanobacteria. A unified concept that evolved, therefore, during the last 1-2 decades signifies the essentiality to develop NH₄⁺ derepressible herbicide resistant cyanobacterial mutants for algalization of the wet agricultural fields with the improved cyanobacterial strains to be a viable and efficient photobio-N fertilizer (Vaishampayan et al. 1996).

Favourably, the high genetic elasticity of some of the N₂-fixing cyanobacterial taxa, their response to physical and chemical mutagenesis have helped their genetic improvement in relation to nitrogenase derepression and herbicide resistance for their prospective use in economical rice culture.

Unusual strains of Anabaena that fix nitrogen at higher than usual environmental levels of combined nitrogen may be useful as study organisms in discovering ways to deactivate the regulatory switch that normally inhibits expression of nitrogen-fixation genes. Efforts to achieve herbicide resistance in cyanobacteria are focused upon studies of mutations in the photosynthetic protein D-1 (encoded by gene psbA), because a point mutation in amino acid “264” of the homologous protein in higher plants is associated with increased herbicide resistance.

The process of identifying such mutants involves treating cyanobacterial cells with a mutagenizing agent or using other methods for randomly “knocking out” gene function. The pool of resulting mutants or potential “knock outs” is then screened for the desired phenotype.

In field studies it has been found that ammonium N-fertilizers at concentration as low as 0.0002 mM markedly repressed nitrogen fixation by cyanobacteria. Combined N inhibition of N₂-fixation is generally thought to result from effects exerted at the level of protein synthesis involving repression of nitrogenase or at least some proteins of the N₂-fixing system. Protons (H₂ evolution) and N₂ are not the only substrates that can be reduced by nitrogenase. Many low molecular weight compounds, e.g., acetylene, azide, cyanide can be reduced by nitrogenase.

The two nitrogenase substrates, i.e., azide and cyanide, have been tested as possible selective reagents to either isolate nif mutants or mutants derepressed for nitrogenase synthesis with the excess of NH₄⁺. In the selection of derepressed mutants, the wild type would be killed on cyanide or azide supplemented medium with NH₄⁺ as the N source.

A derepressed mutant is, therefore, forced to evolve nitrogenase even in NH₄⁺ medium in order to detoxify azide or cyanide by reducing it to NH₄⁺. This is the technique preliminarily employed in the production of derepressed mutants of Nostoc muscorum and an isolate of Anabaena azollae.

A mutation for carbendazine and amitrole resistance may have adversely affected NH₄⁺ assimilating enzyme system as much as to inactivate the control factor for the repression of heterocyst formation and N₂ fixation. The mutants have shown liberation of the unassimilated NH₄⁺ in the exogenous medium at a nearly doubly enhanced rate as compared to the parents.

This is the reason how rice culture with this genetically improved derepressed cyanobacterial mutant has been 40-50% more beneficial as compared to the results obtained with the wild type cyanobacterial inoculation in case of both normal- yielding and high-yielding rice varieties.

Cyanobacteria are apparently most suitable organisms to study the effects of herbicides on photosynthesis, in view of their ‘higher-plant-type’ typical oxygenic photosynthesis on one hand and ‘bacteria type’ prokaryotic gene organization on the other, whereby results on herbicide-cyanobacterial interactions may be extrapolated for the plant system. Monuron and diuron, the phenylurea herbicides have been toxic to cyanobacterial photosynthesis.

Following genetic improvement through mutagenesis, conferring resistance of an agro-chemical, to a N₂-fixing cyanobacterium, two classes of Nostoc muscorum and one class of Nostoc linckia mutant, resistant to a copper fungicide, blitox were isolated. These mutated cyanobacteria were proved to be resistant to heavy metals for repeated cell generations making first report of heavy metal resistance in cyanobacteria.

The isolation of mutants resistant to heavy metals used as pesticides/herbicides for uninterrupted nitrogen fixation in the treated fields. Mutants resistant to a non-essential heavy metal compound, mercuric chloride—a widely used fungicide—and two organo-pesticides— carbendazine and amitrole—were isolated. Of these, the latter two were found to be doubly advantageous in view of their derepressed N₂ fixation in a combined medium.

(ii) Establishing artificial functional association:

Tremendous success has been achieved in establishing artificial functional association between plant seedlings/calli/ protoplast cultures and N₂ fixing cyanobacterial filaments through the application of basic tissue culture technique. Nostoc muscorum, Anabaena variabilis, Plectonema boryanum and Calothrix elenkinii are among the few N₂ fixing cyanobacterial species successfully practised in these studies, using Nicotiana tabacum, Panex ginseng, Medicago sativa, Daucus carota as the hosts.

These studies provide reasonable scope to use pesticide- resistant cyanobacteria as the endophytic inoculants counterselected with rice protoplasts/ pollen cultures/calli as the macrosymbiotic partner for this purpose.

The resultant associative complex is expected not only to thrive in the pesticide-treated fields, but also to internally generate enough NH₄⁺ through biological N₂ fixation in close proximity to the developing seedlings/regenerant of the grain crop for its absolute targetted use. Herbicide-resistant gene or group of genes may be transferred from cyanobacteria to the chloroplasts of cereal plants, such as rice. The latter are grown in haploid state through tissue culture or the genes could be transferred to isolated protoplasts.

(iii) The coding sequence for Saccharomyces cerevisiae copper metallothionein (CUPI) was transformed and integrated into the genome of Synechococcus. The integrated CUPI gene was transcribed and produced a protein product with the metallothionein characteristics in the transgenic cyanobacterium and this property could be used for metal recovery and bioremediation of aquatic environment.

(iv) Biological Insecticide:

Various proteins have been identified as possessing insecticidal activities—α-amylase, lectins and insecticidal proteins from Bacillus sp. (most potent). Use of transgenic cyanobacterium to control insects is a new technology. Larvicidal activity is due to production of an endotoxin. δ-endotoxin genes were cloned into cyanobacteria which inhabit the breeding zone and are used by the larvae as food. Various combinations of the genes cry ivA, cry ivD from B. thuringensis were introduced into Anabaena which displayed toxicity against thirdinstar larvae of Aedes aegypti.

(v) Transfer of Important Genes:

Some of the important genes like cat (production of chloramphenicol), lac Z (acetyl transferase), luc (β-galactosidase), lux A & B (luciferase) have been transferred into the cyanobacterial cells. Schmetter et al. transferred the luc genes from Vibrio harveyi into Anabaena. The contranscribed lux A & B genes from V. harveyi & V. fischeri have been transferred into Anabaena. Natural occurrence of two herbicide resistance genes have been transferred from Gloeocapsa to Nostoc muscorum.

(vi) Gamma-L inolenic Acid:

The richest source of Gamma-linolenic acid (GLA) is the cyanobacterioum Spirulina platensis. In the strain BP of this species, the GLA content increased from 1.2 to 1.6% when cultivated under light-dark (12h/12h) cycles. Cultivation under light-dark condition was found to be preferable to cultivation under continued light.

The GLA level reached 2.4% in the derived mutant Z19 when cultivated outdoor. This is the highest level reported in any alga. Molecular cloning of desaturases of Spirulina is currently underway to produce bacteria with 45% GLA.

(vii) A cyanobacterial delta-6-desaturase gene has been expressed in a transgenic plant. D6D gene catalyses the production of both GLA and Stearidonic acid (SA) from LA (linolenic acid). SA is used in the production of oil films, special waxes and plastics and is not produced in any significant amount in any oil seed. Transgenic tobacco plants carrying the D6D gene were produced and GLA and SA accumulated at low levels in the leaves.

No significant amounts of GLA or SA were detected in seeds of transgenic tobacco plants containing D6D constructs. It is suggested that further research to improve GLA and SA accumulation in seed storage lipids could involve the use of tissue specific promoters, limiting of competing activities and possibly better control of compartmentalization to allow more effective targeting of fatty acids in seed oils (Muruta et al. 1996).

(viii) The gene crt O, encoding beta-C-4 oxygenase which converts β-carotene to canthaxanthin was cloned from the green alga Haematococcus plurialis and transferred crt O to Synechococcus PCC 7942. This cyanobacterium contains a β-carotene hydroxylase gene and normally accumulates β-carotene and zeaxanthin.

The genetically engineered cyanobacterium produced astaxanthin as well as other ketocarotenoids. The results confirm that crt O can function in cyanobacteria in conjunction with the intrinsic carotenoid enzymes to produce astaxanthin. These results provide the first evidence of genetic manipulation of a plant-type carotenoid byosynthesis pathway towards the production of novel carotenoids (Hasker et al. 2001).

(ix) Transgenic Arabidopsis thaliana and Nicotiana tabacum plants that express ict B, a gene involved in HCO₃^– accumulation within the cyanobacterium Synechococcus sp. PCC 7942, exhibited significantly faster photosynthetic rates than the wild types under limiting—but not under saturating—CO₂ concentrations. Under conditions of low relative humidity, growth of the transgenic A. thaliana plants was considerably faster than the wild-type.

This enhancement of growth was not observed under humid conditions. There was no difference in the amount of Rubisco detected in the wild-types and their respective transgenic plants. Following activation in vitro, the activities of Rubisco from either low or high humidity grown transgenic plants were similar to those observed in the wild- types.

In contrast, the in vivo Rubisco activity, i.e. without prior activation, in plants grown under low humidity was considerably higher in ict B expressing plants than in their wild- types, suggesting that the concentration of CO₂ in close proximity to Rubisco was higher. This may explain the higher activation level of Rubisco and enhanced photosynthetic activities and growth in the transgenic plants. These data indicated a potential use of ict B for the stimulation of crop yield (Lieman-Huruitz et al. 2001).

(x) Polyhydroxyalkanoets (PHAs) are among the most investigated biodegradable polymers in recent years. PHAs are superior to other biodegradables because of the large number of different monomer constituents that can be incorporated. PHAs are produced by various microorganisms.

Many of these bacteria when fed with suitable carbon sources can produce PHAs upto about 30% the cell dry weight (CDW). Some can accumulate up to 90% of CDW. In cyanobacteria PHA content is usually about 5% of CDW. Sudesh et al. (2001) investigated various aspects of PHA biosynthesis in the cyanobacterium Synechoystis PCC 6803. PHA biosynthesis can be improved by introducing multicopies of heterologous PHA synthase gene.

A staining and freezefracture electron microscopy revealed the presence of many PHA inclusions in the cell cytoplasm. Based on the sizes and number of these inclusions, the amount of PHAs produced by cyanobacteria is comparable to that produced by most bacteria. This investigation shows that the PHA synthesizing ability of cyanobacteria may in fact be quite similar to that by most bacteria in nature (Sudesh et al. 2001).

2. Biotechnological Application in Other Groups of Algae:

(i) Immobilization of Algal Cells:

The Major Fields Envisaged for the Utilization of Immobilized Algae are as follows:

(a) Accumulation and removal of waste products in aqueous systems,

(b) biosynthesis and biotransformation of different natural products such as polysaccharides, enzymes etc.,

(c) production of ammonia,

(d) production of photosynthetic oxygen in combined bacteria-algae systems,

(e) production of hydrogen.

It is well known that several algal species develop impressive capabilities of accumulating certain compounds, for instance heavy metals but also nitrogen (in the form of ammonia) and phosphorus (as orthophosphate), from the environment.

It can be shown that species of Scenedesmus, immobilized in carrageenan. are able to remove within a few hours high percentages of phosphate and ammonia from typical urban secondary effluents at a similar rate to free living cells. Similar results were obtained on the removal of heavy metals in industrial effluents, where the immobilizing matrix seems to protect the algae to a certain degree against toxic effects of the metal ions.

Most of the studies on immobilized algae deal with their use for biosynthesis and transformation of valuable biological compounds such as enzymes, polysaccharides, NADPH, amino acids and hydrocarbons. Glycolic acid production is a characteristic of all plants that fix CO₂ via the Calvin cycle. Studies on free-living algal cells have demonstrated that the excretion of this compound occurs widely among photosynthetic organisms.

In Chlorella cells immobilized in Calcium alginate gel, glycolate production could be maintained over a period of six months. Other research groups have demonstarted the photoproduction of NADP by Nostoc muscorum, immobilized in polyurethane foam, the continuous production of amino acids over 10 days by different mutants of cyanobacteria, encapsulated in alginate, the long term release of sulfated polysaccharides from polyurethane-entrapped Porphyridium cruentum or the production of hydrocarbons by Botryococcus braunii. Dunaliella sp. is a marine microalga well-known for its potential to produce glycerol and β-carotene.

Reported results suggest significant release of glycerol by this alga depending on parameters such as temperature, light intensity and salinity. Studies on Dunaliella tertiolecta, immobilized in Ca-alginate beads, showed to produce significant amounts of glycerol, even in hypersaline media (up to 4M NaCl) over a period of several months. In addition to the metabolites mentioned above, the production of other compounds such as pigments, vitamins, growth stimulants and antibiotics are projected as potential products from immobilized algae.

Another application of immobilized algae is the production of ammonia. Its production by N₂-fixing cyanobacteria, immobilized in alginate matrix, was described by using either inactivation of glutamine synthetase by L-methionine-D, L-sulfoximine deficient strains, showing a yield of 40% of fixed nitrogen excreted as ammonia (Kanaiyan, 2001).

Production of oxygen for 25 days was reported for Chlorella vulgaris and Scenedesmus obliquus after immobilization in urethane prepolymer, and a period of over six months for Chlorella emersonii entrapped in alginate gel. Oxygen production was also described for the immobilized red alga Porphyridium cruentum.

(ii) Genetically Altered Algae Extract Metals:

Algae that make metal-grabbing protein show promise for cleaning up toxic sediments. Batches of the single celled Chlamydomonas reinhardtii are being grown and genetically altered at Ohio State University, where scientists have successfully extracted copper, zinc, lead, cadmium, mercury, nickel and other metals from contaminated water.

The genetically altered algae could be turned into heavy metal sponges, either living or freeze-dried. The research indicates that some metals bind to the altered algae, while other nontoxic metals remain in the water. The studies found that calcium and potassium which scientists want to keep in any cleaned-up waters, did not interfere with attempts to get cadmium to bind to the algae.

When the acidity level of the medium holding metal-laden algae was increased the metal came off. That would make recycling possible for metals now considered contaminants suitable only for disposal. It also could offer a new way to get at desirable metals. Work is also going on the algae’s prospects for the mining industry. Research is under way into methods for encapsulating the engineered algae for use in large-scale tests and in gold mining.

(iii) Alternative Petroleum:

A variant of normal photosynthesis is possible in which the enrgy-rich product is molecular hydrogen, the primary substrate consumed is water, and O₂ is generated as a by-product. A detailed account is given by Elias (1988). This process in its in vitro and in vivo forms has been referred to as biophotolysis of water. It was observed that the green alga Scenedesmus could evolve molecular hydrogen in a nitrogen atmosphere under both light and dark condition. Dark and light mediated synthesis of H₂ was reported in Chlamydomonas moewusii. H₂ evolution was demonstrated in heterocystous nitrogen fixing Anabaena cylindrica cultures.

Apart from green algae and cyanobacteria the only other biologically based H₂-producing system based on the photosynthetic water splitting system is the reconstituted in vitro system composed of isolated chloroplasts, ferredoxin and hydrogenase. All these three systems have been shown to be capable of the sustained simultaneous photoevolution of H₂ and O₂.

The potential difference required to split water in photosynthesis is generated in the photosynthetic reaction centers of PS I and II. O₂ is produced in the intrathylakoidal space (lumen) and H₂ is evolved in the extrathylakoidal space (stroma). When certain algae such as Scenedesmus or Chlamydomonas are placed in a CO₂ free anaerobic atmosphere, they are capable of synthesising the enzyme hydrogenase and evolving H₂.

Unlike H₂ production in green algae which is mediated by hydrogenase, H₂ production in N₂ fixing cyanobacteria is mediated by nitrogenase and in an ATP requiring process. Under appropriate physiological conditions, certain freshwater and marine green algae are capable of splitting water to H₂ and 0₂ in a sustained steady state reaction. In these algae, the gaseous- fuel producing reaction can be driven by light.

(iv) Porphyra Culture:

The cultivation of P. tenera and P. yezoensis starts with carpospores. Carpospores are used to produce a stock of free living Conchocelis-phase cultures. Large scale culture of Conchocelis-phase on a shell substrate are grown from two sources—(i) diploid carpospores (ii) vegetative fragments of Conchocelis-stage maintained in culture.

In the tray culture of the Conchocelis phase of Porphyra, Oyster shells inoculated with vegetative fragments are hung in pairs from rods. Inoculation is done during spring. During the summer months when temperature reaches 26°-30° C in culture condition, light levels are reduced and nutrient level is shifted to higher P:N ratio.

This induces the formation and maturation of sporangia. Release of conchospores can be induced by manipulating the cultural conditions. Temperature is dropped to 5°-10° C, light is reduced or day-length is shortened. Cultures are kept in these conditions for 4-6 days and when exposed to full sun massive simultaneous release of spores is achieved.

As the shells are kept in deep tanks, a spore suspension is created as spores are released. Nets wound on large reels are partially immersed in the tanks and rotated. Thus, sufficient spores adhre to the net. There are alternative methods also. Nets are then moved to the field for nursery cultivation.

The plants can be grown by traditional pole or semi floating culture or they may be placed on nursery frames. In the first method the nets are placed horizontally at a carefully chosen tidal height to achieve an optimum tidal exposure regime.

When the plants on the nets reach 2-3 cm in length, the nets are dried, rolled up and frozen (at -20° C). Once the plants reach a size of 2-3 cm they no longer need to be dried and may be placed in floating rafts of various configurations. There they grow to a size of 15-20 cm in a few weeks and are harvested. The harvested Prophyra is quickly brought ashore for processing, or it is stored in a saltwater holding tank or frozen until processed.

(v) Seaweed Biotechnology:

An account of seaweed tissue culture is given by Evans and Butler (1990) and Nambisan (1999). Seaweeds are being used by mankind for several purposes – food, feed, manure, important phycocolloids and medicinal purposes. Most seaweeds are being harvested from naturally existing seaweed beds, resulting in over- harvested populations and slow regeneration which does not meet the demand.

Several countries such as China, Japan, Korea, Chile, Vietnam and India have adopted aquaculture methods including pond culture, bottom culture, net culture and raft culture to augment production. The need to apply techniques of modern biology such as gene cloning and genetic engineering—which have been effectively used in higher plant systems for the improvement and better utilisation of algal resources—has been mooted.

A strain of Porphyra yezoensis was genetically engineered to introduce into it bacterial nitroreductase gene nfsl which can remove 10mg/l TNT from seawater to detoxify it.

(a) Seaweed Tissue Culture:

Over the past couple of decades a number of studies have been undertaken with seaweed tissue culture. Work on Chondrus crispus is one of the first such studies. Subsequent studies were on phycocolloid producing algae Gelidium, Laminaria, Gracilaria and on edible species like Porphyra, Eucheuma.

Most tissue culture works have been carried out with pseudoparenchymatous and parenchymatous thalli of all the three divisions. In the red and brown algae, development of callus from intact explants has been mostly described in cases where the thallus is made up of several cells.

In red seaweeds, callus may arise from cortical cells (Gracilaria verrucosa) but more often arises from the medullary cells. In brown seaweeds too, the medullary cells are associated with callus growth. Calli have been reported to resume growth upon excision from the explant.

It has been demonstrated that under appropriate conditions, seaweed polysaccharides can be produced by callus culture, including agar from Pterocladia capillacea, but whether this would ever be a cost effective production method for polysaccharides is doubtful. There are reports that agar can be successfully produced by callus culture of algae such as Gelidium amansi and Gracilaria confervoides. 100 gm of dried callus cells were reported to produce 75 gm of agar.

This compares with 29% and 26% agar yields, respectively, from cystocarpic and tetrasporic plants of Gracilaria verrucosai. Genetic engineering, may eventually be successful in altering expression of existing genes, e.g., incorporation of high expression promoters with replicons involved in agar production could increase agar yields significantly.

(b) Protoplast Isolation:

The starting point for application of the new technology to seaweeds is successful isolation of viable protoplasts from diploid and haploid tissues under aseptic conditions. Protoplasts have been isolated and cultured in all the three classes of seaweeds. In the case of the green seaweed Enteromorpha intestinalis enzymatic digestion of the walls released viable protoplasts which evolved oxygen in the light at a rate similar to that of vegetative cells. Regeneration of protoplasts of Enteromorpha and Ulva into new thalli has been reported.

In the brown and red seaweeds regeneration of protoplasts into callus and into new plant is recorded in several species including commercially important Porphyra, Gracilaria and Chondrus crispus. Protoplasts have some potential in secondary metabolite production. Cultured protoplasts of Kappaphycus alvarezii have been reported to secrete carageenan fragments. Protoplasts could be used as seed stock for macroalgal culture. Heteroplasmic fusions have been reported in the green seaweeds, such as Ulva, Enteromorpha and Monoslroma and in red alga Porphyra. Fusion has been brought about either by the use of Polyethylene glycol or elctrofusion technique.

Of twenty-one red algal genera studied, four were found to contain circular dsDNA plasmids. Some of these have been isolated and studied and one 3.5 Kbp plasmid from Gracilaria lemaniformis was sequenced to reveal two potential ORFs. In this species, plasmids are present in a high copy number per cell and may provide useful vectors for algal transformation.

Seaweed Biotechnology is Important because of the following reasons:

(i) in the generation of sufficient amounts of selected strains of marine macroalgae for cultivation,

(ii) in the use of DNA sequences or molecular probes to prove equivalence of raw material,

(iii) in the development of engineered polysaccharides in the non-consumable markets.

(vi) Biotechnolgy with Chlamydomonas:

Methods have been developed for transforming the Chlamydomonas plastid and mitochondrial genomes as well as nuclear genes. These have allowed the identification of previously unknown genes and the study of their expression and have been particularly useful in elucidating the molecular biology of photosynthesis (Stevens and Purtan, 1997).

The experimental advantages of Chlamydomonas are many (i) the ease with which it can be cultured in the laboratory and the ability to combine facile genetics with the techniques of modern molecular biology (ii) it can grow photoautotrophically or heterotrophically, which permits the isolation of mutants unable to perform photosynthesis, (iii) it is haploid in vegetative phase, allowing any mutation to be expressed, (iv) exogenous DNA can be introduced into the nuclear, chloroplast or mitochondrial genomes, (v) a reporter gene has been developed as used to dissect the regulation of various promoters.

More recently, a gene has been isolated that has the potential to serve as a homologous dominant selectable marker for nuclear transformation by conferring resistance to a herbicide. Knowledge gained from utilizing Chlamydomonas as a model system for studying chloroplast genetics and molecular biology might make it possible someday to manipulate this organelle in crop plants.

The chloroplasts of plants contain many copies of their genome per cell. Thus it may be possible to amplify proteins by inserting their genes in the chloroplast DNA rather than in the nuclear DNA. To do this, however, one must know how the chloroplast DNA and protein synthesizing systems function.

There are multiple factors that control the transcription and translation of a protein from the chloroplast that would need to be adjusted in order to use the chloroplast as a factory for the manufacture of foreign proteins. C. reinhardtii has been used as a model system for understanding physiological and genetic processes in green plants. ATP is synthesized by ATP synthase in the mitochondria and chloroplast as a result of proton motive force that accumulates during electron transport.

The proton gradient in mitochondrion, which is responsible for the proton motive force, can be dissipated by a family of molecules known as uncoupling proteins. A nitrate reductase mutant expressed a low level of uncoupling protein as detected by flowcytometry. This strain was cotransformed with a plasmid encoding nitrate reductase and a construct with genes encoding an uncoupling protein fused to a Chlamydomonas optimized GFP.

These transformants were screened by nitrate reductase-utilization, flurometry and western analysis. Flowcytometry of transformants detected increased uncoupling protein expression. These results suggest a means of manipulating energy metabolism in the cell.

(vii) Diatoms and Nanotechnology:

Diatoms produce diverse three-dimensional structures that may be of use in the manufacture of components for nanotechnology as an alternative to present linear lithographic techniques. Diatoms allow direct fabrication of three-dimensional structures instead of layer by layer manipulation.

Another advantage is the exponential growth of diatom cells by cell division. The degree of versatility in design and composition coupled with the ability to perform manipulations (changes in thin architecture or degree of solidification, substitution of silicon with magnesium) may allow the study of diatoms to take center stage in nanotechnology and biotechnology.

Nanotechnology—The name nano comes from the size of molecules which are measured in nanometers—or one billionth of a meter. The dimension of a single atom is ten fold smaller. Any technology developed at the atomic, molecular or macromolecular range of approximately 1-100 nanometers to create and use structures, devices and systems that have novel properties. Here, a precise molecule by molecule exchange is administered to control products and byproducts in the development of fundamental structures.

Silicon is taken up by diatom cells as the ionized form of monosilic acid, Si (OH)₃O^–. Silicon is packaged by the Golgi into vesicles that are transported to the silicon deposition vesicle by microfilaments. Silicon-laden vesicles fuse with silicon deposition vesicle which has a large number of condensation points on its inner surface that initiate precise positioning of polymeric silicic acid.

During valve formation, cytoskeletal elements would move the trans-silicalemma proteins around in a genetically determined choreography, condensing silicic acid at precise locations to produce the complex valve architecture. Another significant development that attracted people in nanotechnology when all silicon atoms in diatom valve was replaced by magnesium atoms. This was done by placing diatom frustules in a magnesium gas at 900°C for 4 h.

Nanotechnology technique involved in semiconductor industry is tedious and involves building three dimensional structures layer by layer. At present, features are etched onto circuit board using light. The wave length of light limits the smallest size that can be achieved.

For future generation engineers need to get denser features on to computer chips. Experts in diatom biology say that diatoms are natural lithographers in the nanometer range. If the mechanism of laying down of silica in micro lines is properly understood then scientists will be able to simulate it.

The protein that diatoms use to direct silica deposition could be useful to the semiconductor industry. Diatoms have uniform nanoscale pore structure and are chemically inert and biocompatible. Diatom cells can be loaded with drug which will be leached out into the blood stream. By incorporating ferromagnetic particles within the diatom structure it might be possible to use a magnet to guide the drug to the right organ.

(viii) Molecular Farming in Algae:

A significant development in the field of algal biotechnology is molecular farming in microalgae. Molecular farming in microalgae is to generate biomolecules valuable to medicine or industry that are difficult or even impossible to produce in another way or which require high production cost in other systems. One field of activity in this regard is the large scale production of antibodies in algal systems.

A recombinant human monoclonal IgA antibody has successfully been expressed and assembled in C. reinhardtii. To achieve high expression in transgenic algae and simplification of antibody purification required optimization of the codons of the corresponding gene and fusion of the IgA heavy chain to the variable region of the light chain by genetic engineering using a flexible linker.

This way, antibody production can become not only much more convenient but also cheaper. Significantly, expression in an organism without immune system allow expression of antibodies that would otherwise interfere with the immune system of the host animal used in conventional antibody production.

Transgenic Algae to Deliver Antigen to Animals:

In aquaculture, due to dense population, infections disease is a great threat. The fishes may be treated with antibiotic, however, 80% of antibiotic may pass through the fish and resistant bacteria may grow. Alternative strategy to combat this situation is prophylactic administration of an antigen composition. A delivery system is provided for delivering peptides to a host animal.

The Delivery System is a Transgenic Algae that Comprises a Transgene which Comprises:

(i) A polynucleotide encoding at least one peptide—an antigenic determinant of pathogen,

(ii) A promoter for driving expression of the polynucleotide in the algae and

(iii) All other genetic elements required for transcription. This transgenic algae can be orally administered.

(ix) Some Other Achievements:

Diatom Phaeodactylum tricornutum has been successfully transformed (Durrahay et al. 1995, 1996). In diatoms the major goal has been genetic manipulation of lipid metabolism for biotechnological applications, but transformation success also pave the way for molecular dissection of photosynthesis and other aspects of diatoms that differ for green algae.

The giant multicellular green alga Acetabularia, Chara and the green microalgae Dunaliella and Chlorella have also been the subject of transformation experiment. Hebrew University researchers are focusing on a unique strain of Dunaliella, which grows well over a wide range of salinities.

The only difference between the algae grown in low molarity (0.1 M) and high molarity (3.0 M) salt solutions is the appearance of one extra polypeptide at high salt concentration. The researchers hope to clone the corresponding gene and transfer it to non-salt tolerant Dunaliella to see if it can improve their salt tolerance. If successful, tests in higher plants would follow. Increased salinity is a major problem throughout modern irrigation intensive agriculture.