CA2249103A1 - Novel hyaluronic acid produced from algae - Google Patents

Abstract

A novel hyaluronic acid, chlamyhyaluronic acid is described. Also described are methods for producing and isolating chlamyhyaluronic acid, and uses of chlamyhyaluronic acid. The method of producing the acid involves specifying the cultural conditions for the cultivation of phototrophic green algae under specific conditions to obtain encapsulated cells, which accumulate in their capsules a mucopolysaccharide similar to the animal and bacterial hyaluronic acid. Also taught are methods of extracting the capsules, uses for preparations made from the capsules, and methods of partially purifying the chlamyhyaluronic acid from the capsules.

Description

B&P File No. 9157-004/MG
Title: Novel Hyaluronic Acid Produced from Algae BACKGROUND OF THE INVENTION
Technical Field This invention relates to a novel hyaluronic acid and the production of the hyaluronic acid from algae.
Brief Description of the Prior Art Algae have been used as culturable sources of biomedically useful compounds (Parker, Bruce C. (ed.). Journal of Applied Phycology, 6:
10 pp. 91-98 (1994)). The synthesis of such compounds, which may be secondary metabolites, depends on the culture growth conditions. If optimal growth conditions with respect to nutrient availability, pH vaIue, irradiance, light quality, aeration and temperature are maintained, microalgal cells double or quadruple their cellular components and 15 divide. This can result in the continuous production of healthy growing and reproductive generations, as demonstrated in Chlamydomonas segnis Ettl (Badour, S.S., et al., Canadian Journal of Botany, 51:67-72 (1973); Foo, S.K., and Badour, S.S. Canadian Journal of Botany, 55:2178-2185 (1973);
Badour, S.S., et al. Journal of Phycology, 13:80-86 (1977); Badour, S.S.
20 Journal of Phycology, 17:293-299 (1981); Harris, Elizabeth (ed). The Chlamydomonas source book, Academic Press Inc. (1989)~.
In nature, numerous microalgae have been shown to produce mucopolysaccharide cell coverings in the form of highly hygroscopic capsules (Kinzel, Helmut. Unterschungen uber die Chemie 25 und Physikochemie der Gallertbildungen von SuI~wasser algen.
Ostereichische Botanische Zeitschrift, 100:25-79 (1953)). These capsules are metachromatic, i.e. they exhibit a colour different from that of the basic dye (e. g. toluidine blue) used to stain them, because they are negatively charged. The precise culture conditions that induce the formation of 30 capsules and the production of mucopolysaccharides in green microalgae, however, have never been specified (Badour, S.S. Excess light evokes cell encapsulation in Chlamydomonas segnis Ettl. Abstracts of the International Meeting on Molecular Biology, Biochemistry and Physiology of Chloroplast Development. Philipps Universitat, Marburg, Germany (1995); Allard, B., and Tazi, A. Phytochemistry, 32:41-47 (1993); Starr, R.C., and Zeikus, J.A. Journal of Phycology (Supplement) 23: 8(1987)).
ChZamydomonas species are haploid, biflagellate unicellular organisms. Chlamydomonas segnis has been used as an experimental organism by Badour et al, who have described its morphology and ultrastructure, and studied its growth physiology, biochemistry and photobiology (e. g. Badour, S.S., et al., Canadian Journal of Botany, 10 51:67-72 (1973); Foo, S.K., and Badour, S.S. Canadian Journal of Botany, 55:2178-2185 (1973); Badour, S.S., et al. Journal of Phycology, 13:80-86 (1977);Badour, S.S. Journal of Phycology, 17:293-299 (1981); Badour, S.S. et al., Journal of Phycology (supplement) 8:16 (1972); Badour, S.S., and Irvine, B.R. Botanica Acta, 103: 149-154 (1990); Badour, S.S., and Tan, C.K.
15 Zeitschrift fur Pflanzenphysiologie, 112: 287-295 (1983); Badour, S.S., and Tan, C.K. Plant and Cell Physiology, 28: 1485-1492 (1987); Badour, S.S.
Ribulose-bis-phosphate carboxylase in Chlamydomonas. In: Handbook of Phycological Methods. Vol. II, pp. 209-216. Edited by Hellebust, J.A. and Craigie, J.S. Cambridge University Press (2978)).
Hyaluronic acid, also known as hyaluronan, is a mucopolysaccharide made up of alternating 1,4-linked residues of hyalobiuronic acid, forming a gelatinous material. Hyaluronic acid is a naturally occuring heteropolysaccharide consisting of alternating residues of D-glucuronic acid and N-acetyl-D-glucosamine. It is a linear polymer of 25 high molecular weight, up to about 8 to 13 million.
Hyaluronic acid is used as a fluid replacement to correct pathological conditions in the eye and in the joint, and has been used to facilitate wound protection and healing (Urman, B. and Gomel, V., Fertil.
Steril. 56(3):568-570, (1991); Avid, A.D. and Houpt, J.B., J. Rheumatol.
30 21(2):297-301 (1994); and Adams, M.K. et al., Osteoarthritis Cartilage 3(4):213-225 (1995); King et al., in Surgery 109:76-84, (1991)). Hyaluronic acid and fractions thereof have been used in ophthalmic surgery or as therapeutic, auxilliary and substitutive agents for natural organs and tissues. It has been found to be particularly useful in the keatment of arthropathies, wound healing and cystisis. Hyaluronic acid is also used as a gum in food industry and pharmaceuticals, and as a source of mucilage in cosmetics and skin-care products (See for example U.S. Patents Nos.
4,141,973 and 5,442,053 reviewing the technical literature describing isolation, characterization and uses of hyaluronic acid).
Hyaluronic acid and its salts, hereafter collectively referred to as hyaluronic acid, has been obtained from various sources including 10 human umbilical cords, rooster combs, bovine joints, whale cartilages and certain bacterial cultures, such as various Streptococcus, as well as Pasteurella multocida and Pseudomonas aeruginosa. Various methods of culturing microorganisms to prepare hyaluronic acid have been taught (see for example: Japanese laid-open patent application No. 58-56692; Kjem 15 and Lebech, Acta. Path. Microbiol. Scand. Sect. B. 84:162-164, 1976). For example, enriching oxygen in the culture medium (U.S. Patent No.
4,897,394), inhibiting hyaluronidase in the microorganisms (U.S. Patent No. 4,782,046), or adding aromatic compounds to the culture medium (U.S. Patent No. 4,885,244), have been described to facilitate the yield of 20 hyaluronic acid from microorganims.
However, the above-mentioned methods of extracting high hyaluronic acid from animal tissues or microorganisms have various disadvantages, making it difficult to obtain hyaluronic acid effectively and in abundance.
For example, hyaluronic acid present in animal tissues is present only in trace amounts, and it forms a complex with proteins or other mucopolysaccharides. According to Dorfman and Cifonelli, Methods in Enzymology, III, pgs 20-27 (1957), the various procedures employed in the preparation of acid mucopolysaccharides from 30 mammalian tissues involve the following basic steps: (1) extraction, (2) removal of protein, (3) precipitation, and (4) final purification.

CA 02249l03 l998-09-30 Complicated and delicate purification and extraction processes may degrade the hyaluronic acid.
Purifying hyaluronic acid from cultured microorganisms which are capable of producing hyaluronic acid has an advantage in that the purification process tends to be simpler than the aforementioned methods for extraction from animal tissues. This is because a protein-free medium may be used for culturing microorganisms to obtain hyaluronic acid. However, these methods have a disadvantage that the amount of hyaluronic acid produced per culture volume is low, and the use of 10 bacteria can give rise to the need for the removal of toxins as pointed out in the Biology of Hyaluronan, Ciba Foundation Symposium, 143, pgs 265-280(1989).
In view of the above, there is a need for a non-bacterial and non-animal source for production of a hyaluronic acid or hyaluronic acid-like compound in order to reduce purification costs associated withpreparing hyaluronic acid by bacteria or by the extracellular matrix of tissues from animals.
SUMMARY OF THE INVENTIC)N
The present invention relates to a novel hyaluronic acid derived from a green alga, termed chlamyhyaluronic acid, or chlamyhyaluronan, and a method for preparing same.
Broadly stated, the present invention provides an isolated chlamyhyaluronic acid produced by a green alga.
The chlamyhyaluronic acid may be characterized by having various properties. In one embodiment, a solution of the sodium salt of chlamyhyaluronic acid has (a) has an acetyl group peak of about 1.7599 ppm on a 500 MHz nuclear magnetic resonance spectra when in a solution with deuterium oxide at 300K; (b) has carbohydrate peaks of about 3.7052 ppm and about 3.6008 ppm on a 500 MHz nuclear magnetic resonance spectra when in a solution with deuterium oxide at 300K; (C) forms a complex with bivalent copper and said complex shows an absorption band at 232 nm; (d) develops turbidity with serum at an acid pH and forms stable colloidal suspensions; and (e) is degraded by the specific enzyme hyaluronate lyase (EC 4.2.2.1) from Streptomyces hyalurolyticus as well as by hyaluronidase (EC 3.2.1.35) from bovine testes.
The present invention further comprises algal cell encapsulations containing chlamyhyaluronic acid.
The present invention also provides a method for the production of chlamyhyaluronic acid comprising culturing an alga of the phylum Chlorophyta under stressful conditions for a period of time sufficient for the alga to produce extracellular capsules containing 10 chlamyhyaluronic acid.
The term "stressful conditions" as used herein means conditions that alter the normal growth of the algae and causes the cells to divert from the regular metabolic pathways to adjust for survival. Certain cells, such as algal cells described herein, produce extracellular protective 15 structures or capsules in response to stressful conditions.
The term "capsule" as used herein means a layer or layers of mainly mucopolysaccharides external to but contiguous with the algal cell wall. Such capsules may reach 50 fold to over 200 fold of the algal cell volume. These capsules contain compounds that protect the cells from 20 excessive light (particularly blue light and UV), catalyze the breakdown of oxygen free radicals, and stop invasion by predators. The present inventor has found that chlamyhyaluronic acid can be a major component of these capsules, and in particular can be a major component of Chlamydomonas segnis capsules.
These capsules can be extracted from the cultures in the form of partially dried thin layers of caked encapsulated Chlamydomonas segnis Ettl (CENC). Because of its hygroscopic nature, CENC may impart moisture to the skin and protect it from reactive oxygen radicals due to its chlamyhyaluronic acid and catalase contents.
In one embodiment, an alga of the phylum Cholorphyta is cultured at a temperature of from about 15~C to about 35~C, and exposed to excessive light. In response to the stress induced by the excessive light, the chlorophyte produces capsules containing chlamyhyaluronic acid, for the purposes of protecting the organism from such light.
The term "excessive light" as used herein means the light intensity, i.e., irradiance, at which the inception of cell encapsulation in 5 algal cultures exposed to air, aerated with either air or air enriched with carbon dioxide, is detectable by metachromasy.
In a preferred embodiment, the excessive light has a blue:red spectral ratio of higher than one. The term "blue light" as used herein means irradiances with spectral range between 320 nm and 500 nm which 10 includes bluish green light, blue light, violet light and ultraviolet A of 320 nm to 380 nm. The term "red light" as used herein means irradiances with a spectral range from 600 nm to 700 nm and does not include far red light.
In another embodiment, stressful conditions are imposed on 15 the algae to facilitate the production of chlamyhyaluronic acid by the use of boron deficient nutrient medium. In another embodiment, stressful conditions are imposed on the algae to facilitate the production of chlamyhyaluronic acid by the use of low salt (2-6 mM) nutrient medium.
In another embodiment, stressful conditions are imposed on the algae to 20 facilitate the production of chlamyhyaluronic acid by the use of the application of carbon dioxide enriched air (0.1% to 5% CO2 by volume), during the exponential phase of growth and the encapsulation phase.
In one embodiment of the invention, the alga selected for culture is of the genus ChZamydomonas. In a preferred embodiment, the 25 alga selected is Chlamydomonas segnis. In a further preferred embodiment, the alga is Chlamydomonas segnis Ettl. The alga may be obtained from the stock cultures of UTEX, culture No. 1919.
In an embodiment of the invention the algal cells are cultured to produce large extracellular capsules in mass cultures generally 30 within a relatively short period of time. Under specified physical and cultural conditions described herein, the photoautotrophic growth of algae capable of producing chlamyhyaluronic acid can be regulated. As a result, immobile encapsulated cells with high yields of chlamyhyaluronic acid in their capsules may be obtained.
The present invention further comprises the method for extracting the chlamyhyaluronic acid from the culture. A partially 5 purified chlamyhyaluronic acid may be obtained from the extracellular capsules of the alga. The extracted algal chlamyhyaluronic acid may be partially purified for use as substitute or supplement for animal and bacterial hyaluronic acid, which has well established therapeutic and cosmetic utility.
The present invention also provides pharmaceutical compositions comprising chlamyhyaluronic acid. The pharmaceutical composition can be used in various applications where hyaluronic acid is normally used. For example, the composition can be used as a fluid replacement to correct pathological conditions in the eye and in the joint, to facilitate wound protection and healing, as therapeutic, auxiliary and substitutive agents for natural organs and tissues, particularly in the treatment of arthropathies, ophthalmic conditions, and cystitis. The pharmaceutical composition can also be used in the cosmetic industry for example in skin-care products. Additionally, the invention includes a composition comprising chlamyhyaluronic acid for use in the food industry, for example as a gum.
The present invention further comprises the use of the algal cell encapsulations containing chlamyhyaluronic acid as a compound in a topical preparation to protect skin from reactive oxygen radicals or to moisturize skir .
BRIEF DES~RIPTION OF THE DRAWINGS
Figure 1 depicts the changes in the levels of blue, red, and far red spectra with increasing irradiances from a combination of cool-white fluorescent and standard incandescent lamps.
Figure 2 shows the absorbance spectra of acetone extracts from shallow cultures of ChZamydomonas segnis Ettl, at (a) late exponential phase of growth, (b, c and d) early and mid encapsulation phase, and (e) late encapsulation phase.
Figure 3 shows the growth curves of deep cultures of Chlamydomonas segnis Ettl produced in the low salt boron-deficient medium, and in the high salt nutrient medium but without adding boric acid.
Figure 4 shows photomicrographs of encapsulated cells of Chlamydomonas segnis Ettl at the end of encapsulation phase, stained either with toluidine blue (a and b) or alcian blue (c).
Figure 5 depicts growth curves of Chlamydomonas segnis Ettl in shallow cultures.
Figure 6 depicts the relationship between the dilution index and the level of irradiance required for the production of encapsulated algal biomass.
Figure 7 shows photomicrographs of Chlamydomonas segnis Ettl stained with toluidine blue: (a) as capsule free cells at mid exponential phase of growth, (b) as cells with metachromatic extracellular matrix at the beginning of encapsulation, (c) as encapsulated cells with polymorphic capsules, and (d) as spaced cells due to capsule enlargement.
Figure 8 shows transmission electron micrographs, of Chlamydomonas segnis Ettl cell sections; (a) at mid exponential phase of growth showing a chloroplast with interthyalkoidal starch grains in a capsule free cell, and (b) starch sheath around the pyrenoid, (c) at late encapsulation phase showing the stratified structure of the capsule 25 surrounding a cell with large starch grains within a fenestrated chloroplast and large dark lipid droplets, and (d) the relative thickness of the capsule.
Figure 9 shows scanning electron micrographs of desiccated cells of Chlamydomonas segnis Ettl at mid-encapsulation phase, (a) coated with thick mantles of mucopolysaccharides, (b) in a fractured layer of dried 30 extracellular mucopolysaccharide material released in the culture medium, and (c and d) embedded in the thick matrix of this material.

Figure 10 shows photomicrographs of: (a) immobilized spaced cells of Chlamydomonas segnis Ettl at mid encapsulation phase, (b) the autofluorescence of the capsule material and (c) encapsulated cells stained with alcian blue, or (d) toluidine blue to visualize the capsule.
Figure 11 depicts the retarding effect of the presence of one ,uM boric acid (+ Boron) on the inception of the encapsulation phase.
Figure 12 depicts the inhibitory effect of aerating Chlamydomonas segnis Ettl cells with 5% CO2 in N2 instead of 5% CO2 in air (volume by volume) on the inception of the encapsulation phase.
Figure 13 shows photomicrographs of: (a) a sample of caked encapsulated Chlamydomonas segnis (CENC) homogenate, (b) a two-gram slightly moist pellet of CENC, (c) the size increase of the moist pellet of CENC during water imbibition, and (d) three samples of CENC during hydration exhibiting colour shades.
rigure 14 depicts the apparent viscosity of ;% C~NC
homogenate in water, compared to 1% solution of either xanthan or guar in water, as a function of shear rate.
Figllre 15 shows photomicrographs of samples from CENC
homogenate stained with toluidine blue, (a) before extraction, (b) after 20 extraction with the buffered saline solution, and (c) exhibiting the acapsular appearance of CENC cells after extraction.
Figure 16 depicts the increase in absorption around 230 nm of chlamyhyaluronic acid after incubation with about 70 units of hyaluronate lyase (upper graph), compared to a control sample without the said 25 enzyme (lower graph).
Figure 17 depicts the increase in absorption around 230 nm of chlamyhyaluronic acid after incubation with about 980 units of hyaluronidase (upper graph), compared to a control sample without the enzyme (lower graph).
Figure 18 shows a print-out of a cellulose thin layer chromatogram of the products formed by the degradation of chlamyhyaluronic acid (c and d), and hyaluronic acid from human umbilical cord (e and fl with hyaluronate lyase at 37~C for six hours, and the absence of such products (a and h) or their reduced level (b and g) in samples without enzyme or with heated enzyme, respectively.
Figure 19 shows increases in the degradation products indicated in Figure 18, as a result of extending the incubation period to twelve hours at 37~C.
Figure 20 shows a print-out of a chromatogram produced as described in Figure 18, except that hyaluronidase is the enzyme used.
Figure 21 depicts the reduced viscosity of chlamyhyaluronic 10 acid (potassium salt) as a function of concentration, using a Ubbelohde viscometer, and the intrinsic viscosity is determined by extrapolating to zero concentration with an asymptotic model.
Figure 22 depicts the reduced viscosity of hyaluronic acid (sodium salt) from human umbilical cord, as a function of concentration.
15 DETAILED DES~RIPTION OF THE INVENTION
As hereinbefore mentioned, the present invention relates to a novel hyaluronic acid derived from a green alga, termed chlamyhyaluronic acid, and a method for its production.
Chlamyhyaluronic Acid In one aspect, the present invention provides isolated chlamyhyaluronic acid. The present inventor extracted, partially purified, and identified the capsular metachromatic mucopolysaccharide in the caked encapsulated Chlamydomonas segnis Ettl. The partially purified mucopolysaccharide (referred to as Chlamyhyaluronic acid) was found to 25 depolymerize in a similar fashion as the hyaluronic acid from human umbilical cord by the action of hyaluronate-lyase from Steptomyces hyalurolyticus (EC 4.2.2.1), as well as hyaluronidase from bovine testes (EC3.2.1.35). Chlamyhyaluronic acid is metachromatic, ultraviolet-absorbent, and gives fairly stable colloidal suspensions, i.e. turbidity, when 30 mixed with serum albumin at pH 4. The turbidity is reduced or prevented upon depolymerization by either one of the aforenamed enzymes, with simultaneous production of reducing low molecular weight molecules and increase in absorbance at 230 nm. The products of chlamyhyaluronic acid degradation, visualized on thin layer chromatograms match those from human umbilical cord hyaluronic acid.
The chlamyhyaluronic acid may be characterized by having 5 various properties. In one embodiment, a solution of the sodium salt of chlamyhyaluronic acid (a) has an acetyl group peak of about 1.7599 ppm on a 500 MHz nuclear magnetic resonance spectra when in a solution with deuterium oxide at 300K; (b) has carbohydrate peaks of about 3.7052 ppm and about 3.6008 ppm on a 500 MHz nuclear magnetic resonance spectra 10 when in a solution with deuterium oxide at 300K; (c) forms a complex with bivalent copper and said complex shows an absorption band at 232 nm; (d) develops turbidity with serum at an acid pH and forms stable colloidal suspensions; and (e) is degraded by the specific enzyme hyaluronate lyase (EC 4.2.2.1) from Streptomyces hyalurolyticus as well as 15 by hyaluronidase (EC 3.2.1.35) from bovine testes.
In another aspect, the present invention provides capsules containing chlamyhyaluronic acid.
The term "capsule" as used herein means a layer or layers of mainly mucopolysaccharides external to but contiguous with the algal cell 20 wall. Such capsules may reach 50 fold to over 200 fold of the algal cell volume, due to the high water content of the capsule.
Capsules such as those produced by Chlamydomonas segnis Ettl are easily visible by light microscopy after staining wet smears from liquid cultures or agar slants with a metachromatic dye, i.e. they are 25 chromotropes. These capsules contain compounds that protect the cells from excessive light (particularly blue light and UV), catalyze the breakdown of oxygen free radicals, and stop invasion by predators. The present inventor has found that chlamyhyaluronic acid can be a major component of these capsules, and in particular can be a maJor component 30 of Chlamydomonas segnis capsules. In contrast, well characterized Chlamydomonas reinhardtii (Harris, Elizabeth (ed). The Chlamydomonas source book, Academic Press Inc. (1989)), which is widely used in genetic, cytological and biochemical research, does not produce capsules under the stressful cultural conditions designed for Chlamydomonas segnis.
These capsules can be extracted from the cultures in the form of partially dried thin layers of caked encapsulated Chlamydomonas segnis 5 Ettl (CENC). Because of its hygroscopic nature, CENC may impart moisture to the skin and protect it from reactive oxygen radicals due to its chlamyhyaluronic acid and catalase contents.
Preparation of Chlamyhyaluronic Acid In another aspect, the present invention provides a method 10 for the production of chlamyhyaluronic acid comprising culturing an alga capable of providing extracellular capsules containing chlamyhyaluronic acid under stressful conditions for a period of time sufficient for the alga to produce extracellular capsules containing chlamyhyaluronic acid.
The term "stressful conditions" as used herein means 15 conditions that alter the normal growth of the algal cells and causes the cells to divert from the regular metabolic pathways to adjust for survival.
For photoautotrophic alga, stressful conditions include culture conditions under which algal cultures at the lag phase, exponential phase, or stationary phase of photoautotrophic growth are unable to utilize all the 20 absorbed irradiance for biosynthetic activity directed to cell growth and division, but are capable of using the excessive irradiance to synthesize protective secondary metabolites for preserving cell viability. Examples of stressful conditions include nutrient deficiency, low or high nutrient concentration, increased acidity or alkalinity, dim or excessive light with 25 unbalanced spectral coll.position, suboptil..al or relatively high temperature and/or carbon dioxide tension. These conditions force the cells to adjust for survival and divert the regular metabolic pathways to biosynthetic routes that lead to the active production of secondary metobolites in order to protect the cells from the harmful effects of the 30 stressful environment. Certain cells, such as algal cells described herein, produce extracellular protective structures or capsules in response to stressful conditions.

CA 02249l03 l998-09-30 In one embodiment, an alga of the phylum Cholorphyta is cultured at a temperature of from about 15~C to about 35~C, and exposed to excessive light. In response to the stress induced by the excessive light, the chlorophyte produces capsules containing a secondary metabolite, for the purposes of protecting the organism from such light. The present inventor has found that this secondary metabolite is chlamyhyaluronic acid.
The term "excessive light" as used herein means the light intensity, i.e., irradiance, at which the inception of cell encapsulation in 10 algal cultures exposed to air, aerated with either air or air enriched with carbon dioxide, is detectable by metachromasy.
In a preferred embodiment, the excessive light has a blue:red spectral ratio of higher than one. The term "blue light" as used herein means irradiances with spectral range between 320 nm and 500 nm which includes bluish green light, blue light, violet light and ultraviolet A of 320 nm to 380 nm. The term "red light" as used herein means irradiances with a spectral range from 600 nm to 700 nm and does not include far red light.
In another embodiment, stressful conditions are imposed on the alga to facilitate the production of chlamyhyaluronic acid by the use of boron deficient nutrient medium. In another embodiment, stressful conditions are imposed on the alga to facilitate the production of chlamyhyaluronic acid by the use of low salt (2-6 mM) nutrient medium.
In another embodiment, stressful conditions are imposed on the alga to facilitate the production of chlamyhyaluronic acid by the use of the application of carbon dioxide enriched air (0.1% to 5% C ~2 by volume), during the exponential phase of growth and the encapsulation phase.
The alga used to prepare the chlamyhyaluronic acid includes the species Tetraspora, Gloeomonas, Chlamydomonas segnis, Chlamydomonas gymnogama, Chlamydomonas pallidostigmatica, Chlamydom onas augustae, Chlamydom onas intermedia, Chlamydomonas sajao Levin and Chlamydomonas corrosa. These are CA 02249l03 l998-09-30 eukaryotic unicellular green algae containing chlorophyll a and b, with 2 flagella (motile) or without flagella (non-motile), or colonial (coenobia), possessing a pyrenoid or pyrenoids associated with the chloroplast and storing starch.
In one embodiment of the invention, the alga is selected from the genus Chlamydomonas. In a preferred embodiment, the alga is Chlamydomonas segnis. In a further preferred embodiment, the alga is Chlamydomonas segnis Ettl. The alga may be obtained from the stock cultures of UTEX, culture No. 1919.
In an embodiment of the invention the algal cells are cultured to produce large extracellular capsules in mass cultures generally within a relatively short period of time. Under specified physical and cultural conditions described herein, the photoautotrophic growth of algae capable of producing chlamyhyaluronic acid can be regulated. As a result, 15 immobile encapsulated cells with high yields of chlamyhyaluronic acid in their capsules may be obtained.
The present invention further comprises the method for extracting the chlamyhyaluronic acid from the culture. A partially purified chlamyhyaluronic acid may be obtained from the extracellular 20 capsules of the algae.
Applications of Chlamyhyaluronic Acid The chlamyhyaluronic acid of the invention may be used in a variety of applications, including applications for which hyaluronic acid is known to have utility.
For example, chlamyhyaluronic acid may be used in pharmaceutical applications, including use in wound treatment, replacing or supplementing biological fluids, such as fluids in the eye or the joints, retarding cancer development, relieving pain, or treating cystitis.
Chlamyhyaluronic acid also has cosmetic applications, including use as an 30 ultra-violet ray screening agent or as a moisturizer.
Further, some applications of chlamyhyaluronic acid may be utilized without isolating chlamyhyaluronic acid from its cellular capsules. Caked encapsulated algae containing chlamyhyaluronic acid may be used in some therapeutic or industrial, pharmaceutical or cosmetic applications which do not require a high degree of purity of chlamyhyaluronic acid. On hydration, cell encapsulations containing 5 chlamyhyaluronic acid produces highly viscous solutions with rheological properties comparable to that described for high polymeric hyaluronic acids used for medical, pharmaceutical, or cosmetic purposes.
The use of chIamyhyaluronic acid or cell encapsulations containing chlamyhyaluronic acid as a non-bacterial and non-animal 10 source for production of hyaluronic acid-like agents may reduce purification costs by eliminating the need for the removal of toxins from bacterial capsules as pointed out in the Biology of Hyaluronan, Ciba Foundation Symposium, 143, pgs 265-280 (1989), or for the fermentation of various components associated with hyaluronic acid in the extracellular 15 matrix of tissues from human, bovine or ovine organs. This is because the algal capsule components may be almost completely extracted by saline phosphate buffer solutions, leaving intact cells inside ghost capsules.
The novel algal hyaluronic acid extracted by the method described herein may have a range of molecular weights and may not be 20 ultrapure. Its molecular weight can be increased to match the commercial animal or bacterial hyaluronic acid, by improving the extraction methodology to increase the yield of the high molecular weight fraction in cell encapsulations containing chlamyhyaluronic acid extracts. Moreover, the physicochemical properties of cell encapsulations containing 25 chlamyhyaluronic acid extract can be modified for medical and cosmetic purposes.
(1) Pharmaceutical Compositions The chlamyhyaluronic acid of the invention and capsules containing the chlamyhyaluronic acid may be formulated into 30 pharmaceutical compositions for adminstration to subjects in a biologically compatible form suitable for administration in vivo. By biologically compatible form suitable for administration is meant a form CA 02249l03 l998-09-30 of the substance to be administered in which any toxic effects are outweighed by the therapeutic effects. The substances may be administered to living organisms including humans, and animals in a therapeutically effective amount. Administration of a therapeutically 5 effective amount of the pharmaceutical compositions of the present invention is defined as an amount effective, at dosages and for periods of time necessary to achieve the desired result. For example, a therapeutically effective amount of a substance may vary according to factors such as the disease state, age, sex, and weight of the individual, and 10 the ability of peptide to elicit a desired response in the individual. Dosage regime may be adjusted to provide the optimum therapeutic response.
For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation.
The active substance may be administered in a convenient manner such as by topical or transdermal application, injection (subcutaneous, intravenous, etc.), oral administration, inhalation, or rectal administration. Depending on the route of administration, the active substance may be coated in a material to protect the compound from the 20 action of enzymes, acids and other natural conditions which may inactivate the compound.
The compositions described herein can be prepared by per se known methods for the preparation of pharmaceutically acceptable compositions which can be administered to subjects, such that an effective 25 quantity of the active substance is combined in a mixture with a pharmaceutically acceptable vehicle. Suitable vehicles are described, for example, in Remington's Pharmaceutical Sciences (Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., USA
1985). On this basis, the compositions include, albeit not exclusively, 30 solutions of the substances in association with one or more pharmaceutically acceptable vehicles or diluents, and contained in CA 02249l03 l998-09-30 buffered solutions with a suitable pH and iso-osmotic with the physiological fluids.
Compositions for injection include, albeit not exclusively, chlamyhyaluronic acids in association with one or more pharmaceutically 5 acceptable vehicles or diluents, and contained in buffered solutions with a suitable pH and iso-osmotic with the physiological fluids. Any pharmaceutically suitable diluent can be used in the composition for injections: distilled water, physiological or a salt solution, and/or a buffer solution. The composition for injections may be prepared by 10 conventional volume-weight procedures. A certain amount of chlamyhyaluronic acid is diluted to the necessary volume with a diluent or solvent. The solution is then filtered through sterilized filters, bottled or ampouled. The resultant solution is a stable transparent liquid, and does not contain any chemical or other impurities.
Solid form preparations for oral administration can be made in the form of tablets, powders, or capsules. It may contain a medium for the active substance and other additives, including dyes, aromas, etc.
The compositions and treatments are indicated as therapeutic agents or treatments either alone or in conjunction with other therapeutic 20 agents or other forms of treatment.
In one embodiment, chlamyhyaluronic acid of the invention or capsules containing the chlamyhyaluronic acid may be formulated into pharmaceutical compositions for adminstration to subjects for wound healing. Chlamyhyaluronic acid facilitates the formation of stable 25 colloidal suspensions in pharmaceuticals. As with other known hyaluronic acids, chlamyhyaluronic acid reacts with proteins to form either a granular precipitate or a stable "mucin" clot, i.e. it forms stable colloidal suspensions with acidic serum. This property, as well as its antibacterial activity, point to the efficacy of chlamyhyaluronic acid as an 30 agent for wound healing. Chlamyhyaluronic acid may be used to reduce tissue fibrosis in injured tissue caused by, for example, skin burns, ulcers, ruptured tympanic membranes and abraded cornea.

Accordingly, the present invention provides a pharmaceutical composition for reducing tissue fibrosis comprising chlamyhyaluronic acid in admixture with a suitable diluent or carrier.
The present invention also provides a method for reducing tissue fibrosis comprising administering a therapeutically effective amount o~ a chlamyhyaluronic acid to a patient in need thereof.
In another embodiment, chlamyhyaluronic acid of the invention and capsules containing the chlamyhyaluronic acid may be formulated into pharmaceutical compositions for adminstration to 10 subjects for the treatment of cystitis.
Accordingly, the present invention provides a pharmaceutical composition for reducing cystitis comprising chlamyhyaluronic acid in admixture with a suitable diluent or carrier.
The present invention also provides a method for reducing cystitis 15 comprising administering a therapeutically effective amount of a chlamyhyaluronic acid to a patient in need thereof.
In another embodiment, the chlamyhyaluronic acid of the invention and capsules containing the chlamyhyaluronic acid may be formulated into pharmaceutical compositions for adminstration to 20 subjects as a biological fluid replacement or biological fluid supplement.
For example, chlamyhyaluronic acid or preparations containing chlamyhyaluronic acid may be used as a fluid replacement to correct pathological conditions in the eye or the joints. Like hyaluronic acid, chlamyhyaluronic acid polymers may be very large and can displace a large 25 volume of water. This property makes them excellent lubricators and shock absorbers. Chlamyhyaluronic acid or preparations containing chlamyhyaluronic acid may be used in ophthalmic surgery or as therapeutic, auxiliary and substitutive agents for natural organs and tissues, and may be particularly useful in the treatment of arthropathies.
Accordingly, the present invention provides a pharmaceutical composition for biological fluid replacement or biological fluid supplement comprising chlamyhyaluronic acid in admixture with a suitable diluent or carrier. The present invention also provides a method for replacing or supplementing biological fluids comprising administering a therapeutically effective amount of a chlamyhyaluronic acid to a patient in need thereof.
In another embodiment, chlamyhyaluronic acid of the invention and capsules containing the chlamyhyaluronic acid may be formulated into pharmaceutical compositions for administration to subjects to treat cancer. Research indicates that hyaluronic acid may play a role in cancer development and progression. Hyaluronic acid is involved 10 in both cell transformation and cancer cell metastasis. Paradoxically, in large quantities it can also retard cancer development. Thus chlamyhyaluronic acid may be particularly useful as a cancer treatment, and adjunct to cancer treatment, or a drug delivery vehicle for cancer treatment. Hyaluronic acid, and by extension, chlamyhyaluronic acid, may 15 have particular utility in treating basal cell skin carcinoma, actinic keratoses, precancerous lesions resulting from sun exposure, melanoma, ovarian cancer and breast cancer .
Accordingly, the present invention provides a pharmaceutical composition for cancer treatment comprising 20 chlamyhyaluronic acid in admixture with a suitable diluent or carrier.
The present invention also provides a method for treating cancer comprising administering a therapeutically effective amount of a chlamyhyaluronic acid to a patient in need thereof.
In another embodiment, chlamyhyaluronic acid of the 25 invention and capsules containing the chlamyhyaluronic acid may be formulated into pharmaceutical compositions for adminstration to subjects for the purposes of pain relief. Thus in this embodiment, chlamyhyaluronic acid may have pain relief utility, for example, in arthritis treatment.
Accordingly, the present invention provides a pharmaceutical composition for pain relief comprising chlamyhyaluronic acid in admixture with a suitable diluent or carrier. The present CA 02249l03 l998-09-30 invention also provides a method for relieving pain comprising administering a therapeutically effective amount of a chlamyhyaluronic acid to a patient in need thereof.
In any of the above-noted pharmaceutical uses, chlamyhyaluronic may be particularly useful when used in conjunction with a biologically active protein.

(2) Cosmetics In another embodiment, chlamyhyaluronic acid of the invention and capsules containing the chlamyhyaluronic acid may be 10 formulated into cosmetic compositions. Because of its hygroscopic nature, thin layers or films of cell encapsulations containing chlamyhyaluronic acid impart moisture to the skin. As chlamyhyaluronic acid facilitates the formation of stable colloidal suspensions, it may also be used as a source of mucilage in cosmetics and skin-care products.
Accordingly, the present invention provides a cosmetic composition comprising chlamyhyaluronic acid in admixture with a suitable diluent or carrier.
Like hyaluronic acid, chlamyhyaluronic acid is extremely hydrophilic, and may be able to mix with and hold in place several hundred times its weight in liquid. Thus chlamyhyaluronic acid may facilitate the ability of skin to hold its moisture, making it a useful moisturizing ingredient in cosmetics.
Accordingly, the present invention provides a moisturizing composition for moisturizing mammalian skin, hair or nails, comprising chlamyhyaluronic acid in admixture with a cosmetically suitable vehicle, diluent or carrier.
The chlamyhyaluronic acid of the invention and capsules containing the chlamyhyaluronic acid may be formulated into ultra violet ray blocking compositions suitable for topical administration.
30 Chlamyhyaluronic acid and catalase components of the capsules protect the skin from reactive oxygen radicals. Furthermore, chlamyhyaluronic acid's skin adhesive characteristics contribute to its cosmetic utility as a potential ultra-violet ray screening agent.
Accordingly, the present invention provides a sunscreen composition for the photoprotection of mammalian skin or hair, 5 comprising chlamyhyaluronic acid in admixture with a suitable vehicle, diluent or carrier.

(3) Foodstuffs The chlamyhyaluronic acid of the invention and capsules containing the chlamyhyaluronic acid may be formulated into 10 compositions in an edible form for use in foodstuffs.
In one embodiment, chlamyhyaluronic acid of the invention and capsules containing the chlamyhyaluronic acid may be formulated into gum compositions for use as a gum additive to foodstuffs.
Chlamyhyaluronic acid may be particularly useful as a gum for the food 15 industry because it facilitates the formation of stable colloidal suspensions.
Accordingly, the present invention provides a composition for gelling a foodstuff comprising chlamyhyaluronic acid in admixture with a suitable diluent or carrier. The present invention also provides a method for gelling a foodstuff comprising contacting the foodstuff with an 20 effective amount of chlamyhyaluronic acid.
In another embodiment, chlamyhyaluronic acid of the invention and capsules containing chlamyhyaluronic acid may be formulated into compositions for use as a preservative in foodstuffs. The present inventors have found that homogenates of cell encapsulations 25 containing chlamyhyaluronic acid prepared at room temperature in open air and stored over a year at 4-6~C have not been the subject of fungal or bacterial contamination. Hence, the homogenates are bacterio- and/or fungi-static and may be of benefit for the food industry as a preservative.
Accordingly, the present invention provides a composition 30 for inactivating enzymes, microorganisms or spores in a foodstuff comprising chlamyhyaluronic acid in admixture with a suitable diluent or carrier. The prf~sent invention also provides a method for inactivating CA 02249l03 l998-09-30 enzymes, microorganisms or spores in a foodstuff comprising contacting the foodstuff with an effective amount of chlamyhyaluronic acid.

(4) Biosensor The chlamyhyaluronic acid of the invention and capsules containing the chlamyhyaluronic acid may be formulated for use in electronic applications. Because of its photonic property, chlamyhyaluronic acid may also be used as a matrix biopolymer and/or a biosensor for future electronic applications as recently suggested by Angell, Science, 267, pgs 1924- 1935 (1995) and Meerholz, K. et al., The Spectrum, 8, 10 pgs 1-6(1995).
Accordingly, the present invention provides a matrix biopolymer or a biosensor comprising chlamyhyaluronic acid in admixture with a suitable matrix or solution. The present invention also provides a method for sensing occurrences photonically, comprising detecting changes in a detectable amount of chlamyhyaluronic acid.
EXAMPLES

Selection of Algae The phylum Chlorophyta, also known as the division Chlorophycota or the class Chlorophyceae, includes Tetraspora, Gloeomonas, Chlamydomonas segnis, Chlamydomonas gymnogama, Chlamydomonas pallidostigmatica, Chlamydomonas augustae, Chlamydomonas intermedia, Chlamydomonas sajao Levin and Chlamydomonas corrosa. These are eukaryotic unicellular green algae containing chlorophyll a and b, with 2 flagella (motile) or without flagella (non-motile), or colonial (coenobia), possessing a pyrenoid or pyrenoids associated with the chloroplast and storing starch.
Chlamydomonas segnis, isolated by S. Badour in 1969, was deposited in 1971 under # 1919 in The Culture Collection of Algae at Indiana University, which subsequently moved to the University of Texas (UTEX) at Austin (Starr, R.C., and Zeikus, J.A. Journal of Phycology (Supplement) 23: 8(1987); Starr, R.C. and Zeikus, J.A., Journal of Phycology (supplement) 29:19-20 (1993)). Another culture of ChZamydomonas segnis Ettl was deposited in 1985 under #1.79 in the Center of Algal Collection at the Institute of Plant Physiology, University of Gottingen, Germany (Schlosser, U.G. SAG- Sammlung von Algenkulturen at the University of 5 Gottingen, Botanica Acta, 107:129 (1994)). Chlamydomonas segnis Ettl is also formerly known as Tetraspora species having accession No. ATCC
30631, Gloeomonas species, Chlamydomonas gymnogama, Chlamydomonas pallidosfigmatica, Chlamydomonas intermedia, Chlamydomonas sajao Levin, which have been assigned accession No.
10 1919, 1638, 1343, 1905, 222, and 2277 according to the list of Richard, C., Starr and Jeffrey, A., Zeikus in Journal of Phycology supplement to volume 29 of April 1993. Because of nomenclatural uncertainty, two Chlamydomonas species named Chlamydomonas augustae and Chlamydomonas corrosa, which release extracellular polysaccharides into 15 the surrounding media, as reported by Allard, B. and Tazi, A., Phytochemistry, 32,pgs 41-47 (1993), may later be identified as Chlamydomonas segnis Ettl. In 1980, two cultures of Chlamydomonas segnis Ettl, the composition of a high salt nutrient medium and description of a method used for producing synchronous cultures of this 20 alga were sent to the Ecotoxicology group of the National Research CounciI of Canada, Ottawa. (Weinberger, P., et al., Canadian Journal of Botany 65: 696-702 (1987); Dechacin, C., et al., Exotoxicology and Environmental Safety 21: 25-31 (1991)).
In nature, Chlamydomonas segnis Ettl grows in shade (i.e. at 25 irradiance between 10-25 Wm-2), in soil ditches and roadside pools with relatively high salt concentrations (15-30 mM). For active growth in the laboratory, it requires all the essential macro- and micro-nutrients common to most land plants (Salisbury, F.B., and Ross, C.W. Plant Physiology, Wadsworth Publishing Company, Belmont California (1992) 30 pp.l16-135). Therefore, low-salt nutrient media coupled with high irradiances would constitute stressful cultural conditions for this particular microalga.

UTEX culture No.l919, provided on proteose agar slants was selected for use in the examples described herein. UTEX culture No.1346, a Chlamydomonas segnis Ettl isolate of H. Ettl appears identical to UTEX
culture No.l919, as it produces encapsulated cells when grown under the culture conditions for the production of chlamyhyaluronic acid described herein.
Secondary stock cultures were aseptically prepared for the inoculation of the low salt boron-deficient nutrient medium used herein for the mass cultivation of Chlamydomonas segnis Ettl. For this purpose, 10 inocula from the stock cultures of UTEX culture No.l919 were transferred under sterile conditions to 1.6% agar slants of the inorganic low salt boron-deficient nutrient medium in 16-20 ml capacity test tubes with loosened screw plugs to allow the diffusion of air-CO2. These stock cultures are then kept either at room temperature facing diffused day light 15 from west-located windows, or placed in a growth chamber at 18-20~C and irradiance of 8-10 Wm-2 using cool white or day light fluorescent lamps 400-700 nm)-Liquid precultures of about 150 mls, prepared by inoculating the Woods Hole MBL pH 7.2 nutrient medium with Chlamydomonas 20 segnis Ettl from the agar slants described above, were usually used as the stock cultures. These precultures were maintained at 18-20~C and low irradiances of 8-10 Wm-2, and diluted every two to three weeks to a cell density of about 2X106 cells ml-l. Suspensions of the algae between 25 mls and 50 mls secured from a liquid stock preculture with a cell number of 25 about 4X106 ml-l were used to inoculate one litre of the low salt boron-deficient nutrient medium adapted in this novel method for the production of CENC.
EXAMPLE Z
Selection of Nutrient Medium Chlamydomonas segnis Ettl, like many other unicellular green microalgae, grows photoautotrophically in any mineral nutrient medium as long as it contains all the essential macro- and CA 02249l03 l998-09-30 micro-elements, particularly in high salt nutrient media as those listed in Methods in Enzymology (edited by A. San Pietro), 23 part A, pgs 29-96 (1971), or recommended by Kuhl, A. and Lorenzen, H., Methods in Cell Physiology (edited by D. M. Presscott), 1, pgs 159-187(1964).
In laboratory cultures, synthetic nutrient media generally contain the 18 essential elements oxygen, carbon, hydrogen, nitrogen, potassium, calcium, phosphorus, magnesium, sulfur, chlorine, iron, manganese, copper, boron, zinc, molybdenum, silicon, and nickel for higher plants or cobalt for microalgae. A balanced nutrient medium 10 containing the essential macro- and micro-elements required for cell growth and division, even for a short period of time is generally at a pH
value between 6.0 and 7.5, and osmotic potential between about -0.126 MPa and about -0.01 MPa, at temperatures within about 20~C to about 30~C
[Salisbury, F.B., and Ross, C.W. Plant Physiology, Wadsworth Publishing 15 Company, Belmont California, pp. 116-135, 226-233 and 240-241 (1992);
Nichols, H. Wayne. In: Handbook of Phycological methods Vol. I, pp. 8-23.
Edited by Stein, R. Janet. Cambridge University Press (1973); Starr, Richard C. In: Methods in Enzymology, Vol. 23, Part A, pp. 29-52. Edited by San Pietro, A. Academic Press (1971)]. Water soluble salts are used to provide a 20 variety of balanced nutrient media which become exhausted at the end of the exponential phase of the algal growth.
A low salt nutrient medium with a total salt concentration lower than 6.0 mM either during the lag phase, exponential phase, or the stationary phase of the algal culture supports the algal growth and cell 25 division in cultures, even for a short period of time, or sustains the photosynthetic capacity of the algal cells in the culture to synthesize and accumulate secondary metabolites. The use of low-salt nutrient media would shorten the time required for the algal culture to attain the stationary phase of growth, during which the cells neither grow nor divide 30 due to the lack of one or more essential elements. Photoassimilation of inorganic carbon, however, continues so that polysaccharides as well as organic acids are synthesized and then excreted (Badour, S.S. Excess light CA 02249l03 l998-09-30 evokes cell encapsulation in Chlamydomonas segnis Ettl. Abstracts of the International Meeting on Molecular Biology, Biochemistry and Physiology of Chloroplast Development. Philipps Universitat, Marburg, Germany (1995); Allard, B., and Tazi, A~ Phytochemistry, 32:41-47(1993)) Boron-deficient cells are expected to have an inelastic cell wall that cannot undergo normal cell-stretching growth as reported for higher plants (Hu, Hening, and Brown, Patrick H. Plant Physiology, 105:
681-689 (1994)). Cell wall expansion and plasticity which are associated with cell growth and division, require the formation of borate-ester 10 cross-links with mucopolysaccharides (e. g. pectin ) in the cell wall.
Inhibition of cis-diol boric acid complexes due to boron deficiency would lead to decreases in cell plasticity and cell growth and may result in the formation of mucilaginous thickened cell walls (Hu, Hening, and Brown, Patrick H. Plant Physiology, 105: 68l-689 (1994); Raven, J.A. New 15 Phytologist, 84: 231-249(1980)).
The nutrient medium designated Woods Hole MBL pH 7.2 (Nichols, H. Wayne. Growth media- freshwater. In: Handbook of Phycological methods Vol. I, pp. 8-23. Edited by Stein, R. Janet. Cambridge University Press (1973) pp.l6-18 and table I-3),is low in salt (5.725 mM, or 20 1.725 mM without added Tris-HCl buffer) and boron deficient. The term boron-deficient medium or boron-deficiency in the context of mass cultivation of algae is used herein to mean a nutrient medium in which a generation time shorter than 48 hours is obtained, or one in which inhibited algal cell growth is completely restored and cell encapsulation 25 may be retarded by adding only either boric acid or its sodium or potassium salts to the medium at a final concentration of one millimolar or less.
This medium was selected and modified to produce cultures of ChZamydomonas segnis Ettl with reduced exponential growth, and cells 30 with thick mucilaginous matrix (Handbook of Phycological Methods (edited by Janet R. Stein), 1, pgs 16-18 (1973)).

In particular, the medium was modified by excluding vitamins, sodium carbonate, sodium silicate, and reducing the concentration of Tris-HCI from 4000 ,uM to 500 ,uM as seen in TABLE 1. As a result, the initial total salt and buffer concentration of the modified nutrient medium becomes 1.975 mM instead of 5.725 mM. The decrease in the concentration of the Woods Hole MBL pH 7.2 nutrient medium brings about a drop in its osmotic potential from about -0.029 MPa to about -0.01 MPa at temperatures between 20~C and 30~C; thereby increases the rate of water movement into the algal cell.
0 Chlamydomonas segnis Ettl is considered a soil microalga adapted to relatively high salt surrounding media. The use of the modified low salt boron-deficient nutrient medium, designated B in TABLE 1, was selected to add more stress to the algal cells already exposed to excessive light and accelerate cell encapsulation. In constrast, the high 15 salt nutrient medium of Kuhl and Lorenzen, which is slightly modified by Badour, S. and Waygood, E. R., Phytochemisltry, 10, pgs 967-976 tl971), shown in TABLE 1, column C, and has almost 10-fold the total salt concentration of the modified Woods Hole MBL pH7.2 nutrient medium, will exert its effect as a stress factor for the said microalga in light only 20 when salt becomes depleted.
Figure 3 shows the growth curves of deep cultures of Chlamydomonas segnis Ettl produced at 25~C in the low salt boron-deficient rnedium given in TABLE 1, column A, and the high salt nutrient medium given in TABLE 1, column C, but without adding boric 25 acid. All cultures were bubbled with air and exposed to 30 Wm-2 for 72 hours, followed by aeration with 5% CO2 in air and increasing irradiance to 60 Wm-2 for the low and high salt-A cultures, and to 135 Wm-2 for the high salt-B culture. The term deep culture is used herein to mean a culture in which the growing algal suspension has a depth between about 30 10cmandaboutl2cm.
The results presented in Figure 3 show that deep cultures of Chlamydomonas segnis Ettl grown in the high salt boron-deficient CA 02249l03 l998-09-30 medium with the initial salt concentration of 19.350 mM due to boric acid exclusion, require at least twice as much irradiance as those grown in the low salt boron-deficient medium to induce cell encapsulation. The pronounced decline of cell number in the low salt boron-deficient culture after being exposed to 60 Wm-2, compared to the slight decrease of cell number in the high salt (B) boron-deficient culture which receives 135 W m -2, indicate that the algal cells' encapsulation rate in the former culture is 2.5 times greater than in the latter culture. The term "encapsulation rate" is used herein to mean the decrease in the cell 10 number, determined by a hemacytometer, per unit time, i.e. per hour(s), that takes place in algal cultures at early or late stationary phases of growth, as a result of cell encapsulation and capsule enlargement that causes progressive cell-spacing and consequently decreases in cell counts within the hemacytometer chamber.
Importantly, the phase of cell number decrease represents the encapsulation phase. During this phase the enlargement of the algal capsule, which is only visl~ali7etl by metachromatic dyes, causes the spread of the greenish algal cells within the chamber of the hemacytometer used for counting the cells. As a result, fewer cells are counted due to the increase in capsule size, and a comparison of the ascending slopes seen in Figure 3 would reflect the rate of encapsulation and the relative average size of the capsule.
Figure 4 shows photomicrographs, with a scale bar of 20 ,um, of encapsulated cells of Chlamydomonas segnis Ettl at the end of encapsulation phase, stained either with toluidine blue (a and b) or alcian blue (c), from (a) low salt (5.725 mM) boron-deficient culture at pH 7.2 enriched with 10 mM sodium nitrate and 4.5 mM dipotassium hydrogen phosphate, and (b) high salt (19.35 mM) boron-deficient culture at pH 4.5, and (c) low salt (5.725 mM) boron-deficient culture at pH 5Ø As seen from the photomicrographs in Figure 4, the encapsulated cells of Chlamydomonas segnis Ettl vary in the size and structure of the capsule, CA 02249l03 l998-09-30 which are influenced by the salt concentration and composition of the nutrient medium as well as the level and duration of irradiance.
Low salt nutrient media can be prepared by diluting high salt nutrient media with water. For example, when the high salt nutrient medium given in TABLE 1, column C, is diluted by a factor of five, its initial salt concentration drops from 19.352 mM to 3.87 mM and the initial boric acid concentration becomes 0.3,uM instead of 1.5,uM.
In an alternate embodiment, one can select glucose as an additional source or as the only source of carbon for phototrophic cultures 10 of the alga for the production of encapsulated cells in low salt boron-deficient nutrient medium. Provision of glucose may lower the activity of Rubisco, the enzyme which catalyzes the fixation of carbon dioxide and the formation of the organic material needed for cell encapsulation. As a result, the blue: red spectral ratio greater than one will not be required.
Instead, a blue: red spectral ration less than one would be appropriate for active glucose uptake and chlamyhyaluronic acid production. However, the use of glucose may require one to maintain sterile conditions throughout the culturing process.
Under the stressful conditions of the low salt boron-deficient nutrient medium when glucose alone (i.e. in carbon dioxide-free air) is used for the production of CENC in liquid, semi-solid, or solid cultures in light, the cultures are photoorganic cultures. When air (i.e. with about 0.03% carbon dioxide) is provided to these cultures instead of carbon dioxide-free air, the cultures are photomixokophic cultures.

Selection of Light When photoautotrophic plants such as algae are exposed to excessive light, the absorbed but not utilized quanta can destroy the photosynthetic apparatus due to over-excitation (Long, S.P., and Humphries, S. Annual Review of Plant Physiology and Molecular Biology, 45: 633-622 (1994)). Utilization of quanta will decrease when an essential element is deficient or unavailable for the growing cells.

However, the detrimental effect of high, i.e. excessive, light is minimized through energy dissipation and photoprotection (Deemmig-Adams, B. and Adams, W.W. III, Annual Review of Plant Physiology and Molecular Biology, 43: 599-625 (1992)).
As previously stated, "excessive light" is the light intensity at which the inception of cell encapsulation in algal cultures exposed to air, aerated with either air or air enriched with carbon dioxide, is detectable by metachromasy.
There is no standard minimal level of irradiance for all 10 cultures. Cell encapsulation usually occurs in dim day-light in aged cultures, as for example in the immobilized algal cells which are maintained on agar slants for relatively long period of time in presence of air. Dim light, e.g. irradiances of 2 to 5 Wm-2, may be excessive for cultures deprived of some essential macro- or micro-elements.
Imposition of intense light on low-salt cultures of Chlamydomonas segnis Ettl, which normally thrives at relatively low irradiances in a high salt medium (Badour, S.S., et al. Journal of Phycology, 13:80-86 (1977); Badour, S.S. Journal of Phycology, 17:293-299 (1981); Badour, S.S., and Irvine, B.R. Botanica Acta, 103: 149-154 (1990)), was found to encourage the cells to develop mechanisms for photoprotection. The ability of the alga to produce the compounds required for such mechanisms may be linked to the carboxylase activity of ribulose-1,5-bisphosphate carboxylase-oxygenase (Rubisco) and the levels of its substrates (Salisbury, F.B., and Ross, C.W. Plant Physiology, Wadsworth Publishing Company, Belmont California, pp. 226-233 (1992)).
In Chlamydomonas segnis Ettl, the carboxylase function of Rubisco reaches its highest level of specific activity at the stationary phase of growth (Badour, S.S. In:Handbook of Phycological Methods. Vol. II, pp.
209-216. Edited by Hellebust, J.A. and Craigie, J.S. Cambridge University Press (1978)), and blue rather than red light increases such activity (Badour, S.S. et al., Journal of Phycology (supplement) 8:16 (1972)). In other algae, blue light also enhances the synthesis of photosynthetic pigments and CA 02249l03 l998-09-30 proteins (Laudenbach, B., and Pirson, A., Archiv fur Microbiology, 67:
226-242 (1969); Wallen, D.G., and Geen, G.H.. Marine Biology, 10: 44-51 (1971)). The protein that activates rubisco, i.e. rubisco activase, functions in light and loses activity in darkness or dim light (Salisbury, F.B. and 5 Ross, C.W., Plant Physiology, Wadsworth Publishing Company, Belmont, California, pp.240-241 (1992)). Therefore, the present inventor selected excessive light with higher blue:red spectral ratios to ascertain its effect on the developmental changes in Chlamydomonas segnis Ettl for photoprotection.
The term "blue light" is used herein to mean irradiances with spectral range between about 320 nm and about 500 nm, which includes bluish green-light, blue-light, violet-light and ultraviolet A of about 320 nm to about 380 nm, and the term "red light" is used herein to mean irradiances with spectral range between about 600 nm to about 700 nm, 15 which does not include far red.

Regulation of Carbon Dioxide Carbon dioxide is the inorganic substrate used by Rubisco to form the organic acids which provide the carbon skeleton for the 20 biosynthesis of cellular compounds (Badour, S.S., and Tan, C.K. Zeitschrift fur Pflanzenphysiologie, 112:287-295 (1983)). These compounds are required for the growth of the algal cells as well as for developmental changes that would occur in response to stressful environments such as excessive light. For the enhancement of carbohydrate and protein 25 accumulations, CO2 tensions higher than those at air levels should be continuously supplied to the cells by bubbling the algal cultures with 0.1-5% CO2 (by volume) enriched air (Badour, S.S., et al., Canadian Journal of Botany, 51:67-72 (1973); Badour, S.S., et al. Journal of Phycology, 13:80-86 (1977); Badour, S.S. Journal of Phycology, 17:293-299 (1981)). To 30 minimize the escape of CO2 from the nutrient medium at relatively higher temperature, ChZamydomonas segnis Ettl cultures were kept at 20-30~C. To avoid increases in bicarbonate and carbonate ions which CA 02249l03 l998-09-30 prevail in alkaline media bubbled with CO2, the culture medium must be maintained at around pH 7. At such pH and temperature range, Chlamydomonas segnis Ettl cells exhibit maximal growth and biosynthetic activity (Badour, S.S., et al. Journal of Phycology, 13:80-86 (1977); Badour, S.S. Journal of Phycology, 17:293-299 (1981); Badour, S.S., and Irvine, B.R. Botanica Acta, 103:149-154 (1990); Badour, S.S., and Tan, C.K.) Zeitschrift fur Pflanzenphysiologie, 112:287-295 (1983); Badour, S.S., and Tan, C.K. Plant and Cell Physiology, 28:1485-1492 (1987)).
Continuous aeration of Chlamydomonas segnis Ettl cultures 10 from the start, by air enriched with CO2 (0.1 to 5% C Oz)~ compared to aeration only by air (0.03% C ~2)~ enhances the accumulation of carbohydrates and proteins within the relatively large algal cells as reported by Badour et al., J. of Phycology, 13:80-86 (1977) and Badour, J. of Phycology, 17:293-299 (1981). The carbohydrates and proteins, which start to accumulate in such cultures at early exponential phase of growth are probably used at late exponential phase of growth for the synthesis of intracellular carotenoids among other components, e.g. lipids, instead of extracellular mucopolysaccharides within the cell capsules for photoprotection. An example is shown in Figure 2, which depicts the increase in carotenoid content as indicated by the absorbances of acetone extracts between 420 nm and 480 nm from shallow cultures aging in low salt boron-deficient nutrient medium bubbled with 5% C ~2 in air at 25~C
and about 40 Wm-2. Figure 2 shows the absorbance spectra (normalized at 662 nm) of acetone extracts from shallow cultures of Chlamydomonas segnis Ettl, using Hewlett Packard Diode Array Spectrophotometer Model 8452 A, at (a) late exponential phase of growth, (b, c and d) early and mid encapsulation phase, and (e) late encapsulation phase. In contrast, their counterparts which are continuously bubbled with air, i.e. 0.03% C ~2~
produce relatively small cells with significantly less carbohydrate and protein contents, but with higher catalase activity and ascorbate/
dehydroascorbate metabolism than the large cells produced in 5% C ~2 probably for photoprotection against oxygen free radicals generated in light at air levels of CO2, as pointed out by Raven, J. A. et al.,Biological Reviews, 69, pgs 61-94 (1994). As the air-bubbled shallow cultures age, the cells show little increases in their low carotenoid content and slowly enter 5 the encapsulation phase. These cultures exhibit a greenish hue. In 5%
CO2 -bubbled shallow cultures, the cells continue to actively increase their carotenoid content as they age and quickly enter the encapsulation phase.
These cultures exhibit a yellowish hue. The term shallow culture is used herein to mean a culture in which the growing algal suspension has a 10 depth between about 3 cm and about 4 cm.
Accordingly, for the production of large capsules and regardless of the cell size of Chlamydomonas segnis Ettl, the mineral nutrient medium, after being inoculated, is aerated with only air until about mid-exponential phase of growth, i.e. at cell numbers between 3 and 15 4 X106 cells ml-l depending on the inoculum size, and then 5% CO2 in air is introduced to lower the oxygen tension and stimulate the accumulation of the mucopolysaccharides in the algal capsule.
Exposure of Chlamydomonas segnis Ettl cells growing in a low-salt medium to high CO2 concentrations will lead to an initial 20 enhancement of cell growth and division concomitant with a rapid progressive reduction of the nutrients in the culture medium. At later stages when the cells enter the stationary phase of growth due to nutrient depletion, CO2 will be actively photoassimilated as long as the carboxylase function of Rubisco is maintained, e.g. by blue light. In other words, the 25 continuous provision of high CO2 will enable Chlamydomonas segnis Ettl to utilize more of the absorbed excessive light to synthesize intra- and extra-cellular compounds, and thereby minimize over-excitation and mitigate the destruction of the chloroplast.

Selection of Apparatus The apparatus required for the cultivation of photoautotrophic microalgae is basically a combination of four 5 constituents, namely culture vessels, a temperature controlled space (e.g. a chamber or tank), lighting source and aeration equipment. The process of mass cultivation of Chlamydomonas segnis Ettl described herein specifies the physical and cultural conditions under which the aforenamed microalga produces algal biomass of encapsulated cells at low cost within a 10 relatively short period of time.
Routinely four-litre deep cultures of Chlamydomonas segnis Ettl were grown in the modified low salt boron-deficient nutrient medium given in TABLE 1, column B, using round vessels of 6 litres capacity at 25~C, exposed to irradiance of about 30 Wm-2 and bubbled with air for 72 15 hours, followed by increasing irradiance to about 60 Wm-2 and switching aeration to 5% CO2 in air for 72 hours. Shallow cultures of aged Chlamydomonas segnis Ettl were produced under the same cultural conditions, using low form Fernbach flasks. Shallow cultures of Chlamydomonas segnis Ettl were produced under the same cultural 20 conditions, but with continuous aeration from the start with either air or 5% CO2 in air for 150 hours.
Round glass vessels (6 likes capacity) fitted with inlet and outlet tubing for aeration are used for producing deep cultures of four litres algal biomass per vessel. In order to demonstrate that under the 25 same physical and cultural conditions, Chlamydomonas segnis Ettl cells grow and age faster in shallow than in deep cultures, low-form Fernbach flasks of about 3 litres capacity were also used to obtain one to one and a quarter litre of algal biomass per flask.
In an alternate embodiment, encapsulated cells of Chlamydomonas segnis Ettl can be produced on solid nutrient substrates, as for example on 5 to 10 mm thick layers of 1.6-2.0% agar dissolved in low salt boron-deficient nutrient medium, using flat trays exposed to natural day-light or cool fluorescent light in air.
Any of the temperature-controlled chambers with circulated filtered air and fluorescent lamps designed for growing green plants may 5 be employed in the process of the present invention. For example, Conviron growth chambers from Controlled Enviroments Limited, Winnipeg, Canada, equipped with cool-white 1500 General Electric (GRO
and SHO Wide Spectrum), or cool-white Sylvania GTE
(GRO-LUX-WS-very high output) fluorescent lamps, and standard 10 incandescent lamps (40-60 W) were used. Alternately, cool-white or day-light fluorescent lamps (CRI 75, 6500K) from DURO-TEST
Corporation, North Bergen, New Jersey 07047, may be used. Figure 1 depicts the changes in the levels of blue, red, and far red spectra with increasing irradiances from cool-white fluorescent and standard 15 incandescent lamps.
In order to check that the excessive light applied to Chlamydomonas seg~is Ettl cultures for the production of CENC provides blue:red spectral ratios higher than one, a plant growth photometer, IL 150 from International Light Inc. (Dexter Industrial Green, Newburyport, 20 Massachusettes 01950) was used to measure the levels of blue (400-500 nm), red (600-700 nm) and far red (700-800 nm) spectra radiating from the fluorescent and incandescent lamps at various irradiances. Irradiances (Wm-2) were measured with a LI-185B radiometer using a LI-COR, Inc.
pyranometer sensor from LI-COR, Inc., Lincoln, Nebraska 68504. As 25 shown in Figure 1, the cool-white fluorescent and incandescent lamps of the Conviron chamber provide irradiances of up to 145 Wm-2, and the high irradiances between 90 Wm-2 and 145 Wm-2 are due to small increases in the red and far red spectra rather than the blue spectrum.
Since standard incandescent lamps, e.g. with CRI 98 and 3200 K have a 30 blue:red ratio of about 0.5 including the violet and ultraviolet spectra as part of the blue, their use is limited to radiating dense cultures with algal suspensions more than 4 cm in depth. Thus, incandescent lamps are used CA 02249l03 l998-09-30 to supplement dense Chlamydomonas segnis Ettl deep cultures with irradiances higher than 90 Wm-2, which offset cells' self-shading by furnishing sufficient red light for active photosynthesis without virtually affecting the preexisting blue:red ratio of four.
Conventionally, compressed air is used for the aeration of microalgal cultures. The air pressure is regulated to allow gentle agitation of the algal suspension, and thereby maximize the cells' exposure to incident light. The air current is filtered to prevent culture contamination by dust particles and air borne fungal and bacterial spores. Continuous 10 aeration provides about 0.03% (volume by volume) CO2, which is the only source of carbon for photoautotrophic algal cells bubbled with air. Gas regulators, gauges and gas needle valves for regulating and measuring gas pressure from Matheson of Canada, Ltd. (Whitby, Ontario) and Union Carbide (Linden Division) were employed in the present invention.
15 Suitable flow rates of air or gas mixtures were judged by minimal water evaporation from the culture nutrient medium, and measured by Matheson flowmeters Model 665 with a tube Model No. 603 and steel float for air and nitrogen gas, and a tube Model No. 601 and steel float for CO2.
To obtain, for example, 5% C ~2 enriched air, or nitrogen gas (volume by 20 volume), CO2 at a flow rate of 100 ml/min was mixed with air or nitrogen gas at a flow rate of 2000 ml/min using the Matheson flowmeter Model 665. The regulated gas or gas mixture was directed to the culture vessel inlets via rubber or plastic tubing, and distributed to each culture vessel by a manifold. Air, CO2 enriched air, or nitrogen gas was introduced into 4 25 litres of the nutrient medium at a flow rate of about 1000 ml/min.

Culturing Chlamydomonas segnis Ettl for the Production of CENC
The efficient production of CENC is facilitated by the use of minimal concentrations of the nutrients in the medium coupled with 30 high inocula and low levels of irradiance to limit the microalgal growth and cell division. This approach accelerates cell entry into the encapsulation phase, i.e. the production of encapsulated algal biomass at small cost. The example shown in Figure 5 provides strong evidence in support of this thesis.
In contrast, when shallow cultures of Chlamydomonas segnis Ettl were grown in the high salt nutrient medium given in TABLE 1, column C (19.352 mM), and continuously exposed either to low irradiance of 30-35 Wm-2 or high irradiance of 58-65 Wm-2, and aerated with air for 72 hours followed by 150 hours aeration with 5% CO2 in air, the resulting algal biomass with a greenish hue was virtually deprived of encapsulated 10 cells.
However, algal growth in the high salt nutrient medium after being diluted to 3.87 mM and receiving the same type of aeration and level of irradiances as the high salt cultures, resulted in yellowish algal biomass. Cells' encapsulation occurs in such diluted high salt cultures 15 after relatively longer period~ of time, i.e. 22q hours or more, compared to about 120 hours in cultures produced with the modified Woods Hole pH
7.2 nutrient medium given in TABLE 1, column B. Furthermore, the encapsulation rate is comparatively low, and the encapsulated algal biomass is of poor quality due to predominance of large cells with small 20 capsules.
Greenish algal samples, secured from various cultures of Chlamydomonas segnis Ettl during the encapsulation phase, settled after standing at 4-6~C within 48-72 hours, compared to the yellowish algal samples which settled after 120-240 hours depending on their oil-content 25 levels.
Therefore, the greenish encapsulated algal biomass rather than the yellowish ones can easily settle and separate from the surrounding liquid medium; thereby allowing the removal of the clear supernatant by suction using vacuum devices.
Figure 5 depicts growth curves of Chlamydomonas segnis Ettl in shallow cultures, inoculated from a growing liquid precultures to give the initial cell density of about 0.1 x 106 cells ml-l, and bubbled with air for 48 hours followed by aeration with 5% CO2 in air for 48 hours at irradiance of (a) 18 Wm-2, (b) 42 Wm-2, and (c) 60 Wm-2, at 25~C in Woods Hole MBL
pH 7.2 medium (5.725 mM) as given in TABLE 1, column A (closed circles), and in the said nutrient medium after modification (1.975 mM) as given in TABLE 1, column B (open circles).
Thus, regardless of the level of irradiance, the use of the modified Woods Hole MBL pH 7.2 nutrient medium with a concentration of 1.975 mM instead of 5.725 mM has little effect on the onset time (around the 72nd hour) and rate of encapsulation, as indicated by the rate of 10 decrease in cell number between the 72nd and the 96th hour and highlighted by the use of a polynomial curve fit. At the high irradiances of 42 Wm-2 and 60 Wm-2, the number of cells in the nutrient medium with the initial concentration of 5.725 mM is about 133% greater than that with the lower concentration of 1.975 mM, probably due to the strong 15 buffering action of the high concentration of Tris-HCL used as given in TABLE 1, column (A). However, there appears to be a relationship between the level of irradiance and the initial concentration of the nutrient medium. This is because cultures produced in the modified nutrient medium (1.975 mM) are not significantly affected by increasing 20 irradiance from 18 Wm-2 to 42 Wm-2 and 60Wm-2, whereas those produced in the Woods Hole MBL (5.725 mM) show about 20 to 25%
increases in cell number after 72 hours. In other words, relatively high irradiances are required to promote Chlamydomonas segnis Ettl growth and particularly cell encapsulation in low salt boron-deficient nutrient 25 media with initial salt/nutrient concentration greater than 2.00 mM.
Figure 6 depicts the inverse relationship between the dilution index and the level of irradiance required for efficient (i.e. within a relatively short period of time), production of encapsulated algal biomass in shallow and deep cultures of Chlamydomonas segnis Ettl.
From the example shown in Figure 6, the dilution index, i.e.
the ratio of cell number (in 106 ml-l) at the onset of the encapsulation phase to the initial salt/nutrient concentration in mM of the medium, is used in the present invention to adjust irradiance level for the efficient production of encapsulated algal biomass in shallow and deep mass cultures of Chlamydomonas segnis Ettl. When the dilution index is about 0.52, i.e. the cell number ml-l (x106) is about half that of the initial nutrient 5 concentration (mM), the relatively high irradiance of 150 Wm-2 will be effective for the rapid induction of cell encapsulation in deep cultures of the said microalga. On the other hand, if the cell number ml-l (x106) of the same culture increases such that the dilution index becomes about 6.00, 40 Wm-2 will be sufficient to induce cell encapsulation.
The increase in the cell number occurs at the expense of nutrient utilization, which leads to a decrease in the salt/nutrient concentration of the medium. The resulting nutrient depleted medium acts as a stress factor, and under these stressful conditions the photoautotrophic algal cells become incapable of utilizing all the energy absorbed from incident light for ~rowth and cell division. Instead~ the microalgal cells undergo encapsulation and synthesize mucopolysaccharides within their capsules to offset the effect of excessive light, even at the low irradiances of 10 Wm-2 to 15 Wm-2 in shallow cultures or 20 Wm-2 to 25 Wm-2 in deep cultures.
Chlamydomonas segnis Ettl cells with large extracellular capsules are produced photoautotrophically in shallow and deep cultures within 90 to 150 hours from the time of inoculating a low salt boron-deficient nutrient medium, with an initial total salt concentration between 2 mM and 6 mM, and pH value from 6 to 7.5. (The term photoautotrophic is used herein to mean that the growth and cell division is achieved under illumination by providing only inorganic salts, i.e.
mineral nutrients which are assimilated by the algal cells at the expense of solely light energy via photosynthesis.) The inoculated nutrient medium with initial cell density between 0.1x106 cells and 0.2x106 cells ml-l was supplied with air, i.e. about 0.03% C ~2~ during the lag and mid-exponential phases of growth. At mid-exponential phase, air enriched with 0.1 to 5% CO2 (volume by volume) is provided to the cultures, which are placed after inoculation in a growth chamber at a temperature between 20~C and 30~C, and exposed to irradiances from fluorescent lamps with blue:red spectral ratios greater than one. During the first 72 hours of incubation and depending on the initial cell density, the inoculated nutrient medium was exposed to irradiances between 15 Wm-2 and 30 Wm-2 in case of shallow cultures, and to irradiances between 25Wm-2 and 30Wm-2 in case of deep cultures.
During the exponential phase of growth, when the cell density is between 3X106 cells and 4X106 cells ml-l, irradiances are increased 10 to a level within the 60 Wm-2 to 150 Wm-2 range for the induction of cell immobilization and encapsulation. Irradiances greater than 60 Wm-2 are applied to the cultures when the ratio of the cell number (in 106 ml-l) at the onset of the encapsulation phase to the initial salt concentration of the nutrient medium (in mM), i.e. the dilution index, is less than three.
15 The Encapsulation Phase The encapsulation phase is the stationary phase of growth or the second stationary phase of growth followed by a phase of decline in cell number. This is akin to the death phase discussed in some microbiology and phycology teachings. Thus, the early- and mid-encapsulation phases 20 discussed herein correspond to early and late stationary phases of growth.
However, the decline of cell number at the end of the stationary phase of growth in Chlamydomonas segnis Ettl cultures is due solely to enlargement of the capsules which encase viable cells.
Figure 7 shows photomicrographs, with a scale bar of 40 ,um, 25 of Chlamydomonas segnis Ettl stained with toluidine blue; (a) as capsule free cells at mid exponential phase of growth, (b) as cells with metachromatic extracellular matrix at the beginning of encapsulation, (c) as encapsulated cells with polymorphic capsules, and (d) as spaced cells due to capsule enlargement.
The encapsulation phase in Chlamydomonas segnis Ettl cultures commences when the capsule-free cells, with diameters between 6 ~m and 8 ,um at the end of the exponential phase of growth, as seen in CA 02249l03 l998-09-30 Figure 7, excrete layers of metachromatic polysaccarides that form relatively small capsules with diameters between 12 ,um and 18 ,um. Such capsules enlarge and attain various shapes depending on the density of the algal biomass. Polymorphic and large capsules with diameters ranging from 30 ,um to 80 ,um prevail by the end of the encapsulation phase. The volume of the capsule may reach over 200-fold that of the algal cells due to their high water content. When samples of the encapsulated algal biomass are dehydrated during the fixation process required for the preparation of ultra-thin sections for transmission electron microscopy (as 10 described by Badour et al, Canadian J. of Botany, 51, pgs 67-72 (1973)), the size of the capsule decreases by almost 98% as shown from the capsule in Figure 8 (c) and (d), with a diameter between 9 ,um and 10 ,um.
Figure 8 shows transmission electron micrographs, with a scale bar of 4 ,um, of Chlamydomonas segnis Ettl cell sections; (a) at mid 15 exponential phase of growth showing a chloroplast with interthylakoidal starch grains in a capsule free cell, and (b) starch sheath around the pyrenoid, (c) at late encapsulation phase showing the stratified structure of the capsule surrounding a cell with large starch grains within a fenestrated chloroplast and large dark lipid droplets, and (d) the relative thickness of 20 the capsule.
Furthermore, the scanning electron micrographs of desiccated samples of encapsulated Chlamydomonas segnis Ettl cells in Figure 9 reveal the heavily coated cells with mantles or thick chlamyses embedded in the dried thick matrix of extracellular polysaccharides. Figure 9 shows 25 scanning electron micrographs of desiccated cells of Chlamydomonas segnis Ettl at mid encapsulation phase, (a) coated with thick mantles of mucopolysaccharides, (b) in a fractured layer of dried extracellular mucopolysaccharide material released in the culture medium, and (c and d) embedded in the thick matrix of this material.
Visualization of the polysaccharide(s) content of the capsule in the immobilized cells of the said microalga shown in Figure 10 is achieved by autofluorescence due to the ability of the capsule material to dissipate ultraviolet A, and by the use of metachromatic dyes as toluidine blue (1% in borax) or solutions of alcian blue in water.
Figure 10 shows photomicrographs,with a scale bar of 60 ,um, of: (a) immobilized spaced cells of Chlamydomonas segnis Ettl at mid encapsulation phase, (b) the autofluorescence of the capsule material using excitation filters UV 330-380 nm and a Nikon epifluorescence microscope, and (c) encapsulated cells stained with alcian blue, or (d) toluidine blue to visualize the capsule.
Figure 11 depicts the retarding effect of the presence of one 10 ~M boric acid (+ Boron) in the low salt boron-deficient nutrient medium (- Boron) given in TABLE 1, column A, on cell encapsulation as indicated by the downturn of the cell number in shallow cultures of Chlamydomonas segnis Ettl, incubated at 25~C, bubbled with air for 72 hours at 25 Wm-2 to 30 Wm-2, and thereafter with 5% CO2 in air (volume 15 by volume) at 60 Wm-2 (high light). The example presented in Figure 11 shows that in shallow cultures the encapsulation phase, which is indicated by the downturn in cell number after 96 hours incubation at 25 Wm-2 to 30 Wm-2 followed by 60 Wm-2 at the 72nd hour, is delayed for 96 hours when boric acid or sodium borate is included in the low salt boron-deficient 20 medium, given in TABLE 1, column A, at a final concentration of one micromolar. Probably, in the boron repleted medium mechanisms other than cell encapsulation are operating for photoprotection under the stressful cultural conditions caused by nutrient depletion after 96 hours incubation at 60 Wm-2.
Figure 12 depicts the inhibitory effect of aerating Chlamydomonas segnis Ettl cells with 5% CO2 in N2 instead of 5% CO2 in air (volume by volume) at the end of the exponential phase of growth, on the encapsulation phase in shallow cultures provided with the low salt boron-deficient nutrient medium given in TABLE 1, column A, incubated 30 at 25~C, bubbled with air for 72 hours at 25 Wm-2 to 30 Wm-2, followed by 60 Wm-2 (high light) and aeration with 5% CO2 in air, which is replaced by 5% CO2 in N2 at the 96 hour to remove ambient oxygen. The graph in Figure 12 provides strong evidence that cell encapsulation and capsule enlargement are linked to the presence of air levels of oxygen. This is because bubbling shallow cultures of Chlamydomonas segnis Ettl by the end of the exponential phase of growth with 5% CO2 in nitrogen gas 5 instead of 5% CO2 in air (volume by volume) at 60 Wm-2 restores celI
division as indicated by the almost linear increase in cell number.
Another observation not shown in Figure 12 is that switching off light and maintaining the culture in darkness for 48 to 72 hours instead of bubbling the culture with 5% CO2 in nitrogen gas also restores cell 10 division. This suggests that air levels of oxygen promote the microalgal cell encapsulation in continuously illuminated nutrient depleted cultures of Chlamydomonas segnis Ettl, i.e. under conditions of excessive light.
Thus, cell encapsulation in cultures of the microalga is a means for protection against oxygen radicals which are generated through 15 the p~rtial reduction of oxygen ll.olecule~ during ph~to~yl.'hetic elec~on transport as explained in Plant Physiology Textbooks, (e.g. Mohr and Schopfer, Plant Physiology, Springer-Verlag, pgs 173-175 (1995)). Such electrons are not used for biosynthetic activity towards cell growth and division in nutrient depleted media. The high activities of photosystems I
20 and II as well as catalase at the early phase of cell encapsulation, shown inTABLE 2, support this conclusion and point to the potential use of CENC, harvested at early to mid encapsulation phases, as an effective ingredient for protection against oxygen radicals.

25 l~xtraction of CENC from the Algal Medium For the production of encapsulated algal biomass of Chlamydomonas segnis Ettl with large capsules, deep cultures were grown in the modified Woods Hole MBL nutrient medium pH 7.2 as given in TABLE 1, column B, using the aforedescribed method of culturing the said 30 microalga. When the greenish microalgal biomass becomes viscous due to the formation of large metachromatic capsules, i.e. at mid encapsulation phase, the microalgal suspension was withdrawn from the culture vessels into glass or plexiglass standing settling containers, using a vacuum device as an aspirator pump. The encapsulated microalgal biomass was then left for 48 hours to 96 hours, depending on the cell density, at 4-6~C to separate from the liquid medium and settle at the bottom of the container forming 5 the microalgal slurry. The latter can also be obtained by centrifugation, if, for example, a high speed refrigerated centrifuge with large rotor and plastic centrifuge bottles of 500 ml capacity is available. A vacuum device, however, may be required for the removal by suction of the supernatant and thereafter the microalgal slurry for further processing.
Thus, to one volume of the microalgal slurry four volumes of 95% ethanol were added to give a final ethanol concentration of 70% to 80%, depending on the thickness of the said slurry.
The mixture was then left either at room temperature or at 4-6~C to settle for about 4 to 6 days, to allow the extraction of chlorophylls, 15 accessory pigments and ethanol soluble cellular compounds, as well as the dehydration of the mucopolysaccharide(s) of the microalgal capsule. As a result, the supernatant of the ethanol-microalgal slurry mixture turns greenish or yellowish green in colour, and the encapsulated microalgal slurry forms a white to yellowish coloured pellet at the bottom of the 20 settling container. After removing the supernatant by suction, the solid pellet of the achlorophyllous dehydrated encapsulated microalgal biomass was resuspended in about half- to one-litre 95% ethanol to wash out residual pigments and ethanol-soluble cellular compounds. The dehydrated, relatively clear and moist encapsulated microalgal biomass 25 was collected after the removal of the supernatant by suction, centrifugation, or by filtration using glass fiber, teflon, or nylon filters with pore size between one and three micrometer, depending on the thickness of the said biomass. The microalgal crust obtained by filtration, and the compact moist biomass secured by centrifugation or scooped out after 30 removing the supernatant, is the caked encapsulated Chlamydomonas segnis Ettl, i.e. CENC, which is the raw source for algal hyaluronic acid-like substance.

CA 02249l03 l998-09-30 Charact~ri~tion of CENC
Greenish suspensions of Chlamydomonas segnis Ettl encapsulated cells, harvested from cultures exposed to excessive light (60 Wm-2 to 150 Wm-2) for 24 to 72 hours, i.e. at early to mid-encapsulation phase, can readily settle in 75% to 80% ethanol within 48 to 72 hours at 4~C
to 6~C or at room temperature. Yellowish suspensions of the said microalga, obtained after prolonged exposure to excessive light, i.e. at late encapsulation phase, require more time to settle in ethanol due to the accumulation of lipid droplets in chloroplasts. The ethanol dehydrated encapsulated cells of Chlamydomonas segnis Ettl yield a moist cake, which has at a concentration of 1.0% in water (dry weight per volume) higher viscosity than the commercial gums xanthan and guar, and exhibits a degree of shear thinning similar to the pseudoplastic xanthan gum solution.
Figure 13 shows photomicrographs of: (a) a sample of CENC
homogenate, (b) a two-gram slightly moist pellet of CENC,(c) the size increase of the moist pellet of CENC during water imbibition, and (d) three samples of CENC during hydration exhibiting colour shades.
As shown in Figure 13 (a), CENC is highly hydrophilic and forms a stable colloidal homogenate when blended with water. The example photographed in Figure 13 (b) and (c) shows that a two-gram pellet of a slightly moist preparation of CENC imbibes about 200 ml of distilled water within 24 to 48 hours at room temperature. The colour and texture of CENC may vary according to the age of the microalgal biomass harvested during the encapsulation phase. The orange colour of the CENC sample number 3 in Figure 13 (d) may be due to the presence of extraplastidic carotenoids, as reported by Burczyk et al., Planta, 151, pgs 247-250 (1981), which may accumulate at late encapsulation phase.
Prolonged desiccation of the moist CENC, e.g. for 96 hours using anhydrous calcium sulfate of the Hammond Dreirite Company, will abolish its ability to imbibe water and rehydrate.

CA 02249l03 l998-09-30 Figure 14 depicts the apparent viscosity of 1% CENC
homogenate in water, compared to 1% solution of either xanthan or guar in water, as a function of shear rate, determined by a Bohlin Viscometer (Bohlin Reologi, Lund, Sweden).
As seen in Figure 14, viscosity measurements for CENC
homogenate in distilled water point clearly to its high viscosity with shear thinning similar to the pseudoplastic xanthan gum solution. CENC
homogenate is a gel, which does not show thixotropy and has higher viscosity than the commercial gums xanthan and guar. Furthermore, 10 CENC homogenates prepared in open air and stored for two weeks at room temperature or for over a year at 4-6~C have not been the subject of fungal or bacterial contamination, as judged from the absence of microbial growth. This observation suggests that CENC must be at least bacterio-and/or fungi-static.

Extraction of Chlamyhyaluronic acid from CENC
By use of the process of the present invention, removal of protein from the precipitated material is not necessarily required because of the relatively low absorption of its aqueous solutions at 280 nm.
20 Furthermore, the protein content of the said material does not exceed 0.5%
of its weight as determined by the method of Bradford, Analytical Biochemistry, 72, pgs 248-254 (1976), using Coomassie Brilliant blue G 250.
Furthermore, electrophoresed aqueous solutions of 2-5 mg ml-l of the precipitated capsular extract on a 6% polyacrylamide gel (as described by 25 Badour and Kim, Canadian J. of Botany, 66, pgs 1750-1754 (1988)), and stained with 0.5 % alcian blue dissolved in water, migrated as homogeneous streaks along two thirds the length of the gel matrix with blue coloured portion at the top, and faint blue towards the bottom. This suggests that the CENC precipitated extracts consist of the same acid 30 mucopolysaccharide but with different molecular weights, the largest at the top and the smallest at the bottom of the streak. Similar observations have been reported by DeAngelis et al., J of Biological Chemistry, 268, pgs CA 02249l03 l998-09-30 14568-14571 (1993) for purified hyaluronic acid extracts from transformed bacteria.
Accordingly, the CENC precipitated extract appears to be at least partially pure, and further purification would be mainly focused on the removal of the low molecular weight population and/or inhibiting the degradation of the high molecular weight population during extraction.
Thus, for the extraction of the metachromatic material, which is clearly visualized within the capsules of the CENC preparation by 10 toluidine blue as shown in Figure 15 (a), a batch of eight to twelve grams of the moist CENC, which contains about 50% of its weight as water (TABLE 3), is used. This batch is usually obtained from a four-litre Chlamydomonas seg~is Ettl deep culture after four to six days growth and encapsulation period, depending on the density of the microalgal suspension used for inoculation.
Figure 15 shows photomicrographs of samples from CENC
homogenate stained with toluidine blue, (a) before extraction, exhibiting the metachromatic capsular content, (b) after extraction with the buffered saline solution (0.8 M NaCI dissolved in 0.05 M phosphate buffer of p H 7), exhibiting intact cells inside the ghost capsule with strata indicated by arrows, and (c) exhibiting the acapsular appearance of CENC cells after extraction.
Eight to twelve grams of the aforedescribed ethanol dehydrated moist CENC were homogenized in 200 ml of 0.05 M phosphate buffer, p H7 to 7.5 by a blender or through continuous stirring for one to two hours in a closed container at room temperature. The resulting homogenate was extracted for about two hours by adding either sodium chloride, sodium acetate or potassium chloride under continuous stirring to a final concentration of 0.8 M to 1.0 M. One volume of the solubilized 30 fraction, which is clarified either by sieve filtration or by centrifugation, is precipitated by the addition of four volumes of 95% ethanol. After 24 hours to 48 hours, the clear supernatant is removed through suction using CA 02249l03 l998-09-30 a vacuum device, and the precipitated mucopolysaccharide salt is collected directly as pellets by centrifugation or as fairly thick suspensoids in ethanol, and gently aerated with nitrogen gas to remove traces of ethanol before storage at -16~C. One litre of the encapsulated cells of ChZamydomonas segnis Ettl from deep cultures produced according to the culturing method described herein, yields between 0.38 gram to 0.75 gram of the mucopolysaccaride salt. Although sodium chloride has been routinely used for the extraction of the capsular material as shown in TABLE 3, it may be replaced by sodium acetate for better extraction.
10 Acetate salts were the preferred agents for the extraction of hyaluronic acidfrom vitreous humor, as reported by Karl Meyer in Physiological Reviews, 27, pgs 335-359(1947).
As seen from Figure 15 (b), efficient extraction of the capsular material results in the negative metachromasy of the capsule which otherwise stains with toluidine blue due to the presence of the acid mucopolysaccharide within. Only intact vegetative cells of Chlamydomonas segnis Ettl can be visualized in the extracted samples of CENC, as shown in Figure 15 (C). If capsular metachromasy persists, CENC
homogenate is extracted repeatedly with 0.8 M to 1.0 M of the said salts.
Precipitation of the extracted material is achieved by adding to one volume of the clear salt extracts four volumes of 95% ethanol. The mixture is then left either at room temperature or at 4~C to 6~C for 24 to 48 hours for the complete precipitation of the white mucopolysaccharide salt at the bottom of the settling container.

Partial Purification of Chlamyhyaluronic acid Extract After removing the bulk of ethanol by suction, thick suspensoids of the precipitate in ethanol are transferred into centrifuge tubes for further removal of ethanol by suction after sedimentation, or directly by centrifugation. The wet pellet of the precipitate in the centrifuge tubes, or the wet residue obtained if narrow beakers are used instead of tubes, is placed in a desiccator or a closed container equipped CA 02249l03 l998-09-30 with manifolds designed to allow gentle aeration of the wet precipitates with nitrogen gas at room or slightly higher temperature. The dried mucopolysaccharide salts are then scooped out, sealed and stored at -16~C.
As seen from the example in TABLE 3, one litre of Chlamydomonas segn is Ettl cultures, using the culturing method described in this invention and harvested at about mid encapsulation phase, yields on the average about 2.45 grams of CENC, or 1.22 grams dry biomass that provides 0.57 gram of the mucopolysaccharide salt.

10 ~haracterizatiQn Qf Chlamyhyaluronic acid obtained from Algae Conventionally, the methods used to identify hyaluronic acid or its salts are based on the changes in the properties of this acid mucopolysaccharide in solutions when depolymerized by hyaluronate lyase (EC 4.2.2.1), or by hyaluronidase (EC 3.2.1.35). According to Rapport et al., J. Biological Chemistry, 186, pgs 615-623 (1950), changes in the properties of hyaluronic acid can be measured through: (1) the decrease in viscosity, (2) the loss of ability to form stable colloidal suspension, i.e.
turbidity, with acidified protein solutions at pH 4.2, and (3) the increase in reducing groups, i.e. reducing sugar content of the hyaluronic acid solution.
Because the absolute rate of viscosity decrease varies with different hyaluronic acid preparations and other unknown factors, this method was used herein only to determine the intrinsic viscosity of chlamyhyaluronic acid and that of hyaluronic acid from human umbilical cord for comparison.
The turbidimetric or the turbidity reducing method described by Dorfman, A., Methods in Enzymology, I, pgs 166-173 (1955) and Arvidson, S. O., in: Staphylococci and Staphylococcal Infections, 2, pgs 749-750, edited by Easmon, C. S. F. and Adlam, C., Academic Press (1983) was used. TABLE 4 shows that by increasing the concentration of chlamyhyaluronic acid or the human umbilical cord hyaluronic acid, the turbidity increases almost linearly within the 0.4 mg to 2.0 mg ml-l of chlamyhyaluronic acid and between 0.4 mg to 1.6 mg ml-l of the said hyaluronic acid. The results also show that the fraction in chlamyhyaluronic acid which binds to the protein and causes the turbidity is about 36.7% of that in the umbilical cord hyaluronic acid, as indicated by 5 the difference in absorbance between the two solutions.
Also, samples of chlamyhyaluronic acid solutions of about 2.0 mg ml-l in 0.02 M acetate buffer, pH 5.0, treated with about 70 units of hyaluronate lyase from Streptomyces hyalurolyticus (Sigma, No. H-1136), or with about 980 units of hyaluronidase from bovine testes (Sigma, type 10 rv-s, No.3884) for 6-12 hours at 37~C indicate 40-60% decreases in turbidity. Such decreases are associated with increases in the reducing sugars, as determined by the arsenomolybdate method of Nelson, as described by Ashwell, G., Methods in Enzymology, III, pgs 73-87 (1957). In other examples shown in Figure 16 and Figure 17, the minute amounts of 15 reducing compounds produced within 10 to 45 minutes through chlamyhyaluronic acid depolymerization by the said enzymes can be measured spectrophotometrically by the increase in absorption at 230 nm, as described by Greiling, H., Hoppe-Seylers Zeitschrift fur physiologische Chemie, 309, pgs 239-242 (1957), when the enzymically treated and 20 untreated samples are compared.
Figure 16 depicts the increase in absorption around 230 nm of chlamyhyaluronic acid (0.2 mg ml-l of 0.02 M acetate buffer, pH5.0) after incubation with about 70 units of hyaluronate lyase at 55~C for 45 minutes (upper graph), compared to a control sample without the said enzyme 25 (lower graph), using Hewlett Packard Diode Array Spectrophotometer Model 8452A.
Figure 17 depicts the increase in absorption around 230 nm of chlamyhyaluronic acid (0.8 mg ml-l of 0.02 M acetate buffer, pH 5.0) after incubation with about 980 units of hyaluronidase at 37~C for 10 minutes 30 (upper graph), compared to a control sample without the said enzyme (lower graph), using Hewlett Packard Diode Array Spectrophotometer Model 8452A.

CA 02249l03 l998-09-30 Figure 18 shows a print-out of a cellulose thin layer chromatogram, developed with butanol, acetic acid, and water (50: 15: 35), after exposure to ultraviolet radiation in a Gel Doc 1000 Video Gel Documentation System (BIO-RAD) to visualize the products formed by the degradation of chlamyhyaluronic acid (c and d), and hyaluronic acid from human umbilical cord (e and f) with hyaluronate lyase at 37~C for six hours, and the absence of such products (a and h) or their reduced level (b and g) in samples without enzyme or with heated enzyme, respectively.
Figure 19 shows increases in the degradation products indicated in Figure 10 18, as a result of extending the incubation period to twelve hours at 37~C.
Figure 20 shows a print-out of a chromatogram produced as described in Figure 18, except that hyaluronidase is the enzyme used.
As seen from the thin layer chromatograms in Figures 18-20, ultraviolet absorbing spots from the enzymically treated 15 chlamyhyaluronic acid are almost mirror images of those formed in the enzymically treated hyaluronic acid from human umbilical cord.
However, from Figure 21, it appears from the relatively low intrinsic viscosity of chlamyhyaluronic acid, as compared to that of the human umbilical cord hyaluronic acid in Figure 22, that the majority of the 20 polymer population in the former is of low molecular weight. Improving the method of chlamyhyaluronic acid extraction, e.g. by inhibiting the endogeneous enzymes that could cause its depolymerization during the preparation of CENC, is expected to increase the proportion of high molecular weight fraction in the extract.
Figure 21 depicts the reduced viscosity of chlamyhyaluronic acid (potassium salt) as a function of concentration, using a Ubbelohde viscometer, and the intrinsic viscosity is determined by extrapolating to zero concentration with an asymptotic model.
Figure 22 depicts the reduced viscosity of hyaluronic acid 30 (sodium salt) from human umbilical cord, as a function of concentration, using a Ubbelohde viscometer, and the intrinsic viscosity is determined by extrapolating to zero concentration with a linear model.

Thus chlamyhyaluronic acid (the partially purified sodium and potassium salts of the mucopolysaccharide extracted from CENC after being homogenized in 0.05M phosphate buffer at pH 7-7.5), was found to contain less than 0.5% protein, to be degraded by Streptomyces 5 hyalurolyticus hyaluronate-lyase (EC 4.2.2.1) and to form stable colloidal suspensions with acidic serum albumin, i.e. develops turbidity in a manner similar to animal hyaluronic acid.
Chlamyhyaluronic acid does not appear to be identical to hyaluronic acid obtained from umbilical cord or rooster comb.
Differences between chalmyhyaluronic acid and umbilical cord hyaluronic acid were shown by comparing the 500 MH~ NMR
(nuclear magnetic resonance) spectra of the algal and human umbilical cord sodium salts of hyaluronic acid solutions in D2O (deuterium oxide) at 300K. The peak of the algal hyaluronic acid at 1.7599 ppm was off the peak 15 of acetyl group ~hown at 1.8980 pprlP~ by the huma~. umbilical cord hyaluronic acid. Also, the peaks associated with carbohydrates were 3.2349 ppm to 4.0879 ppm for the human umbilical cord hyaluronic acid, and in the range of 3.4907 ppm to 3.8873 ppm for chalmyhyaluronic acid. More specifically, the carbohydrate region shows five peaks at different ppm, 20 namely at 3.8873 ppm, 3.8125 ppm, 3.7052 ppm, 3.6008 ppm and 3.4907 ppm. The highest peaks are at 3.7052 ppm and 3.6008 ppm, which are different from the human umbilical cord hyaluronic acid carbohydrate peak at 3.7116 ppm.
Differences were also observed between chalmyhyaluronic 25 acid and rooster cori,b hyal-uronic acid. Sol-u.ions of hyaluronic acid prepared from rooster comb form a complex with bivalent copper according to Figueroa et al., Biochemical and Biophysical Research Communications, 74:460-465 (1977), show an absorption band at 238 nm.
Chlamyhyaluronic acid solutions also form a copper-complex, but differ in 30 showing an absorption band at 232 nm.
However, chlamyhyaluronic acid is similar to the hyaluronic acid known in the art. It is degraded by hyaluronate lyase and it reacts CA 02249l03 l998-09-30 with proteins to form either a granular precipitate or a stable "mucin" clot, i.e. it forms stable colloidal suspensions with acidic serum. This property, as well as its antibacterial activity, point to its potential for efficacy as anagent for wound healing.

(~ommercial Production Using the novel process described in this application, Chlamydomonas segnis Ettl can be induced in cultures to produce encapsulated cells with extracellular chlamyhyaluronic acid in its capsules.
10 Such cultures may be utilized commercially by known algal biomass production systems. Such systems provide culture containers or vessels designed to allow light absorbance, aeration and harvesting the algal biomass on a large scale. Existing systems which are used in algal biotechnology, and described in Hydrobiologia, 204-205, pgs 401-408 (1990), Biotechnology Techniques~ 4, pgS 321-324 ~1990)~ Bioresource Technology, 38, pgs 233-235 (1991), and in Biotechnology Advances, 8, pgs 709-728(1990), can be modified to produce mass cultures of encapsulated Chlamydomonas segnis Ettl. The economic feasibility parallels that of mass cultivation of microalgae such as Spirulina and Dunaliella for human nutrition as health food, which has achieved economic success.
Preparation of CENC by a two-step procedure consisting of dehydrating and settling the algal biomass, will provide a relatively clean, inexpensive source of chlamyhyaluronic acid. Partial purification of the dehydrated raw algal preparation would yield on the average about 0.6 g chlamyhyaluronic acid per one litre algal suspension or per 0.98-1.67 g algal dry matter. In contrast, about 1250 grams rooster combs are required for the production of one gram hyaluronic acid by Fermentech UK of Woking, Surrey, as reported in Chemistry and Indus~ry, 11: 374 (1986), and 76 g of dried human umbilical cord would yield 1.5 g of hyaluronic acid as reported in the early study of Meyer and Palmer, Journal of Biological Chemistry, 114, pgs 689-703(1936).

Having illustrated and described the principles of the invention in a preferred embodiment, it should be appreciated to those skilled in the art that the invention can be modified in arrangement and detail without departure from such principles. We claim all modifications 5 coming within the scope of the following claims.
All publications, patents and patent applications referred to herein are incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety.

Composition of the low salt Woods Hole MBL pH7.2 medium before (A) and after mol1ifi~ Afi-ln (B), compared to the high salt nutrient medium (C) routinely used for growing Chlamydomonas segnis Ettl at pH7+0.1.
A (~ i.. B ~ n~n~rAtir~n C C~-)n~l~n~rA~i~n Salt (mM) Salt (mM) Salt (mM) CaCl2-2H2O 0.25 CaCl2-2H2O 0.25 Cacl2 0.10 MgSO4 7H2o 0.15 MgSO4 7H2O 0.15 MgSO4 7H2O 1.00 NaHCO3 0.15 NaHCO3~ 0.00 NaH2PO4.H2O 3.70 K2HPO4 0.05 K2HPO4 0.05 K2HPO4.2H20 4.50 NaNO3 1.00 NaNO3 1.00 KNO3 10.00 Na2SiO3-9H20 0.10 Na2SiO3 9H20~ 0.00 Na2EDTA 0.012 Na2EDTA 0.012 Na2EDTA 0.025 Fecl3 6H2O Q012 Fecl3 GH2O 0.012 FeSO4 7H2O 0.025 (llM) (llM) (llM) CuSO4.5H2O 0.04 CuSO4.5H2O 0.04 CuS04 0.038 ZnsO4 7H2o 0.076 znSO4.7H2O 0.076 ZnSO4.7H2O 0.350 CoCI2.6H2O 0.04 CoCk.6H2O 0.04 CoCI2.6H2O 0.034 MnCI2 4H2O 0.91 Mncl2 4H2O 0.91 MnSO4 4H2O 0.120 Na2MoO4 2H2O 0.025 Na2MoO4.2H2O 0.025 (NH4)6Mo7O24.4H2O 0.0049 Tris-HCI Buffer, Tris-HCI Buffer H3BO3 1.5 pH7.2 4000 pH7.2 500 Vitamins (llg) 101 Vitamins (~lg) 000 Total with ~ Total without L2~ Total (mM) l9.;~iZ
Vitamins (mM) Vitamins & salts marked~ (mM) Rates of photosynthetic oxygen evolution (Ps), activities of photosystem I (PSI), photosystem II (PSII), and catalase (Cat) from low salt boron-deficient shallow cultures of Chlamydomonas segnis Ettl at early (24 h) and late (72 h) encapsulation phases, using the methods adopted by Badour and Irvine, Botanica Acta, 103, pgs 149-154 (1990).

,u mol ~2 mg Chlorophyll-l h-l (n=4) 24h 72h Ps 1016.5 + 99.7 406.4 + 33.6 PSII 1401.0 i 139.4 455.1 ~ 41.9 PSI 1371.9 99.2 431.8 i 62.0 Cat 2591.1 + 149.0 1092.4 +118.4 Yields of the phosphate buffered (pH 7.0)-sodium chloride (0.8 M) extracts of CENC, after being precipitated in about 80% ethanol and dried by gentle aeration with nitrogen gas; values in brackets are the water contents of CENC samples given inpercentage of their wet weights.
Grams per litre of Chlamydomonas segnis Ettl encapsulated culture Example Moist Desiccated Dried Ethanol No. CENC CENC Precipitate 2.07 (52.7) 0.98 0.38 2 2.69 (48.3) 1.39 0.66 3 1.97 (58.4) 0.82 0.47 4 3.08 (45.8) 1.67 0.75 Increased turbidity, measured at 500 nm with Ultroscopic 2000 (Pl~rm~ Biotech) Spectrophotometer, due to the increases in the concentrations of Chlamyhyaluronic acid (ChlamyHA) and human umbilical cord hyaluronic acid (U cord HA), using one ml of hyaluronic acid solutions in 0.02 M acetate buffer, pH5.0, to which ten mls of the acid albumin solution as describedby Dorfman, Methods in Enzymology, I, pgs.l66-173 (1957), are added.

ChlamyHA U cord HA
mg ml-l Absorbance ~hsorb~n' ~ Absorbance Ahsorb~rlce [HA] [HA~

0.4 0.102 0.255 0.261 0.653 0.8 0.211 0.264 0.544 0.680 1.2 0.294 0.245 0.802 0.668 1.6 0.389 0.243 1.060 0.663 2.0 0.526 0.263 1.209 0.605 REFERENCES
Abstract of Japanese Patent (Basic): JP 07101871 A of LION CORP, for Jeffrey Madge, NRC. Canada. February 26 (1996).
Allard, B., and Tazi, A. Phytochemistry, 32: 41-47 (1993).
5 Angell, C. A. Science, 267: 1924-1935 (1995).
Arvidson, S.O. Extracellular enzymes from S~aphylococcus aureus. In:
Staphylococci and staphylococcal infections, Vol. 2, edited by Easmon, C.S.F. and Adlam C., pp. 749-752, Academic Press (1983).
Badour, S.S. et al., Journal of Phycology (supplement) 8:16 (1972).
10 Badour, S.S., et al., Canadian Journal of Botany, 51: 67-72 (1973).
Badour, S.S., et al. Journal of Phycology, 13: 80-86 (1977).
Badour, S.S. Journal of Phycology, 17: 293-299 (1981).
Badour, S.S., and Mortimer, D.C. Correspondence letters regarding Chlamydomonas segnis samples and a method for culturing this alga 15 in a high salt medium (1980-81).
Badour, S.S. Excess light evokes cell encapsulation in Chlamydomonas segnis Ettl. Abstracts of the International Meeting on Molecular Biology, Biochemistry and Physiology of Chloroplast Development. Philipps Universitat, Marburg, Germany (1995).
20 Badour, S.S., and Irvine, B.R. Botanica Acta, 103: 149-154 (1990).
Badour, S.S., and Tan, C.K. The path of carbon in photosynthesis by Chlamydomonas segnis adapting to low and high carbon dioxide tension. Zeitschrift fur Pflanzenphysiologie, 112: 287-295 (1983).
Badour, S.S., and Tan, C.K. Plant and Cell Physiology, 28: 1485-1492 25 (1987).
Badour, S.S. Ribulose-bis-phosphate carboxylase in Chlamydomonas.
In: Handbook of Phycological Methods. Vol. II, pp. 209-216. Edited by Hellebust, J.A. and Craigie, J.S. Cambridge University Press (2978).
Balows, A., Truper, H.G., Dworkin, M., Harder, W. and Schleifer, K-H
30 (Editors). The prokaryotes, Vol. II, pp. 1459-1461, 2nd edition, Springer-Verlag.

Balazs, E. A., and Leshchiner A. Hyaluronate modified polymeric articles. United States Patent # 4,500,676 (1985).
Balazs, E. A., and Leshchiner A. Cross-linked gels of hyaluronic acid and products containing such gels. United States Patent # 4,582,865 5 (1986).
Balazs, E. A., and Leshchiner A. Cross-linked gels of hyaluronic acid and products containing such gels. DIV United States Patent #
4,636,524 (1987).
Balazs, E. A., Leshchiner A., and Band, P. Chemically modified 10 hyaluronic acidpreparation and method of recovery thereof from animal tissues. United States Patent # 4,713,448 (1987).
Balazs, E. A., and Denlinger, J. L. Clinical uses of hyaluronan. In: The Biology of Hyaluronan, Ciba Foundation Symposium 143: pp.265-280, Wiley, Chichester, England (1989).
15 Ben-Amotz, A., et al., Bioresource Technology, 38: 233-235 (1991).
Ben-Amotz, A., and Avron, M. Trends in Biotechnology, 8: 121-126 (1990).
BIO CARE SA: Proven performance in reducing wrinkles. Union Carbide Corp/ Amerchol Corp Publication ID: HAC-003, July, (1994).
20 Biotech News. Chemistry and Industry. Issue number 11, June 2, p.
374 (1986).
1984 Bio-Hyaluronic Acid. Cyber Island of Shiseido for Shiseido Co. Ltd (1995).
Biozyme Laboratories International Limited Home Page. Product 25 Information: Hyaluronic Acid. Webmaster(~biozyme.com, February 8 (1996).
Boyd, R.F., and Hoerl, B.G. Basic medical microbiology, pp. 402-405, 4th edition. Little, Brown and Company, Boston/ Toronto/ London.
Butow, B., et al., Journal of Phycology, 30: 17-22 (1994).
30 Clinical comparison demonstrates Hyanalgese-D has the edge over Voltaren Emulgel in relieving arthritis pain. Canada News Wire for Hyal Pharmaceutical Corporation, Mississauga, Ontario, December 13, (1995).

CA 02249l03 l998-09-30 Cook, J.R. Synchronous cultures: Euglena. In: Methods in Enzymology, Vol. 23, Part A, pp. 74-78; Edited by San Pietro A.
Academic Press (1971).
Complex carbohydrate therapeutic products: Tomorrow's Billion $
market. Genetic Engineering News, January 1: 6-7(1993).
DeAngeiis, P.-L., e~ ai., Journai o~ Bioiogicai Chemistry, 268:
14568-14571(1993).
DeAngelis, P. L., Yang, N., and Weigel P. H. The S~reptococcus pyogenes hyaluronan synthase: sequence comparison and 10 conservation among various group A strains. Biochemical and Biophysical Research Communications, 199: 1-10 (1994).
Dechacin, C., et al., Exotoxicology and Environmental Safety 21: 25-31 (1991).
Deemmig-Adams, B. and Adams, W.W. III, Annual Review of Plant Physiology and Molecular Biology, 43: 599-625 (1992).
Enzyme nomenclature: Recommendations (1972) of the International Union of Pure and Applied Chemistry and the International Union of Biochemistry, pp. 217 and 295, Elsevier.
Foo, S.K., and Badour, S.S. Canadian Journal of Botany, 55: 2178-2185 (1973) Ghosh, P. The role of hyaluronic acid (hyaluronan) in health and disease: interactions with cells, cartilage and components of synovial fluid. Clinical and Experimental Rheumatology, 12: 75-82 (1994).
Ghosh, P., et al., International Journal of Biological Macromolecules, 16:237-244(1994).
Greiling, H. Eine spektrophotometrische Methode fur die Bestimmung der Bakterien-Hyaluronidase. Hoppe-Seylers Zeitschrift fur physiolgische Chemie, 309: 239-242(1957).
Hall, C.L., Yang, B., Yang, X., Zhang, S., Turley, M., Samuel, S., Lange, L. A., Wang, C., Curpen, G. D., Savani, R. C., Greenberg, A. H., and Turley, E. A. Overexpression of the hyaluronan RHAMM is transforming and is also required for H-ras transformation. Cell 82:
19-28(1995).
Harris, Elizabeth (ed). The Chlamydomonas source book, Academic Press Inc. (1989) CA 02249l03 l998-09-30 Hu, Hening, and Brown, Patrick H. Plant Physiology, 105: 681-689 (1994) Hyal Pharmaceutical Corporation press release. Canada News Wire.
July 14 (1995).
Hyal Pharmaceutical Australia Limited (HPAL) releases positive clinical trial results. Canada News Wire for Hyal Pharmaceutical Corporation, Mississauga, Ontario. November 28 (1995).
~yaluronic acid from farmyard to factory (Biotech' 86 report).
Chemistry & Industry, 11: 374(1986).
10 ICN Biomedicals Catalogue. Hyaluronic Acid, p. 359 (1995).
Innovative osteoarthritis treatment subject of Canadian agreement.
Biomatrix, Inc. Doctors Guide (The Internet), September 11, (1995).
Japanese partner to fund further development for Bioniche product, Cystistat (TM). Canada News Wire for Bioniche Inc., London, Ontario.
February 7 (1996).
Kinzel, Helmut. Unterschungen uber die Chemie und Physikochemie der Gallertbildungen von Su~wasser algen. Osterrichische Botanische Zeitschrift, 100: 25-79(1953).
Kreis, T., and Vale R. (editors). Guidebook to the extracellular matrix and adhesion proteins. Oxford University Press (1993).
Kubo, K~, et al., Glycoconjugate Journal, 10: 435-439 (1993).
Laudenbach, B., and Pirson, Archiv fur Microbiology, 67: 226-242 (1969).
Laurent, T. Introduction. The biology of Hyaluronan, Ciba Foundation Symposium, 143:1-5, Wiley, Chichester Publication (1989).
Laurent, T. C, and Fraser, J. R. E. Hyaluronan. Federation of American Societies for Experimental Biology, 6: 2397-2404 (1992).
Laurent, T.C., and Fraser, J. R. E. The properties and turnover of hyaluronan. In: Functions of Proteoglycans, Ciba Foundation Symposium 124, pp. 9-29, Wiley, Chichester, England (1986).
Linker, A. et al., Journal of Biological Chemistry, 213: 237-248 (1955).

CA 02249l03 l998-09-30 Long, S.P., and Humphries, S. Annual Review of Plant Physiology and Molecular Biology, 45: 633-622 (1994).
Meerholz, K., Kippelen, B., and Peyghambarian, N. Photorefractive polymers for photonicapplications. The Spectrum, 8:1-6 (1995).
Metzner, H., Rau, H., and Senger, H. Untersuchungen zur synchronisierbarkeit einzelner Pigmentmangel-Mutanten von Chlorella.. Planta, 65: 186-194 (1965).
Meyer, K., and Palmer, J.W. Journal of Biological Chemistry, 114:
689-703(1936).
10 Moseley, R., et al., Biochimica et Biophysica Acta, 1244: 245-252 (1995).
Moulton, T.P., and Burford, M.A. The mass culture of Dunaliella virid is (Volvocales, Chlorophyta) for oxygenated carotenoids laboratory and pilot plan studies. Hydrobiologia, 204-205: 401-408 (1990).
Nichols, H. Wayne. Growth media- freshwater. In: Handbook of Phycological m-ethods Vol= Ij pp= 8-23. dit~d hy Stein; R JanPt Cambridge University Press (1973).
Ohya, T., and Kaneko, Y. Biochemical Biophysica Acta, 198: 607-609 (1970).
Parker, Bruce C. (ed.). Journal of applied phycology, 6: pp. 91-98(1994).
Rapport, M.M., et al., Journal of the American Chemical Society, 73:
2416-2420(1951).
Raven, J.A. New Phytologist, 84: 231-249 (1980).
Redekop, B. Cancer find hailed. Winnipeg Free Press. July 15 (1995).
Salisbury, F.B., and Ross, C.W. Plant Physiology, Wadsworth Publishing Company, Belmont California, pp . 116-135, 226-233 and 240-241(1992).
Sato, H., et al., Arthritis and Rheumatism, 31(1): 63-71 (1988).
Schlosser, U.G. SAG- Sammlung von Algenkulturen at the University of Gottingen Botanica Acta, 107: 129 (1994).
Scott, J. E. Secondary structures in hyaluronan solutions: chemical and biological implications, Ciba Foundation Symposium 143: pp.6-21, Wiley, Chichester, England (1989).

CA 02249l03 l998-09-30 Searle Canada Licenses Topical Analgesic from Hyal Pharmaceutical Corporation. Canada News Wire for Searle Canada and Hyal Pharmaceutical Corporation, Oakville, Ontario, February 15, (1996).
Shelef, G., and Soeder, C. J.(editors). Algal Biomass: Production and Use, Elsevier, North-Holland, Amsterdam (1980).
Sigma Catalogue of Biochemicals, Organic Compounds and Diagnostic Reagents. Hyaluronic Acid, pp. 549-550(1996).
Stadler, T., Mollion, J., Verdus, M.-C., Karamanos, Y., Morvan, H., and Christiaen, D. Algal biotechnology, Elsevier Applied Science, London 10 and New York (1987).
Starr, R.C., and Zeikus, J.A. Journal of Phycology (Supplement) 23:
8(1987).
Starr, R.C., and Zeikus, J.A. Journal of Phycology (supplement) 29:
19-20(1993).
Starr, Richard C. Algae Cultures- source and methods of cultivation:
In: Methods in Enzymology, Vol. 23, Part A, pp. 29-52. Edited by San Pietro, A. Academic Press (1971) Synvisc (hylan G-F 20). Information pamphlet, Biomatrix Medical Canada Inc. Pointe-Claire, Quebec (1996).
Tammi, R., Agren, U. M., Tuhkanen, A-L., and Tammi, M.
Hyaluronan metabolism in skin. Progress in Histochemistry and Cytochemistry, 29:1-78(1994).
Takigami, S., and Takigami, M. Carbohydrate Polymers, 22:135-160 (1993).
Toha, J., et al., Biotechnology Techniques., 4: 321-324 (1990).
Volk, W.A., Benjamin, D.C., Kander, R.J., and Parsons, J.T. Essentials of medical microbiology, pp. 356-358, 4th edition. J.B. Lippincott Company Philadelphia.
Vonshak, A. Microalgal biotechnology: is it an economic success? In:
Biotechnology: economic and social aspects, edited by: DA Silva, E. J., Ratledge, C., and Sasson, A., pp.70-80, Cambridge University Press (1992).
Vonshak, A., Biotechnology Advances, 8:709-728 (1990).

CA 02249l03 l998-09-30 Wallen, D.G., and Geen, G.H.. Marine Biology, 10: 44-51 (1971).
Weinberger, P., et al., Canadian Journal of Botany 65: 696-702 (1987).
Weissmann, B. Journal of Biological Chemistry, 216: 783-794 (1955).
Wong, S. F., Halliwell, B., Rich~nond, R., and Skowroneck, W. R. The role of superoxide and hydroxyl radicals in the degradation of hyaluronic acid induced by metal ions and by ascorbic acid. Journal of Inorganic Biochemistry, 14:127-134 (1981).

Claims (35)

1. Isolated chlamyhyaluronic acid.

2. Chlamyhyaluronic acid according to claim 1, wherein said chlamyhyaluronic acid is encapsulated.

3. Chlamyhyaluronic acid according to claim 1, wherein said chlamyhyaluronic acid is derived from an alga.

4. Chlamyhyaluronic acid according to claim 3, wherein said alga is a eukaryotic unicellular green alga.

5. Chlamyhyaluronic acid according to claim 4, wherein said alga is from the phylum Chlorophyta.

6. Chlamyhyaluronic acid according to claim 4, wherein said alga is from the genus Chlamydomonas.

7. Chlamyhyaluronic acid according to claim 6, wherein said alga is Chlamydomonas segnis.

8. Chlamyhyaluronic acid according to claim 6, wherein said alga is Chlamydomonas segnis EttI.

9. Chlamyhyaluronic acid according to claim 1, wherein a solution of the sodium salt of chlamyhyaluronic acid (a) has an acetyl group peak of about 1.7599 ppm on a 500 MHz nuclear magnetic resonance spectra when in a solution with deuterium oxide at 300K;
(b) has carbohydrate peaks of about 3.7052 ppm and about 3.6008 ppm on a 500 MHz nuclear magnetic resonance spectra when in a solution with deuterium oxide at 300K; (c) forms a complex with bivalent copper and said complex shows an absorption band at 232 nm; (d) develops turbidity with serum at an acid pH and forms stable colloidal suspensions; and (e) is degraded by the specific enzyme hyaluronate lyase (EC 4.2.2.1) from Streptomyces hyalurolyticus as well as by hyaluronidase (EC 3.2.1.35) from bovine testes.

10. A method for the production of chlamyhyaluronic acid comprising culturing an alga capable of providing extracellular capsules containing chlamyhyaluronic acid under stressful conditions for a period of time sufficient for the alga to produce extracellular capsules containing chlamyhyaluronic acid.

11. A method according to claim 10, wherein said stressful conditions comprise excessive light.

12. A method according to claim 11, wherein said stressful conditions comprise excessive light with a blue:red spectral ratio which is higher than one.

13. A method according to claim 10, wherein said stressful conditions comprise culturing the algae in boron deficient nutrient medium.

14. A method according to claim 10, wherein said stressful conditions comprise culturing the alga in low salt nutrient medium.

15. A method according to claim 12, wherein the alga is a eukaryotic unicellular green algae.

16. A method according to claim 15, wherein said alga is from the phylum Chlorophyta.

17. A method according to claim 15, wherein said alga is from the genus Chlamydomonas.

18. A method according to claim 16, wherein said alga is Chlamydomonas segnis.

19. A method according to claim 18, wherein said alga is Chlamydomonas segnis EttI.

20. A method according to claim 10, further comprising isolating chlamyhyaluronic acid from the algal cells.

21. A method according to claim 10, further comprising adding from about 0.1% to about 5% carbon dioxide in air, during the exponential phase of growth and the encapsulation phase.

22. A method according to claim 10, wherein said alga is maintained at a temperature of from about 20°C to about 30°C.

23. A method according to claim 10, further comprising adding glucose to said culture.

24. Chlamyhyaluronic acid produced by a method according to claim 10.

25. A composition comprising chlamyhyaluronic acid according to claim 1 in admixture with a suitable diluent or carrier.

26. A method of protecting the skin from reactive oxygen radicals comprising applying a composition according to claim 25 to the skin.

27. A method of moisturizing the skin, hair or nails comprising applying a composition according to claim 25 to the skin, hair or nails.

28. A method for reducing tissue fibrosis comprising administering a therapeutically effective amount of a chlamyhyaluronic acid according to claim 1 to a patient in need thereof.

29. A cosmetic composition comprising chlamyhyaluronic acid according to claim 1 in admixture with a suitable diluent or carrier.

30. A sunscreen composition for the photoprotection of mammalian skin or hair, comprising chlamyhyaluronic acid according to claim 1 in admixture with a suitable vehicle, diluent or carrier.

31. A method for gelling a foodstuff comprising contacting the foodstuff with an effective amount of a chlamyhyaluronic acid according to claim 1.

32. A method for inactivating enzymes, microorganisms or spores in a foodstuff comprising contacting the foodstuff with an effective amount of a chlamyhyaluronic acid according to claim 1.

33. A matrix biopolymer or a biosensor comprising chlamyhyaluronic acid according to claim 1 in admixture with a suitable matrix or solution.

34. A method for sensing occurrences photonically, comprising detecting changes in a detectable amount of a chlamyhyaluronic acid according to claim 1.

35. A method for inactivating enzymes, microorganisms or spores on a patient comprising administering a therapeutically effective amount of a chlamyhyaluronic acid according to claim 1 to a patient in need thereof.