WO1993019193A1

WO1993019193A1 - L-ascorbic acid enhanced biomass and its production

Info

Publication number: WO1993019193A1
Application number: PCT/US1993/002430
Authority: WO
Inventors: James Allan Doncheck; Ronald John Huss; Jeffrey A. Running; Thomas J. Skatrud
Original assignee: Bio-Technical Resources
Priority date: 1992-03-18
Filing date: 1993-03-18
Publication date: 1993-09-30
Also published as: CA2091919A1; AU3811793A; CN1080955A; JPH0646834A

Abstract

A microalgal biomass containing a high intracellular level of ascorbic acid (greater than 1.5 % of the dry weight of the cells) is produced by growing a mutagenized L-ascorbic acid-producing microalga. Such a biomass is useful as an animal feed and as an aquaculture fish food or corresponding additive.

Description

TITLE

L-ASCORBIC ACID ENHANCED BIOMASS

AND ITS PRODUCTION

BACKGROUND OF THE INVENTION

1. Field of the Invention:

This invention relates to microalgal biomass that contains a high intracellular level of ascorbic acid, its preparation and its use for animal feed. More particularly, the invention relates to animal feed compositions enhanced with ascorbic acid biomass and their preparation and their use as a fish food.

2. Description of Related Art:

L-ascorbic acid, or Vitamin C, is a water-soluble vitamin widely distributed in the- plant and animal kingdom. It is of major importance in nutrition, maintenance of good health, and the food industry. Nearly all species of animals synthesize L-ascorbic acid and do not require it in their diet. However, humans, other primates, guinea pigs, fruit eating bats, some birds, and fish such as Coho salmon, rainbow trout and carp cannot synthesize the vitamin because they lack a liver enzyme. Such animals develop scurvy if deprived of a dietary source of L-ascorbic acid. To overcome deficiencies, marginal or otherwise, that are detrimental to good health and normal growth (especially in fish farming), it is essential to make available to such an animal an adequate supply of Vitamin C.

Methods for the isolation of Vitamin C from plants, animal tissue and foods are well known and involve extraction, clean up of the extract, and analysis or isolation of the vitamin from solution. Some natural sources from which L-ascorbic acid has been isolated include paprika, Gladiolus leaves, rose hips, persimmon, and citrus fruit. The vitamin can also be synthesized starting with L-xylose, L-galactose or more preferably from low cost, readily available D-glucose. Still another way which can be employed to produce L-ascorbic acid is to use microorganisms. While wild-type microorganisms are known to produce only minor amounts of L-ascorbic acid, progress in yield is being made using Chlorella algae as evidenced by the recent issuance of U.S. 5,001,059.

Loewus, F.A., in L-Ascorbic Acid: Metabolism, Biosynthesis, Function, The Biochemistry of Plants. Vol. 3, Academic Press, Inc., pp. 77-99, 1980, provides a review of the sources and biosynthesis of L-ascorbic acid. Descriptions of production of ascorbic acid in algae may be found in Vaidya e al, Science and Culture (1971) 37:383-384; Subbulakshmi e aL, Nutrition Reports International (1976) 14:581-591; Aaronson et s Arch. Microbiol. (1977) 112: 57-59; Shigeoka et al, J. Nutr. Sci. Vftaminol. (1979) 29:29-307; Shigeoka et aL, Agric. Biol. Chem. (1979) 43:2053-2058: Bayanova and Trubachev, Prikladnaya Biokhimiya i Mikrobiologyia (1981) 17:400-407 (UDC 582.26:577.16); and Ciferri, Microbiological Reviews (1983) 47:551-578.

Production of microalgae for food is known. It is also known that various algae, including Chlorella species, contain vitamins, including Vitamin C. For example, Aaronson et. al., Arch. Microbiol. 112. 57-59 (1977) discloses light-grown

Chlorella contain up to 15 ug Vitamin C per mg of dry weight of cells, i.e., 1.5% by weight.

Renstrom et. al., Plant Sci. Letters, 28:299-305 (1982/1983) discloses that L-ascorbic acid content in Chlorella is reported to be 6-82 u mol/g dry weight for cells grown in light, i.e., 0.11 to 1.44% by weight; also that Chlorella p renoidosa Chick, culture (UTEX) No. 343, dark-grown for 0.5 to 6 days in a glucose-enriched medium produced about 3 u mol of L-ascorbic acid per g dry weight (0.06 wt %) and four-fold this amount (0.24 wt %) in 12 hours when light was supplied, a result similar to those reported in earlier studies.

The prior art heterotrophic processes and microorganisms are not entirely satisfactory for commercial use: The biomasses produced are too deficient in L-ascorbic acid, primarily resulting from the ineffectiveness of the microorganisms employed and the poor utilization of the carbon source under the process conditions involved.

A need exists for improved L-ascorbic acid-producing microorganisms and for improved methods of utilizing them in the production of L-ascorbic acid-enriched biomass.

SUMMARY OF THE INVENTION

An L-ascorbic acid enriched biomass comprising green microalgal cells having an intracellular L-ascorbic acid (L-AA) content greater than 1.5% of the dry weight of the cells, preferably at least about 2% and more preferably at least about 3%.

Other embodiments include: novel high L-AA producing mutagenized

Chlorella pyrenoidosa microalgae identified below; heterotrophic methods for producing high L-AA content biomasses of the microalgae; animal feed compositions comprising the L-AA-enriched biomasses and their use as animal food, including aquaculture fish food capable of providing at least a significant proportion of the daily Vitamin C requirement of the animal.

DESCRIPTION OF THE INVENTION

The microorganisms may vary widely provided they produce L-AA heterotrophically. Preferred are the L-AA-producing green microalgae, especially the so-called high-producers thereof. Organisms showing promise as potential high-producers can be identified using standard fermentation procedures for cell growth accompanied by L-AA production. They are then preferably mutagenized using physical or chemical mutagenizing means, e.g. ultraviolet (U.V.) light, X-rays, N-methyl-N'-nitro-N-nitrosoquanidine, dimethyl sulfate or the like agent known to the art. Mutagenized L-AA overproducers produced by such treatments can be determined with redox dyes. Further, by employing analogs to metabolic intermediates to ascorbic acid or inhibitors of the ascorbic acid synthesis, microorganisms may be selected that are capable of maintaining or increasing L-AA production in the presence of chemical interference.

Preferred progeny of the above procedures are those microorganisms providing improved specific formation of L-AA as measured by milligrams of L-AA per gram of cells (dry weight basis). These progeny may then be further separated into individual clones and further subjected to the above procedures eventually to yield organisms providing still high specific formations of L-AA.

Preferred are the green microalgae of the genus Chlorella, in particular of the species pyrenoidosa, including high L-AA-producing mutagenized strains thereof derived for example from such wild strains as C. pyrenoidosa UTEX 1663 and C. pyrenoidosa UTEX 1230. C. regularis UTEX 1808. C. sorokiniana a.t.c.c. 22521. Ankistrodesmus braunii A.T.C.C. 12744 and Profotheca zopfii UTEX 1438, UTEX is the culture collection of algae at the University of Texas at Austin. One such mutagenized strain derived from UTEX 1663 is C. pyrenoidosa UVlOl-158 having A.T.C.C. accession no. 53170.

Other representative and preferred high producers of L-AA and their parents, themselves high producers and also suitable for use herein, are tabulated along with the applicable method of mutagenesis as follows:

DAP388-19 was derived from DAP300-4, which was derived, in turn, from UV489-5, SUV359-2, SUV296-5, SUV97-1, UV213-4, UV212-11, SUV9-1, UV182 2576, NA5-3, UV137-253, UV101-158, UV29-132, UV18-374, UV12-15, UV3-482, and Chlorella Ppyrenoidosa XJtex 1663.

UV711-1 and EMS156-1 were derived from C115-1, which was derived, in turn, from DAP300-5, UV489-5, SUV359-2, SUV269-5, SUV97-1, UV213-4, UV212-11, SUV9-1, UV182-2576, NA-3, UV137-253. UV101-158, UV29-I32UV18-374, UV12-15, UV3-482, and Chlorella PpyrenoidosaVtex 1663.

C 166-4 was derived from SUV551-3, which was derived, in turn, from DAP389-23, DAP300-5, UV489-5, SUV359-2, SUV296-5, SUV97-1, UV213-4, UV212-11, SUV9-1, UV182-2576, NA5-3, UV137-253, UV101-158, UV29-132, UV18-374, UV12-15, UV3-482, and Chlorella PpyrenoidosaVtex 1663.

NA687-1, UV869-3 and C284-130 were derived from C166-4. NA722-22 was derived from C123-4, which was derived, in turn, from UV609-4, NA528-2 DAP300-6, UV489-5, SUV359-2, SUV296-5, SUV97-1, UV213-4, UV212-11, SUV9-1, UV182 2576, NA5-3, UV137-253, UV101-158, UV29-132, UV18-374, UV12-15, UV3-482, and Chlorella Ppyrenoidosa Utex 1663. C325-2 was derived from C123-2, which was derived, in turn, from UV609-4, NA528-2 DAP300-6, UV489-5, SUV359-2, SUV296-5, SUV97-1, UV2I3-4, UV212-11, SUV9-1, UV182 2576, NA5-3, UV137-253, UV101-158, UV29-132, UV18-374, UV12-15, UV3-482, and Chlorella Ppyrenoidosa Vtex 1663. UV872-26 was derived from NA687-1.

DAPN1010-50 was derived from DAP994-1, which was derived, in turn, from C360-70, C284-130, C284-130, C166-4, SUV551-3, DAP389-23,

DAP300-5, UV489-5, SUV359-2, SUV296-5, SUV97-1, UV213-4, UV212-11,

SUV9-1, UV182-2576, NA5-3, UV137-253, UV101-158, UV29-132, UV18-374, UV12-15, UV3-482, and Chlorella Ppyrenoidosa Utex 1663.

UV232-1 was derived in turn from NA28-4, NA19-3, UVI65-239, UV137-253. UV101-158, UV29-132.UV18-374, UV12-15, UV3-482, and Chlorella Ppyrenoidosa Utex 1663.

The prefixes of the mutant strain names are abbreviateds of the mutagenizing treatment used to create them as follows: C = camphor; DAP = 2,6- diaminopurine; EMS = ethyl methanesulfonate; NA=nitrous oxide; DAPN = DAP and

NA, in succession; SUV = short wavelength ultraviolet light; UV = broad wavelength ultraviolet light.

In each case, the mutagenized offspring is a higher L-AA producer than its parent. Further, each organism produces a higher specific formation of L-AA under the invention process conditions as described below than under conventional conditions.

Depending upon the particular microalgae employed, the process may be a conventional heterotrophic fermentation wherein the organism is grown under unrestricted growth conditions in a growth-promoting medium at an effective temperature, pressure and pH, wherein the medium contains a suitable carbon source such as glucose and molecular oxygen (O₂) dissolved therein in amounts sufficient for the cells to grow to a high cell density accompanied by the production of intracellular L-AA, and wherein growth is maintained until the cells contain a suitably high percent by weight of intracellular L-AA.

Preferably, the process comprises heterotrophically growing the cells under unrestricted growth conditions as above to a high cell density, which may or may not be substantially maximum, allowing the carbon source to become substantially depleted and cell growth to substantially cease, then resuming feed of the carbon source in restricted amounts such that L-AA production continues and results in an enhanced production of L-AA with substantially no increase in cell density, and continuing the restricted growth phase on controlled amounts of carbon source in the pressure of dissolved O~ until the L-AA concentration per unit weight of cell biomass reaches a desired level for the purposes of this invention. Another preferred process comprises heterotrophically growing the cells under unrestricted growth conditions as above, i.e., in the presence of sufficient carbon source and dissolved O₂ at a temperature, pressure and pH effective to result in a high density of cells containing intracellular L-AA, then lowering the dissolved O« content or allowing it to become lowered through normal cell metabolism to a low concentration such that cell growth substantially ceases with L-AA production continuing with substantially no increase in cell density, and maintaining the low OΛ conditions until a desired intracellular L-AA concentration in the biomass is attained.

It will be noted both above process embodiments provide for enhanced utilization of the carbon source for the production of intracellular L-AA. In carrying out the process, a sterile aqueous nutrient culture medium is aseptically inoculated with an actively growing culture of the selected microorganism in amounts sufficient to result after a reasonable growth period (during which growth is normally exponential) in a relatively high cell density. Typical initial cell densities are generally in the range of from about 0.15 to 0.4 g/L based on the dry weight of the cells. Small amounts of an antifoaming agent may be added initially or during the process as needed. The culture medium includes the carbon source, various salts and generally also trace metals.

The carbon source is preferably a source of glucose, including glucose, for reasons of economy, but may be any saccharide or polysaccharide that can be converted m situ to glucose, e.g. molasses, corn syrup, etc. The total amount of glucose source employed can vary broadly depending upon the particular organism and the result desired. Normally, with high L-AA producing organisms such as those described above, the total amount of glucose employed would, if not metabolized, provide a concentration of about 65 to 90, more usually about 75 to 85, and preferably about 80 g L. Usually, about 15 to 30% of the total glucose is added initially. The glucose is normally added initially and during the course of the fermentation along with other additives identified below. During the initial glucose source addition and fermentation period, which is the period of unrestricted cell growth wherein the cells are grown to a relatively high density, for example, about 20 to 45 g L, more usually about 30 to 40 g L, accordingly to provide a basis for a high total L-AA concentration during the carbon-controlled or uncontrolled restricted cell growth step of the invention process for high intracellular L-AA content microalgae.

The amount of glucose source in the fermentor should be a non-repressing non-limiting amount, that is, it should optimally promote and not inhibit or unduly limit cell growth. Optimum non-repressing concentrations of the glucose source may vary from organism to organism, and are readily determined by trial for any particular organism. For example, with C. pyrenoidosa strains the glucose source concentration is generally maintained by timely additions in the 15-30 g/L range, found sufficient to promote cell growth while avoiding glucose inhibition of growth. Carbon source availability to the organism throughout the process can be monitored with known methods. Glucose, for example, can be determined in the supernatant by use of the glucose oxidase enzyme test or chromatography (HPLC). When the carbon source concentration drops, it can be replenished as needed, while ensuring the total concentration remains below growth-repressive levels, which is generally below about 30 g/L on a glucose-equivalent basis.

Desirably, other additives are present initially along with glucose source, notably sources of phosphorus, nitrogen, magnesium, iron and trace metals, as is known in the art; and their concentrations in the nutrient medium are also continually or periodically augmented or replenished by subsequent additions of the same additives in conjunction with the continued addition of the glucose source. The ratio of the concentrations of these additives to the concentration of the glucose source may be the same or different throughout the fermentation.

A typical nutrient medium, its composition and use, are described more fully in U. S. Patent 5,001,059, which disclosure is incorporated herein by reference. The O₂ source is preferably air, but may be molecular O₂ undiluted or diluted with any gas inert to and unreactive with the fermentation components. The O₂ source is conveniently sparged into the fermentation mass, which is preferably agitated to distribute the gas throughout the medium and facilitate solution of O₂ therein. The solubility of the O₂ in the medium, up to the saturation concentration (100% dissolved O₂) is largely a function of the O₂ source flow rate, agitation rate, medium composition, temperature and pressure. For a given medium at a given temperature and pressure the availability of dissolved O₂ to the organism is easily controlled by the agitation rate and the O₂ gas flow rate.

The agitation rate is usually at about 200 to 1000 rpm, while the aeration rate is generally in the range of about 0.1 to 0.6 liters of air per minute (1pm) depending upon the amount of dissolved O₂ desired during any particular stage of the process. During the unrestricted cell growth stage the dissolved O₂ concentration is generally about 20% of the saturation value. Preferably, it is 50% or more.

During the unlimited stage of the O₂-limited process, the O₂ source is fed at a rate such that the concentration is well below the 20% of saturation value and is best kept just above the O% value, nominally around 1%. Whatever the absolute value, the low O« concentration should be sufficient at optimum pH described below to result in an increase in the intracellular L-AA content of the cells without resulting in substantial cell growth in the presence of carbon source. The dissolved O₂ concentration is conveniently monitored with an oxygen probe electrode.

The temperature and pressure should be such as to promote cell growth and L-AA production without destroying the cells. Normally, the temperature is 20 to about 40' C, preferably about 35^ΛC. The pressure is generally atmospheric but may be superatmospheric; the greater the pressure the greater the solubility of air (O₂) in the medium.

The pH can vary widely depending on the ability of the organism to grow, produce and retain intracellular L-AA, i.e. resist secreting it into the aqueous medium. In general, with Chlorella pyrenoidosa and its various strains, the pH is normally in the range of about 6.5 to 8, more usually about 6.9 to 7.5 as it has been found such relatively high pHs retard passage of L-AA from cell to medium; i.e. the cells retain more L-AA than they secrete into the medium. pH can be controlled by adding ammonia gas as needed to increase pH.

ML, also serves as a source of nutrient nitrogen. It can also be controlled by adding as needed a physiologically compatible acid such as phosphoric, acetic, lactic, tartaric or the like to lower pH. Alternatively, the microorganisms can be allowed to lower the pH metabolically, if necessary inasmuch as they inherently produce acid byproducts.

During the high oxygen unrestricted growth phase of the process wherein the pH is relatively high as discussed above intracellular L-AA can increase to as high as 1 to 4% of the dry weight of the cells. The intracellular L-AA content can be increased still further to at least 1.5% preferably at least about 2% and to as high as 5-6% by also maintaining the high pH during the low carbon and the low oxygen restricted growth phases of the processes described above. The higher the intracellular content the better for animal food use is the biomass.

The following examples are intended to illustrate the invention, and are not to be construed as limiting the scope of the appended claims.

EXAMPLE 1 Medium Preparation. A solution of 0.27g monobasic potassium phosphate and 0.23g dibasic sodium phosphate in 600 ml distilled water was heat-sterilized in a one-liter glass feπnentor equipped with means for agitating the medium and feeding to it nutrient components, oxygen source and other agents described below. After the medium had cooled, five ml of a 1.92 g/1 ferrous sulfate solution (pH 2.5) was added through a 0.2 micron sterile filter.

Glucose-Salts Concentrate. The following aqueous nutrient components were separately sterilized, then combined after cooling to a final volume of 104 ml: 80ml containing 56g glucose; 11 ml containing 0.70g trisodium citrate, 0.46g magnesium sulfate and 0.70 ml 96% ILSO, 10 ml containing 1.53g monobasic potassium phosphate and 1.53g dibasic sodium phosphate; and 9.4ml of a trace metal solution whose composition is described in U.S. Patent 5,001,059 incorporated herein by reference.

Nutrient Medium. 20 ml of the glucose-salts concentrate was then added to the phosphate medium.

Cell Growth and L-AA Production. The nutrient medium was heated to and maintained at 35 C, agitation was begun at 450 rpm, air was sparged into the medium at 0.1 liter per min (1pm), the pH was adjusted to 6.9 with anhydrous M , added to the airflow and the medium was inoculated with an actively growing culture of Chlorella pyrenoidosa strain DAPN1010-50 to give an initial cell density of approximately 0.3, i.e. 0.2 g/L. In the first 10 hours, there was a barely noticeable drop in the dissolved

O₂ content of the medium and a slight increase in cell density. At the 20 hour mark, cell growth (g/1 cell density) was on the rise and dissolved O₂ was down to about 70%. After 30 hours, cell density had increased to about 5 g/1 and the O₂ content had decreased sharply to somewhat less than 20%, reflecting the strong uptake of O~ by the increasing mass of growing cells. At this point, the agitation rate was stepped up to nearly 800 rpm and the aeration rate to 0.2 1pm. This resulted in a sharp increase in dissolved O₂ peaking at about 85%, then declining to about 25% over the next 10 hours again reflecting the high cell growth rate, the cell density reaching 14 g/1.

Over the next 10 hours, with agitation and aeration steady at 775 rpm and 0.2 1pm resp., dissolved O₂ dropped to near zero percent, the rate of cell density increase was at a maximum with the cell density at 28 g/L, and the L-AA content increased to 0.8 g/1, substantially all of it intracellular. All during the 50 hour growth period, the glucose concentration was maintained in excess at 20-30 g/1 by addition as needed of the glucose-salts concentrate described in U.S. Patent 5,001,059. As for the pH, it had dropped from 6.9 to 6.8 in 42 hours, during which time it had been maintained at such levels with ML addition to the air flow.

In summary, the intracellular L-AA content of the cells (biomass) by the end of the run was 2.9% based on the dry weight of the cells (0.8/28 X 100).

EXAMPLES 2 - 12

The procedure of Example 1 was repeated substantially as described in a series of runs with a different microalga in each run. The microalgae and the results obtained are tabulated below. The organisms, all high L-AA producing Chlorella pyrenoidosa mutants, are described in Table 1. The first four examples (2 - 5) were run to a cell density of around 20 g/1, the next 7 to a cell density of around 40. Intracellular L-AA contents of the resulting biomasses at selected cell density points in the runs are given in Table 1. TABLE 1

EXAMPLES 2 - 12: L-AA - ENRICHED BIOMASS

L-AA

Ex. Strain Biomass

2 DAP 388-19(a) 1.9

3 DAP 388-19 (b) 1.9

4 EMS 156-1 2.0

5 UV 711-1 2.5

6 C 166-4 (c) 2.4

7 NA 687-1 (d) 2.6

8 NA 722-22 (d) 2.4

9 C 225-2 3.1

10 UV 869-3 1.6

11 UV 872-26 (c) 2.0

12 C 284-130

2.2

(a) Average of 4 runs

(b) Conducted in 14-liter fermentor; all others in 1-liter

(c) Average of 2 runs

(d) Average of 3 runs EXAMPLE 13

Sterilized in a 1L fermentor was 0.6L distilled water, 0.23g dibasic sodium phosphate and 0.27g monobasic potassium phosphate. To the phosphate solution was then aseptically added 11.2mg of ferrous sulfate (heptahydrate) in 5ml distilled water and 20 ml of sterile glucose-salts concentrate prepared as follows with Groups of nutrients sterilized individually and combined after cooling: Group 1 consisting of 56g glucose, food-grade monohydrate (anhydrous basis) in 80ml water; Group 2 consisting of 0.7g trisodium citrate dihydrate, 0.47g magnesium sulfate anhydrous and 1ml sulfuric acid in 10ml water; Group 3 consisting of 0.65g monobasic sodium phosphate, 1.3g monobasic potassium phosphate and 0.6g dibasic sodium phosphate in 10ml water; Group 4 consisting of 9.4ml of the trace metal solution of U.S. Patent 5,001,059.

The temperature was raised to 35 and agitation begun at about 200rpm. Air was.passed through the medium at the rate of 0.2 liters per minute (1pm) and 50ml of Chlorella pyrenoidosa strain UV 101-158 (A.T.C.C. Accession No. 53170) at a concentration of about 0.3g cells/L was added. After 5 hours the agitation rate was increased to 400 rpm, the air flow to 0.4 1pm After 16 hours the agitation rate was raised to 550 rpm, the air flow to 0.6 1pm. The agitation rate was later increased to 700 then 800 rpm as noted in Table 3 while the air flow was steady at 0.6 1pm for the remainder of the run.

The following chart described the conditions and analytical results for the fermentation.

glucose/salts concentrate

+ 20% glucose. This addition was repeated periodically during the course of the fermentation as shown.

** intracellular EXAMPLE 14 fBEST MODE)

The procedure of Example 13 was followed except that (a) 5.6 mg ferrous sulfate, instead of 11.2 mg, was employed in the initial 0.6L distilled water charge; (b) the nutrient solution consisted of: 28g glucose in 40ml distilled water; 0.53g trisodium citrate dihydrate plus 0.2g magnesium sulfate in 20ml; 0.65g each of monopotassium acid phosphate and disodium acid phosphate in 20ml; and 4.7ml of the trace metal solution plus 1ml sulfuric acid in 15ml; and (c) the actively growing culture of C. pyrenoidosa was strain UV 232-1.

The temperature was raised to 35' C, agitation begun at 350rpm, air passed through the medium at 0.2 liters/min (1pm), the pH adjusted to 6.9 with anhydrous ammonia (ML) added to the air flow, and strain UV 232-1 added to the medium. ML was fed throughout the run as nutrient nitrogen source and pH controller. After 6.2 hours the air flow was increased to 0.4 1pm, the agitation to 400 rpm. At 11.8 hours the air flow was raised to 0.6 1pm, where it was held for the remainder of the run, and the agitation rate raised to 650 rpm. After 12.4 hours, 40 ml more of the glucose-containing nutrient solution was added to the fermentor, followed by 20ml more at 24.4 hours and 15 ml more at 26.3 hours; in the meantime, the agitation rate was raised to 750 rpm at 22.8 hours, then adjusted to 800 rpm at 34.8 hours and maintained at that rate for the rest of the run. The glucose content of the medium became depleted at 31.7 hours, at which time the cell density (CD.) was 19.5 and the L-AA concentration was 322 mg/L. Glucose, 2% ml of 10% solution, was fed to the fermentor at 41.7 hrs and every 3 hrs from 41.7 until 94.8 hrs. Cell density and L-AA concentration were followed with time as tabulated below along with calculated L-AA content of the biomass.

TABLE 3

Time CD. L-AA Biomass hr pH al mg L Comments Wt. % L-AA

0 6.9

6.2 6.9

11.8 6.9 40ml glucose at 12.4 hrs, 20 at

22.8 24.4 hrs, 15 at

26.3 hrs.

31.7 6.9 glucose depleted

34.8 7.0 19.5 322 1.7

46.9 7.7 18.7 429 2.3

53.6 7.8 528

58.6 7.8 17.9 613 3.4

70.8 7.9 694

77.7 7.9 17.3 819 4.7

82.8 7.8 915

94.8 7.6 17.6 945 5.4

* 2 ml 10% glucose added at 41.7 hrs and every 3 hrs thereafter vmtil 94.8 hrs. The above procedure was repeated four more times, substantially as described. One run included the addition of Maltrin, a maltodextin, (5g/20ml distilled water) added at 47.3 and 71.2 hours as glucose equivalent carbon source. The % L-AA averaged 5.2% of the dry weight of the biomass over the 5 runs. The method employed for determining L-ascorbic acid is described by

Gran and Loewus, Analytical Biochemistry (1983) 130:191-198. The method is an ion-exchange procedure, employing a 7.8 X 300mm organic acid analysis column, HPX-87 (Bio-Rad Laboratories, Richmond, CA). The conditions are: mobile phase, 0.013 M nitric acid, flow 0.8 rnl/min, pressure 1500 psig, detection, UV 245-254nm. With the above conditions, resolution of L-ascorbic acid and isoascorbic is possible. As noted in the above Examples, all the ascorbic acid under the described process conditions is intracellular.

Cell density (dry weight of cells in grams per liter) is determined as follows: A biomass sample (5ml) is transferred to one weighing pan and 5ml of centrifuged supernatant transferred to a second weighing pan. The pans are dried in a convection oven (105 0 for 3 hours). After cooling in a desiccator, the pan contents are weighed. The grams of cells per liter are determined as: (sample weight-supernatant weight) X 200.

The biomasses, however, may be dried by any of the methods known to the art with care being taken to minimize air oxidation or thermal degradation of the L-ascorbic acid content.

The above examples show that the invention methods and new high L-AA-producing microalgae provide biomasses highly enriched in their Vitamin C content. The vitamin contents range from greater than 1.5 to as high as 5 wt % or more, far exceeding the results of the prior art. Hence, in view of their other well-known nutritional values, they are satisfactory for use as vitamin C enriched animal feed compositions alone or in admixture with other suitable feed compositions. Such feed composition mixtures typically may contain from about 60 to 2,000 grams of L-ascorbic acid per ton of mixture depending on the host animal to be treated, more usually about 320 g/ton.

Having thus described and exemplified the invention with a certain degree of particularity, it should be appreciated that the following claims are not to be so limited but are to be afforded a scope commensurate with the wording of each element of the claim and equivalents thereof.

Claims

1. A biomass comprising green microalga cells having an intracellular content of L-ascorbic acid greater than 1.5% of the dry weight of the cells.

The biomass of Claim 1 wherein the ascorbic acid content is at least about 2% of the dry weight of the cells.

3. The biomass of Claim 1 wherein the ascorbic acid content is at least about 3% by weight of the cells.

The biomass of Claim 1 wherein the microalga is of the genus Chlorella.

The biomass of Claim 4 wherein the microalga is of the species pyrenoidosa.

The biomass of Claim 5 wherein the microalga is a mutagenized strain of said C. pyrenoidosa having A.T.C.C accession number 53170.

7. The biomass of Claim 5 wherein the microalga is at least one mutagenized Chlorella pyrenoidosa strain having the designation UV187-1319, UV232-1, DAP388-19, UV711-1, EMS156-1, C166-4, NA687-1, NA722-22, C225-2, UV869-3, UV872-26 or C284-130.

The biomass of Claim 7 wherein the microalga is at least one of the group C166-4, NA687-1, NA722-22, C225-2, UV872-26, C284,130 and UV232-1.

9. The biomass of Claim 8 wherein the microalga is UV232- 1.

10. A method for producing a biomass composition of Claim 1 which comprises selecting a green microalga capable of growing heterotrophically on a suitable carbon source in the presence of oxygen and potentially capable of producing intracellular L-ascorbic acid in an amount of at least 1.5% by weight of the dry weight of the microalgae, heterotrophically growing cells of the microalga in a growth-promoting aqueous nutrient medium at an effective temperature and pressure and at a pH at which the ascorbic acid produced is predominantly intracellular, said medium containing a suitable carbon source and a source of dissolved molecular oxygen in amounts sufficient for cell growth and

L-ascorbic acid production, maintaining said cell growth until a mass of cells is produced containing intracellular L-ascorbic acid in an amount corresponding to at least 1.5% by weight of the dry weight of the cells, and separating the mass of cells from the aqueous supernatant of the medium.

11. The process of Claim 10 wherein the cell growth is continued until the intracellular ascorbic acid content is at least 2% by weight of the cells.

12. The process of Claim 10 wherein the biomass of cells is dried without substantially lowering the ascorbic acid content.

13. The process of Claim 10 wherein the selection of the microalga comprises:

(a) identifying through heterotrophic growth tests potential high-producing strains of a L-ascorbic acid-producing microalga,

(b) mutagenizing at least one such potential high-producing parent strain,

(c) identifying and isolating at least one mutagenized product strain of said potential high-producing parent that is a higher ascorbic acid producer than the parent,

(d) repeating, if necessary, step (b) on at least one mutagenized product strain of step (c) until there is obtained a mutagenized strain of said microalga capable of producing at least 1.5% intracellular L-ascorbic acid based on the dry weight of the microalga cells.

14. The process of Claim 10 wherein cell growth is continued to a high cell density, the carbon source is allowed to become depleted, the depleted carbon source state is maintained until cell growth substantially ceases and until resumption of feeding of the carbon source in controlled amounts results in the production of additional amounts of intracellular

L-ascorbic acid with little or no substantial increase in cell density, whereby the intracellular L-ascorbic acid content based on the dry weight of the cells is further increased.

15. The process of Claim 10 wherein the temperature is in the growth-sustaining range of from about 20 to about 40^*0, the pressure is atmospheric or superatmospheric sufficient to maintain growth-promoting amounts of molecular oxygen dissolved in the medium and the pH is growth-sustaining and sufficiently high to maintain a high intracellular L-ascorbic acid level in the cells.

16. The process of Claim 15 wherein the temperature is 30 to 40' C, the pressure is at least atmospheric and the pH is about 6.5 to 8.

17. The process of Claim 16 wherein the temperature is 35-38 and the pH is at least about 6.9.

18. The process of Claim 10 wherein cell growth is continued at a pH at which the ascorbic acid produced is predominantly intracellular to a high cell density in the presence of said carbon source and molecular O« in concentrations sufficient to provide such high density growth the concentration of O₂ is decreased or allowed to decrease to a concentration at which cell growth slows and substantially ceases and the production of intracellular L-ascorbic acid continues and increases with little or no increase in cell density, whereby the intracellular L-ascorbic acid content based on cell dry weight is further increased.

19. An animal feed composition comprising the composition of Claim 1.

20. The animal feed composition of Claim 19 mixed with a second animal feed composition in an additive amount providing a physiologically acceptable amount of L-ascorbic acid.

21. The composition of Claim 20 wherein the feed mixture contains from 60 to 2,000 grams of L-ascorbic acid per ton of the mixture.

22. The composition of Claim 19 wherein the animal feed composition is an aquaculture fish food.

23. A method of providing L-ascorbic acid to an animal having a Vitamin C requirement which comprises providing in the diet of said animal sufficient of the biomass of Claim 1 or of the animal feed composition of Claim 19 to provide at least a significant proportion of the daily Vitamin C requirement of said animal.

24. The method of Claim 23 wherein the animal having the Vitamin C requirements is a fish.