CA2091918C - L-ascorbic acid production in microorganisms - Google Patents

Abstract

Improved heterotrophic biosynthetic production of ascorbic acid is obtained using ascorbic acid-producing microalgae in particular of the genus Chlorella and the genus Prototheca, as the microorganism source and growing, the culture undera controlled pattern of carbon source supply. Greatly improved ratios of ascorbic acid to total carbon source supplied as well as enhanced concentrations of ascorbic acid in the fermentor are obtained. C. pyrenoidosa UV 101-158 was deposited at the A.T.C.C. on June 27, 1985 and given Accession No. 53170.

Description

2Cql918 TITLE
I,ASCORBIC ACID PRODUC~ION IN MICROORGANISMS
BACKGROUND OF THE INVENTION

1. Field of the Invention:
This invention relates to a heterollophic process for the improved production of L-ascorbic acid by microorg~nicmc~ in particular rnicroalgae, in 10 nutrient media cont~ining a suitable carbon source. In particular, the invention relates to such process which produces high concentrations of ascorbic acid, preferably high con~e.~ tions per unit weight of cell mass.
The invention also provides new mutagenized microalgae species suitable for use in the invention L-ascorbic acid process.
L~

2. Description of Related Art:
L-ascorbic acid is an important nutrient supplement, which finds wide application, in vil~~ capsules and as a nutrient supplement in foods for both h -...~ and other Vitamin C requiring ~nim~lc L,ascorbic acid is a bulk chemical which is highly price se~ and requires economic and ef~lcient production to be marketable. Therefore, there is suhst~nti~l interest in being able to develop processes employing microorg~nismc which provide for efficient conversion of nutrients resulting in efficient production of L, ascorbic acid.
2s Loewus, F.~, in L-Ascorbic Acid: Metabolism, Biosynthesic, F m~ion, The Biochemistry of Plants, Vol. 3, ~demic 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 et. al., S~ence and Culture (1971) 37:383-384; Subbul~kchmi et. al., Nutrition Reports International (1976) 14:581-591; Aaronson et. al., Arch. Microbiol.
(1977) 112:57-59; Shigeoka et. al., J. Nutr. Sci. Vitaminol. (1979) 29:29-307;
Shigeoka et. al., Agric. Biol. Chem. (1979) 43:2053-2058: Bayanova and Trubachev, Prikl~-ln~ya Biokhimiya i Mikrobiologyia (1981) 17:400-407 s (UDC 582.26:577.16); and Ciferri, Microbiological Reviews (1983) 47:551-578.
The prior art heterotrophic processes are not entirely s~ti~f~ctory for commercial use: their utilization of the carbon source for ascorbic acid production is generally poor and the vitamin is produced in undesirably low 0 concentrations.

SUlVlMARY OF THE INVENTION

It is an object of this invention to provide a process for the heterotrophic biosynthesis of L-ascorbic acid from a carbon source which results in enhanced utilization of the carbon source. It is another object to provide such a process which affords the vitamin in high yields. It is a furtherobject to provide novel high L-ascorbic acid-producing microorg~ni~m~ as new compositions of matter.
Accordingly, the present invention provides an improved process for kascorbic acid production, which process comprises heterotrophically growing cells of an L-ascorbic acid producing microorganism in a nutrient medium cont~ining a carbon source and dissolved oxygen (~2) in amounts suitable for growth and L-ascorbic acid production, allowing the organism to grow in an initial stage to a high cell density accompanied by intra cellular L-ascorbic acid production and the substantial complete depletion of the carbon source, m~int~ining the cells in the subst~nti~lly depleted carbon source state until cell growth substantially ceases and subsequent addition of the carbon source in controlled amounts results in the formation of additional quantities of L-ascorbic acid with substantially little or no increase in cell density, andcontinuing the controlled carbon source addition until a desired increase in L-ascorbic acid production is attained with substantially little or no increase in cell density.
Thus, improved utili7~tion of the carbon source is observed in relation to L-ascorbic acid (L-AA) production, while obtaining an enhanced yield, as evidenced by an increase in total L-AA expressed as mg L-AA/liter of 23~1918 solution, and preferably also by an increase in the specific formation of L-AA
expressed as mgs of L-AA per gram of dry weight of cells.
The present invention further provides a new high L-AA producing mutagenized microalgae, more specifically: Chlorella pyrenoidosa UVl01-158 5 which is derived from C. pyrenoidosa isolate UTEX 1663 and has been deposited at the American Type Culture Collection (ATCC) on 6l27l85 and given Accession No. 53170; Chlorella regularis UV5-280 derived from C. regularis UTEX 1808;
Prototheca zopfii UV3-132 derived from P. zopii UTEX 1438; Ankistrodesmus braunii UV3-132 derived from P. zopii UTEX 1438; Ankistrodesmus braunii UV2-1 0 370 derived from Ankistrodesmus braunii ATCC 12744. UTEX is the Culture Collection of Algae at The University of Texas at Austin.
Further aspects ofthe present invention are as follows:
A method for producing L-ascorbic acid, the method comprising the steps of growing a culture of an organism of the genus Prototheca to obtain a fermentation 1 5 medium con~ining L-ascorbic acid and recovering L-ascorbic acid from the fermentation medium.
A microalgal biomass comprising cells of an organism of the genus Prototheca, the cells comprising greater than 3.5% L-ascorbic acid by dry weight of the biomass.
2 0 A vitamin C enriched animal feed composition, the composition comprising microalgal biomass, the biomass comprising cells of an organism of the genus Prototheca~ the cells comprising greater than about 3.5% L-ascorbic acid by dry weight of the biomass.
The production of these new microalgae compositions of matter and their 2 5 effectiveness for the production of L-AA in the he process of this invention are described more fully below.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

3 0 In general, the invention process involves three key stages: (l) an initial cell growth stage wherein a microorganism, preferably a microalgae, capable of producing L-AA heterotrophically is grown heterotrophically in a fermentor cont~ining an effective carbon source at a first concentration and dissolve ~2 each in amounts suff1cient for the organism to grow to a high cell density accompanied by 3 5 the formation of intra cellular L-AA and the substantially complete depletion of the carbon source; (2) a substantially completely depleted carbon source stage wherein 209i918 3a the cells of the microorganism are allowed to remain in such depleted carbon source state until cell growth substantially ceases, and (3) a controlled carbon sourceaddition stage wherein the carbon source is fed to and m~ints~ined in the fermentor at a second concentration lower than the first concentration and effective to result in the 5 production of additional amounts of L-AA in the presence of dissolved ~2 without resulting in a substantial increase in the density of the cells.
The addition of the carbon source at the lower concentration (at which L-AA
production is favoured over cell growth) can be continued until the ability of the microorganism to produce L-AA is substantially exhausted.

2û?l ~ 8 This point can be determined by monitoring the L,AA cQnce~ ation and cell density with time throughout the process.
Other stages of the process include the harvesting of the cells and the separation and reco.ely of the L,AA subst~nti~lly free of cellular material in 5 accordance with procedures known to the art.
The L,AA product rccovelcd from the cells (biomass) can be utili7ed as such. A1tel..AI;~rely, the ~AA l~dened biom~cc itself can be used as a vil~ C enriched animal feed co~po~;l;Qn or feed supplement including for use in the aqu~lltl--e of fish.
The microo~g~.-;c.. ~ for use in this invention may vary widely provided they are L,AA producers, in particular such or~e~nicmc capable of producing intracellular ~AA heterotrophically. Preferred microorg~nicmc are the ~
AA producing green microalgae, especially for reasons of economy the so-called high-producers thereof, sometimes refelled to as over-producers.
Or~nicmc showing promise as potential high-producers of L,AA can be identified using standard ferme~t~tion procedures for cell growth acco.,.l-anied by L,AA production. They are then preferably mutagenized using physical or chemical mutagenizing means, e.g. U.V. light, x-rays, N-methyl-N'-nitro-N-nitrosoq~l~ni(line dimethyl sulfate or the like agent known 2a to the art. Mutagenized ~AA o.cll,loducers produced by such treatments can be advantageously determined with redox dyes. Further, by employing analogs to metabolic intermç~ tes to ascorbic acid or inhibitors of the ascorbic acid synthecic) microorg~ni~mc may be selected that are capable of m~it~l~;n;ng or increasing ~AA production in the presence of chPmic~l 25 interference.
Preferred progeny of the above procedures are those microorg~nicmc providing improved specific formation of ~AA as measured by milligrams of ~AA per gram of cells (dry weight basis). These progeny may then be further separated into individual clones and further subjected to the above 30 procedures.
One ~ure~lled microolgallislll is of the green microalgae genus Chlorella in particular Chlorella pyrenoidosa strain, UV101-158, a high L,AA
producing mutant descended from strain UTEX 1663, by mllt~g~..;,;,lg W
light. C ,~yrenoidosa W 101-158 has been deposited with the ATCC and 3s given ~cceccion No. 53170. Another preferred C pyrenoidosa strain is W232-1, the highest intracellular ~AA produced to date. Other 23ql91~

re~rcse~ t;ve and suitable species of the genus Chlorella are: Chlorella regularis strain UTEX 1808; and C. regulans W 5-280, a UV generated high L-AA producing mutant of strain IJTEX 1808. Still other suitable L-AA
producing rnicroalgae also able to grow heterotrophically are those belonging s to the genera Prototheca and Ankistrodesmus. Represe~.tative Protothec~
species are P. zopfii, strain UTEX 1438 and P. zopffi W3-132, a W-gene,aled high L,AA producine mutarlt of UTEX 1438. Represent~tive Ankistrodesmus species are A. braunii ATC 12744 and A. braunii UV 2-370, a high L-AA producing W-genel a~ed mutant of ATC 12744.
o It will be noted, in each above case, the mutagenized o~l.ling is higher L-AA producer than its parent org~nicm Furtber, as shown below, each or~anislll produces a higher m~rimllm concentration of L-AA, in terms of mg L-AA/liter of nutAent medium, under the invention conditions as ~fin~d above than under coll~nlional, prior art conditions.
L5 In carrying out the process, a nutrient culture medium is innoculated with an actively growing culture of the microorganism in amounts sufficient to result after a reasonable growth period in a high cell density generally accol.,p~nie~l by a first, generally low concentration of L-A~ Typical initial cell densities are generally in the range of from about 0.15 to 0.4g/L based on the dry weight of the cells. The culture medium inrllldes the carbon source, a variety of salts and generally also trace metals. It also includes a source of molecular ~2. generally air, fed in amounts that are growth-promoting in conjunction with growth-promoting amounts of the carbon source. In other words,both an effective carbon source and ~2 must be available to the microorg~slJ. to achieve growth to a high cell density.
The carbon source is normally a source of L-galactose or D-glucose, preferably glucose, for reasons of economy. The source of glucose may be any carbohyd~ate that can be convel led in situ to glucose, e.g. molasses, corn syrup, etc. The total amount of glucose source employed can vary broadly depen~ling upon the particular organism and the result desired. Normally, with a high L-AA producing organism such as C ~yrenoidosa W 232-1, the total ~ o~-t of ghlcQse source employed would, if not metabolized, provide a conce.ll~lion of about 65 to 90, more usually about 75 to 85, and prcrel~bly about 80 g/l calc~ ted as glul~se Usually, about 15 to 305'o of the total glucose will be added initially, more usually about 20 to 25~o of the total glucose. The glucose is normally added initially and during the ferm~nt~,tion ~, ~ i .~. ,.

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 cell density, generally accompanied by the formation of L-AA, usually in a relatively low s concentration.
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-growth limiting concentrations of the glucose source may vary from organism to organism and 0 are readily determined by trial for any particular org~nicm. For example, with C pyrenoidosa W 101-158 the glucose source concentration is m~int~ined by timely additions in the 15-30 g/l range,~ found sufficient to promote cell growth while avoiding glucose inhibition of growth.
Desirably, other additives are present initially along with glucose source, and their concentrations in the nutrient medium are generally also contiml~lly provided 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.
Among the additives which may have a different ratio to glucose in the amounts added incrementally as compared to the total amounts added to the fermentor are the alkali metal phosphates, e.g.,sodium and pot~ccil]m phosphates, particularly as the dibasic sodium phosphate and the monobasic pot~ccillm phosphate. The total amount of the dibasic sodium phosphate is typically about 1 to 2 total g/l, usually about 1 to 1.5 total g/L and preferably about 1.3 total g/L. The amount initially present in the fermentor of dibasic sodium phosphate is usually about 35 to 50~o, more usually about 40 to 45~o of the total amount of dibasic sodium phosphate added. The total amount of monobasic potassium phosphate is usually about 1.5 to 3g/L more usually about 2 to 2.5g/L. The amount initially present is generally about 40 to 50%
of the total amount, more usually about 45 to 505~o of the total amount.
In addition to the above additives, a biologically acceptable chelating agent, such as trisodium citrate is advantageously added in a total amount of from about 0.8 to 1.2g/L usually about lg/L. Monobasic sodium phosphate generally present in from about 0.8 to Ig/L preferably about 0.95 to Ig/L. A
biologically acceptable mineral acid is added to m~int~in the trace metals in solution and also to neutralize the ammonia that is usually employed as the nitrogen source. Conveniently, conc. sulfuric acid is employed for this purpose in amount of about 1 to 2, more usually about 1.2 to l.5ml/L.
Among the metals, magnesium is present in about 0.1 to 0.2g/L, preferably s about 0.1 to 0.15g/L, particularly as a physiologically acceptable salt, e.g., sulfate. The amount of iron and copper employed is limited, since these metals repress ascorbic acid formation. Iron (ferrous) is present initially in from about 5 to 7mg/L, preferably about 5.5 to 6mg/L and is not included in any subsequent additions. Copper is present in relatively minute amounts, 0 generally from about 1 to 50ug/g of glucose.
The trace metal solution, described below is used in total amount of from about 10 to 15ml/L, more usually about 12 to 14ml/L. Based on glucose, the trace metal solution coprresponds to 0.1 to 0.2ml/g.
Conveniently, a solution is prepared for addition during the course of the fermentation and has the following composition.

Medium formula COMPONENT CONCENTRATION
~Relative to glucose) glucose 1.0 trisodium citrate, dihydrate 0.0125 magnesium sulfate, anhydrous 0.0082 monobasic sodium phosphate 0.0116 monobasic pot~c~ m phosphate 0.0238 dibasic sodium phosphate 0.0121 trace metal ~ lure 0.1675ml/g sulfuric acid 98~o (w/w) 0.0329 30 Nitrogen supplied is by anhydrous ammonia. This is also used as pH control.
Actual N level in media is determined by acidity of media and buffer capacity of media.
The trace metal mixture and solution have the following composition.

Trace Metal Solution COMPONENT CO~CENTRATION
(conc. in stock solution) mg/liter calcium chloride, dihydrate 3102 m~ng~nese (II) sulfate, monohydrate 400 copper (II) sulfate, monohydrate 0.4 cobalt (II) chloride, pentahydrate 40 lo boric acid 160zinc (II) sulfate, heptahydrate 400 sodium molybdate, dihydrate 19 vanadyl sulfate, dihydrate 20 nickel (II) nitrate, hexahydrate 8 sodium selenite 18 The stock solution of trace metals is prepared by dissolving the appropliate amounts of the various compounds in distilled water containing a trace of HCl so that the final volume is one liter and contains 20ml of concentrated HCl. Distilled water is used to ensure the proper ratio of component trace metals.
While a number of the salts are referred to as mono- or dibasic, it should be understood that this is a matter of convenience and not necessity.
These compounds act as buffers and therefore the extent of protonation will 2s vary with the pH of the medium.
In carrying out a fermentation, dibasic sodium phosphate and monobasic sodium phosphate will be dissolved into about 75 to 90% of the total medium, usually about 80 to 90~o of the total medium, to be added to the fermentor.
The solution to be added incrementally during the course of the fermentation is prepared by combining the individual components in proper ratios. The glucose is dissolved in from about 75 to 85%, preferably about 80% of the water to be used. The citrate, magnesium and sulfuric acid components are combined in an aqueous medium containing from about 5 to 3s 15%, usually about 10% of the water, while the phosphates are combined in g about 5 to lS~o, more usually about 10% of the water to be used, followed by the trace metal solution in about 5 to 15%, more usually about 8 to 10% of the total amount of water to be used. To the fermentor cont~ining a portion of the phosphates is added the ferrous salt and about 20% of the above-s prepared ~ cQse-salts concentrate. The ~diti~n is aseptic, so as to avoid the introduction of any foreign microolg~ ...c The nutrient ~nedium is then brought up to the desired telllpcralure.
This may vary with the micro-olgani~ but generally is in the range of about 30 to 40~C, preferably about 35~C and the fermentor ino~ te~l with the 0 inoculant generally to provide from about 0.1 to 0.4g/L initial cell density. A
small amount of antifoaming agent may be added during the process of the fermentation In the first stage of the ferrnentation, the cells are grown to a high cell density so as to provide a basis for an eventual high total production L-AA, and cell growth is co..l;...~ed until the carbon source is subst~nti~lly completely CQ-.C~ Pd, i.e., its glucose equivalent conce~ ion is normally less than 0.1g/L of solution, i.e., cell-free supell~alanl. The time required may vary with the organism but usually involves a period of about 35 to 50hr, more usually about 40 to 4Shr, with a growth rate of about 0.1 to 0.15hr~1.
The pH of the medium can be controlled within desired limits, generally in the range of about 6.5 to 8.0, by the addition of anhydrous ~mmoni~ as nPede~l The m~tlium is advantageously agitated and aerated during this growth period. The agitation rate is usually at about 200 to 1000 rpm, while 2s the aeration rate is generally in the range of about 0.2 to 0.6L of air/min., although this can vary with the olgallislll and the other fermentation conditions. Aeration provides molecular oxygen ~~2) to the me~i-lm, which is neçes~.y for cell growth and the production of L,AA. Other sources of ~2 can be employed, including undiluted ~2 gas and ~2 gas diluted with inert gas other than N2. Whatever the source of ~2, the dissolved ~2 content in the me-l;nm should be controlled during the course of the ferme~t~ion so as to ensure high cell growth and ~igh intr~cçll--l~r content of ~AA. The ~2 content of the nutrient me~ lm can be monitored by standard methods, co~ nlly with an ~2 probe electrode.
3s The glucose available to the microorganism during this initial growth stage can be mo~ ol~d in the supernatant, i.e., cell-free component of the medium, by convenient means, e.g., the glucose oxidase enzyme test, HPLC, or other known method. When the glucose concentration drops, it can be replenished as needed by adding aliquots, e.g., 20% aliquots of the glucose-salts concentrate described above, while ensuring that the total glucose s concentration remains below growth repressive levels, which as noted above is generally below about 30g/L. Glucose availability is m~int~ined until a desired high cell density is attained, which for C. pyrenoidosa UV101-158, for example, is about 35 to 4sg/L preferably about 40g/L with cell density calculated on a dry cell weight basis. At this point, the glucose content of the0 medium is allowed to become substantially completely depleted if it has not already reached this state.
When the desired high cell density is achieved and the glucose content of the medium has become substantially completely consumed, the glucose depleted state is maintained, i.e., the organism is starved for a period of timeduring which the growth of cells substantially ceases and the cell density reaches a m~ximnm. With cessation of cell growth, L-AA production may also substantially cease. In general, however, the cells can utilize stored starch to m~int~in cell functions including L-AA production to some small extent.
The substantially depleted glucose and no growth state is maintained until the organism on again being provided with glucose source, but in controlled amounts, begins to produce additional quantities of L-AA with little or no increase in cell density. The starvation period may range from minutes or less to hours or more and typically is from about 1 to 4 hours, more usually 2 to 4 hours. The optimum starvation time and the o~thllull-amount and rate of feeding glucose source to the fermentor can be determined for any particular organism by adding glucose source in small test quantities and monitoring the effect on L-AA content and cell density with time. When the ratio of the increase in L-AA to the increase if any in the cell density is greater than the m~xi,,,,l,,, ratio of L-AA to cell density during the growth period, such enhanced L-AA production state is continued by feeding glucose source, generally in equal time increments, in an amount and at a rate favoring L-AA formation over cell density increase. The o~thllulll amount and feed rate for any particular microorganism can thus be readily 35 determined by trial. Typically, with CPyrenoi~losa UV-101-158 as the microorganism the feed rate is in the range of about 0.005 to O.O5g glucose/hr/g cells taken as dry weight of cells and the glucose content is m~int~ined below growth promoting concentrations, e.g., below about 0.1g/L
of solution.
s The subject method affords intracellular L-ascorbic acid in high yields, far higher than those from other naturally occurring sources, such as rose hips. Levels exceeding 3.5~o of biomass material can be achieved with levels of 4.05~o and higher att~in~ble. The concentration of L-ascorbic acid can exceed 1.45g/L and can be 3.3g/L or greater. Based on the substrate 0 consumed, molar yields are attained that are at least about 0.01.
T~he following examples are offered by way of illustration and not by way of limitation.

Sterilized in a lL 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 20ml of sterile glucose-salts concentrate prepared as follows with Groups of nutrients sterilized individually and combined after cooling:
Group 1 56g glucose, food-grade monohydrate (anhydrous basis) (80g/L) in 80ml water ****
2s Group 2 0.7g trisodium citrate dihydrate (1.Og/L) magnesium sulfate anhydrous (0.66g/L) and lml sulfuric acid (1.4ml/L in 10ml water Group 3 0.65g monobasic sodium phosphate (0.97g/L) 1.3gmonobasicpot~c~ mphosphate (1.9g/L) 0.6g dibasic sodium phosphate (0.97g/L) in 10ml water ****
Group 4 9.4ml trace metal solution The temperature was raised to 35~C, agitation begun at about 200rpm. Air 5 was passed through the medium at the rate of 0.2 liters per minute (lpm) and SOml of Chlorella pyrenoidosa UV101-158 at a concentration of about 0.3g cells/L added. After 5 hours the agitation rate was increased to 400 rpm, the air flow to 0.4 lpm. After 16 hrs the agitation rate was raised to 550 rpm, the air flow to 0.6 lpm. The agitation rate was later increased to 700 then 800 o rpm as noted in Table 3 while the air flow was steady at 0.6 lpm for the rem~inder of the run.
The following chart describes the conditions and analytical results for the fermentation.

Time Cell Densit~y Ascorbic hr pH G/L Acidmg~/L
Comments 0 6.9 --6.6 0.7 400rpm; airO.4 lpm 16 6.9 3.8 550rpm; airO.6 lpm 21 7.0 9.5 700rpm; add 20ml 24 6.9 14.2 800rpm; add 20ml*
36 glucose depleted 7.1 38.6 538 add 4ml+
7.2 38.6 654 48 add 4ml+
51 7.6 38.1 775 add 2ml+
7.7 37.8 966 68 7.8 1050 add 4ml+
92 7.6 37.2 1292 101 7.3 36.1 1459**
* glucose/salts concentrate + 20~o glucose. This addition was repeated periodically during the course of the fermentation.
* * corresponds to 0.04g L-AA/g cell dry weight or 4~o by weight of dry biomass 2091 91 (~

-- . ,3 The method employed for deterrnining L,ascorbic acid is described by Grun and Loewus, Analytical Biochemistry (1983) 130:191-198. The method is an ion-~oYch~e procedure, employing a 7.8 x 300mm organic acid analysis column, HPX-87 (Bio-Rad Labo.atolies, Richmon~l CA). The conditions s are: mobile phase, 0.013 M nitric acid, flow 0.8ml/min, I,res~ure 1500 psig, detection, W 245-254nm. Wit_ the above conditions, resolution of L, ascorbic acid and ico~ccollJic is possible.
To determine the grarns of cells per liter, he following procedure is employed. A biomass sarnple (5ml) is transferred to one weighing pan and 0 Sml of ~u~e~l~a~t transferred to a second weighing pan. The supernatant is cenlliruged. The pans are dried in a convection oven ( 105~C for 3hrs). After cooling in a desiccator, the pan contents are weighed. The grarns of cells per liter are deterrnined as: (sample weight- supernatant weight) x 200.
Based on the above results, specific follllations based on grams of S ascorbic acid per gram of cell are achieved of at least 0.04 and the molar yield defined as moles of L-ascorbic acid formed per mole of gh-cQse consumed is at least 0.01 or higher. In addition, the ascorbic acid concentration can be raised to at least about 1.5g/L.

The procedure of Example 1 was repeated substantially as described using: Chlorella pyrenoidosa strain UTEX 1663, strain UV101-158, the W-generated mutant of strain 1663 described above, and strain UTEX 343;
Chlorella regularis strain UTEX 1808 and strain UVS-280, a UV-generated mutant of strain 1808, Prototheca zopfii strain UTEX 1438 and strain W3-2s 132, a W-generated mutant of strain 1438 and Ankistrodesmus braunii strain ATCC 12744 and strain W2-370, a UV-generated mutant of strain 12744.
It should be noted the three rnicroalgae genera, namely chlorella, Prototheca and Anhstrodesmus, selected to illustrate the invention in these EYamples, are widely divergent taxonornically and are concidered rep.csc~ t;~,e of L,ascorbic acid-producing heteroLIo~hic rnicroalgae.
Each of the above species was grown to a~o~;...~tely 40 g/L cell density (dry weight bacis) in a fed batch, one-liter stirred jar fermentor.
NutAent l-illogen was supplied by, and pH controlled by, addition of ammonia After the cells had eYh~ncted the gll~cose-based nutAents, they 3s entered a peAod of 1 hr to 3 hrs without additional glucose, after which theywere given 0.3 grams of glucose per gram dry weight of cells every 3 hours (design~te~l in the Table below as glucose pulsing after growth) until twice-daily analyzes indicated that ascorbic acid synthesis had peaked and was in decline. The results of these runs are given in the following Table alongside "Yes" under the subheading "Glucose pulsing after growth".
s Controls runs were conducted with the same strain under the same fed-batch conditions until glucose depletion; however, no additional nutrients were added after glucose depletion. This growth condition is indicated by '~o" under "Glucose Pulsing After Growth".

o Glucose ~ximllm M~xi~ n Pulsing Ascorbic Acid Specific After Concentration Formation Example Micro-or~anism Growth (mg/liter) (mg/g cells!

5 Chlorella pyrenoidosa Control UTEX 1663 No 38 1.07 2 UTEX 1663 Yes 54 1.24 Control UV101-158 No 570 12.9 3 W101-158 Yes 753 17.0 Control UTEX 343 No 38 0.7 4 UTEX 343 Yes 38 0.66 Chlorella regularis Control UTEX 1808 No 15 0.34 5 UTEX 1808 Yes 28 0.38 25Control UV5-280 No 26 0.54 6 UV5-280 Yes 32 0.65 Prototheca zopfii Control UTEX 1438 No 24 0.7 7 UTEX 1438 Yes 56 2.1 30Control UV3-132 No 50 2.5 8 UV3-132 Yes 157 6.8 Anhstrodesmus braunii Control ATCC 12744 No 25 0.56 9 ATCC 12744 Yes 30 0.40 35Control UV2-370 No 49 1.4 UV2-370 Yes 65 1.2 The tabulated results show that an isolate(strain) of each of t_e four species and its corresponding derived high ascorbic acid-prodl-~nE strain grown under the glucose pulsing conditions of the invention produced enh~nred yields of ascorbic acid, expressed as ~ t;.. ascorbic acid s con~e ..~ ion in mg/liter, relative to growth under simple fed batch con~litionC, i.e., with no gll-cQse ~d~iition after growth. The results also show that C pyrenoidosa I~EX 1663 and its high L,AA pro~ ng mutant W101-158, C. regularis ~TEX 1808 and its high L-AA producing mutant W5-280, and P. zopfii UTEX 1438 and its high L-AA producing mutant W3-132 all 0 provide enh~n~ ed specific fo, ..~tionC of L-AA (mg/g cells) under the invention con~itionc~ indicating improved utili7~tion of the carbon source for ascorbic acid production relative to that obtained without ~lucos~addition after the initial growth and glucose depletion stages.
It will be noted C. pyrenoidosa strain 343, in contrast to the other C.
l5 pyrenoidosa strains did not provide an increased ascorbic acid yield under - either set of conditions; also that although each of the Ankistrodesmus braunii strains yielded a higher concentration of ascorbic acid under the invention conditions, neither one afforded improvement in the specific formation of the acid. lt should be noted in this regard, however, that the particular set of 20 conditionc employed after the initial growth and glucose depletion stages hadbeen o~ d for C. p~lenoidosa strain UTEX 1663 with no attempt made to ~ ulate the conditions to enh~nce ascorbic acid production for either C. pyrenoidos~ UTEX 343 or ~ braunii ATCC 12744 or W2-370. It is believed likely ill-p,oved results may be obtained with these o,g~n;~ as 2s well.
EXAMPLE 11 (BEST MODE) The procedure of Example 1 was followed except that (a) S.6 mg ferrous sulfate, instead of 11.2 mg, was employed in the initial 0.6 L ~lictillçd water charge; (b) the nutrient solution concicted o~28 g glucose in 40 ml 30 distilled water; 0.53 g trisodium citrate dihydrate plus 0.2 g m~.es;lsulfatein 20 m~;0.65 g each of monopot~scinm acid phosphate and disodium acid phosphate in 20 rnl; and 4.7 ml of the trace metal solution plus 1 ml sulfuric acid in 15 ml; and (c3 the actively growing culture of was strain W 232-1 of Table I.
3s The te,l,pelal~e was raised to 35~C, ~git~tion begun at 3S0 rpm, air passed through the m~ m at 0.2 liters/min (Ipm), the pH adjusted to 6.9 '~

~091ql~

with anhydrous ammonia (NH3) added to the air flow, and strain W 232-1 added to the medium. NH3 was fed throughout the run as nutrient nitrogen source and pHcontroller. After 6.2 hours the air flow was increased to 0.4 lpm, the agitation to 400 rpm. At 11.8 hours the air flow was raised to 0.6 lmp, where it was held forthe rem~3in(1er of the run, and the agitation rate raised to 650 rpm. After 12.4 hours, 40 ml more of the glucose-co~ il-g nutrient solution was added to the fermentor,followed by 20 ml 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 900 rpm at 34.8 hours and m~int~ined at that rate for the rest of the run.
1 0 The glucose content of the medium became depleted at 31.7 hours, at which time the cell density (C.D.) 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 hours and every 3 hours from 41.7 to 94.8 hours. Cell density and L-AA concentration were followed with time as tabulated below along with calculated L-AA content of the 1 5 biomass.

Time hr. pHC.D. g/l L-AA mg/L Comments L-AA
0 6.9 6.2 6.9 11.8 6.9 40 ml glucose at 12.4 hrs, 20 at 24.4 hrs, 15 at 26.3 hrs 22.8 31.7 6.9 19.5 322 glucose depleted 16.5 34.8 7.0 *
46.9 7.7 18.7 429 22.9 53.6 7.8 528 58.6 7.8 17.9 613 34.2 70.8 7.9 694 77.7 7.9 17.3 819 47.3 82.8 7.8 915 94.8 7.6 17.6 945 53.7 ~, 2 a ~ s The above procedure was repeated four more times, substantially as described. The percent L-AA averaged 5.2% of the dry weight of the biomass over the 5 runs.
Having thus described and exemplif1ed the invention with a certain degree 5 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 (12)

1. A method for producing L-ascorbic acid, the method comprising the steps of growing a culture of an organism of the genus Prototheca to obtain a fermentation medium containing L-ascorbic acid and recovering L-ascorbic acid from the fermentation medium.

2. The method of claim 1 in which at least 24 mg/L of L-ascorbic acid is produced in the fermentation medium.

3. The method of claim 1 in which the culture is grown in a growth promoting medium containing a non-repressing/non-limiting amount of a suitable carbon source.

4. The method of claim 3 in which at least 157 mg/L of L-ascorbic acid is produced in the fermentation medium.

5. The method of claim 1 in which the organism is Prototheca zopfii.

6. The method of claim 5 in which at least 24 mg/L of L-ascorbic acid is produced in the fermentation medium.

7. The method of claim 6 in which the culture is grown in a growth promoting medium containing a non-repressing/non-limiting amount of a suitable carbon source.

8. The method of claim 6 in which at least 157 mg/L-ascorbic acid is produced in the fermentation medium.

9. A microalgal biomass comprising cells of an organism of the genus Prototheca, the cells comprising greater than 3.5% L-ascorbic acid by dry weight of the biomass.

10. The biomass of claim 9 in which the organism is Prototheca zopfii.

11. A vitamin C enriched animal feed composition, the composition comprising microalgal biomass, the biomass comprising cells of an organism of the genus Prototheca, the cells comprising greater than about 3.5% L-ascorbic acid by dry weight of the biomass.

12. The animal feed composition of claim 11 in which the organism is Prototheca zopfii.