JP2815127B2 - Method for producing L-ascorbic acid - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION

1. FIELD OF THE INVENTION The present invention relates to heterotrophic processes for improved production of L-ascorbic acid by microorganisms, especially microalgae, in nutrient media containing a suitable carbon source. In particular, the invention relates to a method for producing high concentrations of L-ascorbic acid, preferably high concentrations per unit weight of cell mass.
The present invention also provides novel mutated microalgal species suitable for use in producing L-ascorbic acid of the present invention.

[0002] 2. Description of the Related Art: L-ascorbic acid is an important nutritional supplement and is widely used as a dietary supplement in both humans and other vitamin C-requiring animals and in vitamin capsules. L-
Ascorbic acid is a very volatile chemical and is a mass-produced chemical that requires economical and efficient production with high marketability. Thus, there is substantial interest in being able to develop methods using microorganisms that provide for efficient conversion of nutrients that efficiently produce L-ascorbic acid.

[0003] Loewus, FA, in L-Ascorbic Acid: Meta
bolism, Biosynthesis, Function, The Biochemistry of
Plants, Nol. 3, Academic Press, Inc., pp.77-99,
1980 describes a paper on the source and biosynthesis of L-ascorbic acid. A description of ascorbic acid production in algae is given by Vaidya et, al., Seience and Cultur
e (1971) 37: 383-384; Subbulakshmi et, al., Nutri
tion Reports International (1976) 14: 581-591; Aa
ronson et, al., Arch. Microbiol. (1977) 112: 57-5.
9; Shigeoka et. Al., J. Nutr. Sci, Vitaminol. (197
9) 29: 29-307; Shigeoka et. Al., Agric. Biol. Che.
m. (1979) 43; 2053-2058; Bayanovaand Trubachev, P.
rikladnaya Biokhimiya i Mikrobiologyia (1981) 17;
400-407 (UDC 582.26; 577.16); and Ciferri, Microbio
logical Reviews (1983) 47; 551-578.

[0004] Heterotrophic processes according to the prior art are not entirely satisfactory for commercial use. The utilization of carbon sources for ascorbic acid production is generally inadequate and the vitamins are produced at undesirably low concentrations.

[0005]

SUMMARY OF THE INVENTION It is an object of the present invention to provide a method for heterotrophic biosynthesis of L-ascorbic acid from a carbon source which enhances the utilization of the carbon source. Another object is to provide a method for obtaining the vitamin in high yield. Still another object is to provide a novel high L-ascorbic acid producing microorganism as a novel composition. More specifically, an object of the present invention is to grow a culture strain of a microorganism belonging to the genus Prototheca (especially prototecazophy) to obtain a fermentation medium containing L-ascorbic acid, and to recover L-ascorbic acid from the fermentation medium. A microalgal biomass comprising cells of a microorganism of the genus Prototheca, the cells comprising L-ascorbic acid; and a vitamin C enrichment comprising the microalgal biomass. An object of the present invention is to provide an animal feed composition.

Accordingly, it is an object of the present invention to provide an improved method for producing L-ascorbic acid. The method in a nutrient medium containing a carbon source and dissolved oxygen (O ₂₎ in an amount suitable for cell proliferation and L- ascorbic acid-producing L-
The cells of the ascorbic acid-producing microorganism are heterotrophically grown, and the organism is allowed to grow in its early stages to a high cell density, with concomitant substantially complete production of intracellular L-ascorbic acid production and carbon sources. Substantial depletion, cell growth is substantially stopped, and the subsequent addition of a controlled amount of a carbon source produces additional amounts of L-ascorbic acid with substantially no or no increase in cell density. The cells were maintained in their substantially depleted carbon source until they yielded the desired increase in L-ascorbic acid production with substantially no or no increase in cell density. It consists of continuing to add the carbon source.

[0007] That is, the improved utilization of the carbon source is L-
Observed for ascorbic acid (L-AA) production.
Increased by total L-AA, expressed as mg L-AA per liter of solution, preferably also by increased ratio production of L-AA expressed as mg L-AA per gram dry weight of cells As a result, an increased yield is obtained.

[0008] The present invention further relates to novel novel high L-AA producing mutated microalgae, more specifically C. pyrenoido.
sa (Chlorella pyrenoidsa) isolate UTEX 1663
Chlorella pyrenoidosa UV 101-158 deposited on June 27, 1985 with the American Type Culture Collection (ATCC) under accession number 53170;
Chlorella regularis UV 5-280 derived from regularis (Chlorella reglaris) UTEX 1808;
Prototheca zopfii UV 3-132 derived from P. zopfii (Prototecazopfi) UTEX 1438; Ank
Ankistrodesmu derived from istrodesmus braunii ATCC 12744
s braunii UV 2-370. UTEX is a culture collection of algae at the University of Texas at Austin.

The production of these novel microalgal compositions and their effectiveness for L-AA production in the method of the present invention are described in detail below. DESCRIPTION OF THE PREFERRED EMBODIMENTS Generally, the method of the present invention comprises three major steps: (1) L-AA
The heterotrophically producing microorganism preferably microalgae that can be, respectively algae contains an effective carbon source and dissolved O ₂ at a first concentration in an amount sufficient to grow to a high cell density fermentation An early cell growth phase in which the cells grow heterotrophically in the tank, while at the same time achieving the production of intracellular L-AA and substantially complete depletion of the carbon source; (2) cell growth is substantially stopped A substantially completely depleted carbon source stage in which the cells of the microorganism are left intact in the depleted carbon source up to this point; and (3) the carbon source is provided and then substantially increases the cell density. stage not to be effective to produce an additional amount of L-AA in the presence of dissolved O ₂ and is the adjusted carbon source added maintaining the fermentor at a second concentration lower than the first concentration provided Consists of

[0010] The addition of a carbon source at relatively low concentrations, where L-AA production is preferred over cell growth, requires L-AA
Can be continued until the ability of the microorganisms that produce the is substantially exhausted. This point can be determined by monitoring L-AA concentration and cell density over time during the process. Other steps of the method of the present invention include harvesting cells and separating and recovering L-AA substantially free of cellular material by techniques known in the art.

[0011] L- recovered from cell mass (biomass)
The AA product can be used as is. Alternatively,
The L-AA-containing biomass itself can be used as an animal feed composition fortified with vitamin C or as a feed supplement, for example for use in aquaculture of fish.

[0012] The microorganisms used in the present invention are L-
A wide variety of AA-producing strains, especially microorganisms capable of heterotrophically producing intracellular L-AA, can be used. Preferred microorganisms are L-AA-producing green microalgae, especially those that are so-called high-producing strains of L-AA and are sometimes called overproducing strains for economic reasons. Organisms with potential as high producers of L-AA can be identified using standard fermentation procedures for cell growth with L-AA production. They may also be physical or chemical mutagenesis means such as UV radiation, X-
It is preferred to mutate using linear, N-methyl-N'-nitro-N-nitrosoguanidine, dimethyl sulfate or similar agents known in the art. Mutated L-AA obtained by such treatment
Overproducing strains can be advantageously determined with redox dyes. In addition, by using analogs of metabolic intermediates to ascorbic acid or inhibitors of ascorbic acid synthesis, microorganisms that can maintain or increase L-AA production in the presence of chemical interference can also be selected.

Preferred progeny of the above procedure are microorganisms which improve the specific production of L-AA as measured by mg L-AA per gram of cells (dry weight basis). These progeny can then be further separated into individual clones and further subjected to the above operations.

One preferred microorganism is the green microalga Chlorella spp., Especially Chlorella pyrenoidosa U.
V 101-158 strain, high LA from UTEX 1663 strain due to mutagenic UV light
A-producing mutant. C.pyrenoidosa UV 10
1-158 has been deposited with the ATCC and has an accession number of 5317.
0 is assigned. Another preferred C. pyrenoidosa strain is UV232-1, which produced the highest intracellular L-AA so far. Other representative and suitable species of the genus Chlorella are Chlorella regularis UTEX 1808
And C. regularis, a UV-generated high L-AA-producing mutant of the UTEX 1808 strain
UV 5-280. Furthermore, other suitable L-AA-producing microalgae capable of heterotrophic growth are Protot
It belongs to the genus heca and the genus Ankistrodesmus. Representative Prototheca species are the UV-produced high L of P. zopfii UTEX 1438 and UTEX 1438.
-AA-producing mutant. Representative Ankistrode
smus species are A. braunii ATC 12744 and high LA
A. braunii UV 2-370, a mutant of ATC 12744, produced by UV with A productivity.

The progeny mutated in each of the above cases is a higher L-AA producing strain than its protozoa. As further described below, each organism has a higher maximum concentration in mg of L-AA per liter of nutrient medium under the conditions of the invention as defined above than under conventional prior art conditions. Produce L-AA.

In practicing the method, the active growth of the microorganism in the nutrient medium, generally with a first, generally low concentration of L-AA, but in an amount sufficient to result in a high cell density after a modest growth period. Plant a sex culture. A typical initial cell density is generally about 0.1 based on the dry weight of the cells.
5 to 0.4 g / L. The medium contains a carbon source, various salts and generally also trace metals. It also contains a source of molecular O ₂ , typically air, provided in a growth promoting amount with a growth promoting amount of the carbon source. That is, the growth achieved up to a high cell density, both available carbon source and O ₂ must be utilized microorganisms.

The carbon source is usually L-galactose or D-galactose.
-Derived from glucose, but glucose is preferred for economic reasons. The glucose source is any carbohydrate that can be converted to glucose in the reaction system, such as molasses,
It is preferably corn syrup or the like. The total amount of glucose used can vary widely depending on the particular organism and the desired result. Typically, in the case of high L-AA producing organisms such as C. pyrenoidosa UV 232-1 the total amount of glucose source used, if not metabolized, is calculated to be about 65-90 g / l, more usually about 75-85 g / l, preferably about 80 g
Per liter. Usually about 15-30%, more usually about 20-25% of the total glucose is added first. Glucose is usually added initially and during the fermentation with other additives as described below. During the initial glucose addition and fermentation period, there is a period of non-restricted cell growth, in which cells grow to relatively high cell densities, and generally at the same time L-AA is usually present at relatively low concentrations. Generated.

[0018] The amount of glucose source in the fermentor should be an uncontrolled / non-limiting amount. That is, the amount should optimally promote cell growth and should not inhibit or unduly limit proliferation. The optimal non-growth limiting concentration of a glucose source may vary from organism to organism, but is readily determined by tests on any individual organism. For example, C. pyrenoidosa UV 101-15
In the case of No. 8, the glucose source concentration is maintained by appropriately adding it in the range of 15 to 30 g / liter. The range has been found to be a concentration sufficient to promote cell growth while avoiding glucose inhibition of cell growth.

Desirably, the other additives are initially present with the glucose source, and their concentration in the nutrient medium is also generally provided continuously by successive additions of the additive with the continuous addition of the glucose source. Is done. The ratio of the concentration of these additives to the concentration of the glucose source may be the same or different during the fermentation.

Additives which can have a different proportion to the amount of glucose added gradually to the total amount added to the fermenter include alkali metal phosphates such as sodium phosphate and potassium phosphate, especially There are those as sodium phosphate dibasic and potassium phosphate monobasic. The total amount of dibasic sodium phosphate is typically about 1-2 total g / l, usually about 1-1.5 total g / l, and preferably about 1.3 total g / l. The amount of sodium phosphate dibasic initially present in the fermentor is usually about 35-50%, more usually about 40-45% of the total amount of sodium phosphate dibasic added. The total amount of monobasic potassium phosphate is usually about 1.5 to 1.5.
3 g / l, more usually about 2 to 2.5 g / l. The amount initially present is generally about 40% of the total
５０50%, more usually about 45-50%.

In addition to the above additives, a biologically acceptable chelating agent such as sodium citrate may be added at about 0.8 to 1.2.
It is advantageous to add in a total amount of g / l, usually about 1 g / l. Monobasic sodium phosphate is generally present at about 0.8 to 1 g / l, preferably about 0.95 to 1 g / l.
It is present in g / l. The addition of biologically acceptable mineral acids keeps trace metals in solution and also neutralizes ammonia, which is usually used as a nitrogen source. Concentrated sulfuric acid is conveniently used for this purpose in an amount of about 1-2 ml / l, more usually about 1.2-1.5 ml / l. Magnesium is about 0.1-0.2g among metals
Per liter, preferably 0.1 to 0.15 g / liter, but is particularly preferred as a physiologically acceptable salt, such as a sulfate. The amounts of iron and copper used are limited because these metals suppress the formation of ascorbic acid. Iron (first) is initially present at about 5-7 mg / l, preferably about 5.5-6 mg / l, and is not included in any subsequent additions. Copper is present in relatively small amounts and generally ranges from about 1 to 1 per gram of glucose.
50 μg.

The following trace metal solutions are used in a total volume of about 10-15 ml / l, more usually about 12-14 ml / l. On a glucose basis, this trace metal solution corresponds to 0.1-0.2 ml / g.

Advantageously, the solution is prepared for addition during the fermentation process and has the following composition:

[Table 1]

The nitrogen supplied is from anhydrous ammonia.
It is also used as a pH adjuster. The real N level in the medium is measured by the acid value of the medium and the buffer capacity of the medium.

The trace metal mixture and the solution have the following composition:

[Table 2]

Stock solutions of trace metals were prepared by dissolving appropriate amounts of various compounds in distilled water containing traces of HCl, the final volume being 1 liter and concentrated HCl.
Contains 20 ml. Use distilled water to ensure the proper ratio of each trace metal component.

Although a number of salts are referred to as monobasic or dibasic, it is to be understood that this is for convenience and is not required. These compounds act as buffers and therefore the degree of protonation changes at the pH of the medium.

In carrying out the fermentation, dibasic sodium phosphate and monobasic sodium phosphate are added to about 75% of the total medium.
Dissolve in ~ 90%, usually about 80-90%, and add to the fermentor.

The solution to be added gradually during the fermentation process is prepared by combining the individual components in the proper ratio. Glucose dissolves in about 75-85%, preferably about 80%, of the water to be used. The citrate, magnesium and sulfuric acid components are combined in an aqueous medium containing about 5 to 15%, usually about 10% water, while the phosphate is about 5 to 15% of the water to be used. , More usually united in about 10%,
The trace metal solution is then about 5-15% of the total water to be used
More usually coalesce in about 8-10%. To the fermenter containing a portion of the phosphate, ferrous salt and about 20% of the glucose-salt concentrate prepared above are added. This addition is made aseptically to avoid the introduction of any foreign microorganisms.

Next, the nutrient medium is brought to the desired temperature. This temperature can be varied by microorganisms, but generally is about 3
It is 0 to 40 ° C, preferably about 35 ° C. Inoculation of the fermenter generally gives an initial cell density of about 0.1-0.4 g / l. Small amounts of anti-foaming agents can be added during the fermentation process.

The cells are grown to a high cell density in the first stage fermentation to build the basis for essentially high total production L-AA, and until the carbon source is substantially completely depleted, ie, Cell growth is continued until the glucose equivalent concentration is typically less than 0.1 g per liter of solution, resulting in a cell-free supernatant. The time required may vary from organism to organism, but usually is around 35
~ 50 hours more usually about 40-45 hours, growth rate about 0.1 ~ 0.15 hours ^-1

The pH of the medium can be adjusted within the desired range by adding anhydrous ammonia, if necessary, but is generally about 6.5 to 8.0.

The medium is advantageously agitated and aerated during this growth period. The stirring speed is usually about 200-1000rp
m, and the ventilation rate is generally about 0.2 to 2 air per minute.
0.6 liters. However, this can vary depending on the organism and other fermentation conditions. Aeration provides molecular oxygen (O ₂ ) to the medium, which is essential for cell growth and L-AA production. It is also possible to use other O ₂ source, it is possible to use an O ₂ gas diluted with O ₂ gas and N ₂ non-inert gas, for example undiluted. There O ₂ source whatever, the dissolved O ₂ content in the medium is let to ensure a high intracellular content acquisition of adjusted during the fermentation process high cell proliferation and L-AA. O ₂ content of the nutrient medium may conveniently be monitored by standard techniques using an O ₂ probe electrode.

The glucose available to the microorganism during this initial growth phase can be monitored in the supernatant, ie in the cell-free component of the medium, by any convenient technique, such as a glucose oxidase enzyme test, HPLC or other known techniques. If the glucose concentration falls, it can be replenished, if necessary, by adding an aliquot of the glucose-salt concentrate, for example a 20% aliquot, the total glucose concentration generally being about 30 g / l as described above. Below, but ensure that it is present below the growth inhibition level. Glucose availability is maintained until the desired high cell density is obtained. For example, C. Pyrenoidosa UV 101-158 has a cell density calculated on a dry cell weight basis of about 35 to
45 g / l, preferably about 40 g / l. At this point, the glucose content of the medium is substantially completely depleted if it has not already reached this state.

When the desired high cell density has been obtained and the glucose content of the medium has become substantially completely depleted, this depleted state of glucose is maintained. That is, the organism becomes starved during that time, cell growth ceases substantially, and cell density reaches a peak. With cessation of cell growth, production of L-AA can also be substantially halted. However, in general, cells can utilize storage starch to maintain cell function, including some small amounts of L-AA production.

Substantially depleted glucose and non-proliferative conditions are maintained until the organism is again supplied with a glucose source, but the organism is adjusted in a controlled amount with little or no increase in cell density. Begin production of additional L-AA. The starvation period ranges from a few minutes or less to several hours or more, but is typically about 1 to 4 hours, more usually 2 to 4 hours.
Time. Optimal starvation time as well as optimal amount or rate of glucose source supply to the fermenter is to add a small test amount of glucose source to each individual organism and monitor the effect on L-AA content and cell density over time. Can be measured by The ratio of the increase in L-AA to the increase in cell density, if present, indicates the increase in L-AA during growth.
When greater than the maximum ratio of AA to cell density, such an enhanced L-AA production state places glucose sources generally at equal time intervals, with L-AA production over cell density increase. It is maintained by feeding in the amounts or proportions given. In this way, the optimal amount and feed rate for any individual microorganism can be readily determined by testing. Typically, C. Pyre as a microorganism
In the case of noidosa UV 101-158, the supply rate is about 0.005 to 0.05 g of glucose per hour per gram of cells treated as dry weight of cells, and the glucose content is less than the growth promoting concentration, for example, about 1 liter of solution. 0.
It is kept below 1 g.

In the method of the present invention, intracellular L-ascorbic acid can be obtained at a much higher yield than that obtained from other natural sources such as rosehips. Levels above 3.5% of biomass material can be achieved, and levels above 4.0% are achievable. The concentration of L-ascorbic acid can exceed 1.45 g / l and 3.3 g
Per liter or more. A molar yield of at least about 0.01, based on the substrate consumed, is obtained.

The present invention will be described below with reference to examples.
The present invention is not limited to them.

Example 1 In a 1 liter fermenter, 0.6 liter of distilled water, 0.23 g of dibasic sodium phosphate and 0.27 g of monobasic potassium phosphate were put and sterilized. Next, ferrous sulfate (heptahydrate) 1 in 5 ml of distilled water was added to this phosphoric acid solution.
1.2 mg and each group of nutrients individually sterilized and combined after cooling as described below Group 1 56 g Glucose, food grade, monohydrate (anhydrous basis) (80 g / l) Dissolve in 80 ml water Group 2 0.7 g trisodium citrate dihydrate (1.0 g /
1 g) Magnesium sulfate, anhydrous (0.66 g / l) and 1 ml sulfuric acid (1.4 ml / l in 10 ml of water) Group 3 0.65 g monobasic sodium phosphate (0.97 g / l) 1.3 g monobasic Potassium phosphate (1.9 g / l) 0.6 g sodium phosphate dibasic (0.97 g / l) dissolved in 10 ml of water Group 4 9.4 ml sterile glucose-salts prepared using trace metal solution 20 ml of concentrated liquid
Was added aseptically.

The temperature was raised to 35 ° C. and the stirring was
Started at rpm. Air was passed through the medium at a rate of 0.2 liters per minute (lpm) and the Chlorella pyrenoidosa UV 101-158 50 m at a concentration of about 0.3 g cells / liter.
l was added. After 5 hours, increase the stirring speed to 400 rpm,
The air flow was 0.4 lpm. After 16 hours, the stirring speed was increased to 5
It was increased to 00 rpm and the air flow was 0.6 lpm. The agitation speed was then increased to 700 rpm and then to 800 rpm as described in Table 3, while the airflow was constant at 0.6 lpm for the remainder of the experiment.

The following table describes the conditions for fermentation and the results of the analysis.

[0042]

[Table 3]

The method used for measuring L-ascorbic acid is
Grun and Loewus, Anelytical Biochemistry (1983) 13
0: 191-198. The method is 7.8 x 300
mm organic acid analysis column, HPX-87 (Bio-Rad Laborato
ries, Richmond, CA). The conditions are as follows. Mobile phase, 0.013M nitric acid,
Flow rate 0.8 ml / min, pressure 1500 psig, detection, UV 24
5-254 nm. Under these conditions, resolution of L-ascorbic acid and isoascorbic acid is possible.

The following procedure is used to determine the number of grams of cells per liter.

The biomass sample (5 ml) is transferred to one measuring pan and 5 ml of the supernatant is transferred to a second measuring pan. The supernatant is centrifuged. Dry each pan in a convection oven (105 ° C. for 3 hours). After cooling in a desiccator, the content of each bread is weighed. The number of grams of cells per liter is determined as (sample weight-supernatant weight) x 200.

Based on the above results, ratio generation based on grams of ascorbic acid per gram of cells is achieved with at least 0.04. The molar yield, defined as the number of moles of L-ascorbic acid produced per mole of glucose consumed, is at least 0.01 or higher. Further, ascorbic acid concentration can be increased to at least about 1.5 g / liter.

Examples 2 to 10 Chlorella pyrenoidosa UTEX 1663 strain, UV
101-158 strain (= UV generated mutant of 1663 strain described above) and UTEX 343 strain; Chl
orella regularis UTEX 1808 strain and UV
5-280 strain (= UV-generated mutant of 1808 strain above), Prototheca zopfii UTEX 143
8 strain and UV 3-132 strain (= UV produced mutant of 1438 strain above) and Ankistrodesm
us braunii ATCC 12744 strain and UV2-
The procedure of Example 1 was repeated using the 370 strain (= UV-generated mutant of the 12744 strain described above) substantially as described.

In these examples the three microalgal genera selected to illustrate the invention, namely Chlorella, Proto
theca and Ankistrodesmus are widely taxonomically divergent and appear to be the genus representing L-ascorbic acid producing heterotrophic microalgae.

Each of the above seeds was grown in a 1 liter stirred jar fermenter of the feed batch to give a cell density (on a dry weight basis) of about 40 g / l. The nutrient nitrogen was supplemented by the addition of ammonia, and the pH was also adjusted by the addition of ammonia. After the cells had run out of glucose-based nutrients, they entered a state without additional glucose for 1-3 hours. The cells were then given 0.3 g of glucose per gram of dry weight of the cells every three hours until the analysis of ascorbic acid peaked and diminished twice daily by analysis (see below). The table shows glucose pulsing after growth (pulsin
g)). In the table below, the results of these experiments are indicated by “Yes” in the subheading “Post-growth glucose pulsing”.

Control experiments were performed using the same strain under the same feed batch conditions until glucose depletion. However, no additional nutrients were added after glucose depletion. These growth conditions are indicated by "No" in the column "Glucose pulsing after growth".

[0051]

[Table 4]

The results in the above table show that each of the four isolates (strains) grown under the glucose pulsing conditions of the present invention.
And its correspondingly induced high ascorbic acid-producing strain has a mg / mg relative to growth in simple fed-batch conditions, ie without any added glucose after growth.
It shows that an increased yield of ascorbic acid was produced, expressed as the highest concentration of ascorbic acid in liters. These results also indicate that C. pyrenoidosa UTEX
1663 and its high L-AA productivity UV101-
158 mutant, C. regularis UTEX 1808 and its UV 5-280 mutant with high L-AA production and P. zopfii UTEX 1438 and its UV 3-132 mutant with high L-AA production Are all under the conditions of the invention with an increased specific production of L-AA (mg / g
Cells), which indicate improved utilization of carbon sources for ascorbic acid production compared to those obtained without glucose addition after the initial growth and glucose depletion steps. doing.

C. pyrenoidosa 343 strain was obtained from other C.
did not increase ascorbic acid yield under any combination of conditions compared to pyrenoidosa strains;
It should be noted that although each of the istrodesmus braunii strains produced higher concentrations of ascorbic acid under the conditions of the present invention, none of them improved the specific production of the acid. However, in this regard, the particular combination of conditions used after the initial growth and glucose depletion steps were optimized for C. pyrenoidosa UTEX 1663, whereas C. pyrenoidosa UTEX 34
3 or A. braunii ATCC 12744 or UV
It will be noted that no attempt was made to manipulate the conditions to increase ascorbic acid production for any of 2-370. It is believed that similarly improved results may be obtained for these organisms as well.

Example 11 (Best Method) (a) 5.6 mg of ferrous sulfate was used during the initial 0.6 liter charge of distilled water instead of 11.2 mg; 28 g glucose in 40 ml; 0.53 g trisodium citrate dihydrate and 0.2 g magnesium sulfate in 20 ml; 0.65 each of monopotassium phosphate and disodium phosphate in 20 ml
g; and 4.7 ml of a trace metal solution in 15 ml and 1 ml of sulfuric acid; and (c) according to the procedure of Example 1 except that the active growth culture was the UV 232-1 strain in Table 1. Was.

The temperature was raised to 35 ° C. and the stirring was
starting with m, air passed through the medium with 0.2 liters / minute (lpm), pH in anhydrous ammonia was added to the air stream (NH ₃₎ 6.
9, and the UV 232-1 strain was added to the medium. NH ₃ was supplied during the experiment as a nitrogen source of nutrients and a pH adjuster. After 6.2 hours, the air flow was increased to 0.6 lpm and the agitation was at 400 rpm. After 11.8 hours, the air flow was increased to 0.6 lpm and maintained for the remainder of the experiment, with the agitation speed increased to 650 rpm. After 12.4 hours, add more than 40 ml of glucose-containing nutrient solution to the fermenter,
Then, after 24.4 hours, more than 20 ml and after 26.3 hours more than 15 ml were added. During that time the stirring speed was 22.
After 8 hours, the speed was increased to 750 rpm, then adjusted to 900 rpm after 34.8 hours, and the speed was maintained for the remainder of the experiment.

The glucose content of the medium became exhausted after 31.7 hours, at which point the cell density (CD) was 19.5 and the L-AA concentration was 322 mg / l. 41.7 of glucose (2 ml of a 10% solution)
After the hour and between 41.7 and 94.8 hours, the fermenter was fed every 3 hours. Cell density and L-AA concentration
The biomass was followed for the times shown in the table below with the calculated L-AA content.

[0057]

[Table 5]

The above procedure was repeated four more times, essentially as described. The percentage of L-AA averaged 5.2% of the dry weight of biomass in five experiments.

While the present invention has been described in detail, the present invention can be further summarized by the following embodiments. 1. The following steps: (a) L-ascorbic acid can be heterotrophically produced in a growth-promoting nutrient medium containing a suitable carbon source and dissolved oxygen in respective amounts sufficient for cell growth and L-ascorbic acid production. (B) growing the organism to a high cell density at an early stage,
Concomitantly producing the first amount of ascorbic acid and then substantially completely depleting the carbon source; (c) (i) substantially halting cell growth and (ii)
The cells are treated in the presence of dissolved oxygen and in the presence of the carbon source until the subsequent addition of a controlled amount of the carbon source results in the production of an additional amount of L-ascorbic acid without substantially increasing the cell density. (D) continuing to adjust the carbon source addition until the desired increase in L-ascorbic acid production is achieved without substantially increasing cell density. -A method for producing ascorbic acid.

2. In step (d), the produced L-
2. The method of claim 1 wherein the ratio of total ascorbic acid to total cell density is greater than that ratio in step (c) when cell growth has substantially ceased and cell density has stopped increasing. 3. 2. The method according to the above 1, wherein the ascorbic acid is recovered from cells substantially free of cellular substances.

4. 2. The method according to the above item 1, wherein the carbon source is present in an uncontrolled amount. 5. 5. The method according to the above item 4, wherein the carbon source is a glucose source. 6. 6. The method according to the above item 5, wherein the glucose source is a saccharide or polysaccharide which is converted into glucose in a reaction system in a nutrient medium during a fermentation process. 7. 6. The method according to the above item 5, wherein the glucose source contains glucose.

8. The preceding paragraph, wherein the microorganism is a green microalgae
The described method. 9. 9. The method according to the above item 8, wherein the microalgae is a species of the genus Chlorella. Ten. 10. The method according to the above item 9, wherein the species is pyrenoidosa. 11． The organism is a mutant strain of C. pyrenoidosa UTEX 1663, designated CV.
11. The method according to the above item 10, which is the strain of a.

12. A strain of Chlorella pyrenoidosa having ATCC accession number 53170. 13. C. py from Chlorella pyrenoidosa by UV irradiation
renoidosa UV 232-1 strain.

────────────────────────────────────────────────── ─── Continued on the front page (72) Inventor Jeffrey A. Running 54220 Wisconsin, USA. North Fifth Street 834 (72) Inventor Thomas J. Skatrad, Wisconsin, USA 53051. Menomonie Foods. Countryside Drive N.76 D.W.15873 (58) Fields surveyed (Int. Cl. ⁶ , DB name) C12P 17/04 CA (STN) REGISTRY (STN)

Claims (2)

(57) [Claims] 1. A method for producing L-ascorbic acid comprising the steps of: growing a culture of a microorganism belonging to the genus Prototheca to obtain an L-ascorbic acid-containing fermentation medium; and recovering L-ascorbic acid from the fermentation medium. 2. The method according to claim 1, wherein the microorganism is prototecazopfi.