JPH0646870A - Microbial production of l-ascorbic acid - Google Patents

Abstract

PURPOSE: To improve the ratio of a supplied carbon source to ascorbic acid in a fermentation tank and to increase the concentration of ascorbic acid in the fermentation tank by culturing a microorganism capable of producing ascorbic acid by a specific method.

CONSTITUTION: Firstly the cell of a microorganisms (preferably Chlorella- pyrenoidosa strain ATCC 53,170, etc., green minute alga) is cultured in a proliferation promoting nutrient medium containing a carbon source and dissolved oxygen dependently on nutrition up to a high-cell density to form ascorbic acid and to completely consume the carbon source. Secondly, the cell is maintained in the presence of dissolved oxygen and in the absence of the carbon source until the cell density reaches the maximum value after the suspension of the cell proliferation. Successively the carbon source is continuously added to the culture system to produce an additional amount of L-ascorbic acid without causing increase in cell density. Glucose is preferable as the carbon source.

COPYRIGHT: (C)1994,JPO

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION

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

2. Description of 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 mass-produced chemical product that is extremely price volatile and requires economical and efficient production that is highly marketable. Thus, there is substantial importance in being able to develop methods using microorganisms that result in efficient conversion of nutrients that produce L-ascorbic acid efficiently.

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 sources and biosynthesis of L-ascorbic acid. For a description of ascorbic acid production in algae, see 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; Bayanova and Trubachev, P.
rikladnaya Biokhimiya i Mikrobiologyia (1981) 17;
400-407 (UDC 582.26; 577.16); and Ciferri, Microbio
Logical Reviews (1983) 47; 551-578.

Prior art heterotrophic methods are not entirely satisfactory for commercial use. Utilization of carbon sources for ascorbic acid production is generally inadequate and the vitamins are produced in 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 utilization of the carbon source. Another object is to provide a method of obtaining the vitamin in high yield. Still another object is to provide a novel high L-ascorbic acid-producing microorganism as a novel composition.

Accordingly, the present invention is to provide an improved method for producing L-ascorbic acid. The method comprises L- in a nutrient medium containing a carbon source and dissolved oxygen (O ₂ ) in amounts suitable for cell growth and L-ascorbic acid production.
The cells of an ascorbic acid-producing microorganism are heterotrophically grown, and the organism is allowed to grow to a high cell density in the initial stage, and concomitantly, intracellular L-ascorbic acid production and substantially complete production of carbon source are completed. Exhaustion, cell growth is substantially arrested and subsequent addition of a regulated amount of carbon source produces an additional amount of L-ascorbic acid with substantially little or no increase in cell density. Cells were maintained in their substantially depleted carbon source until resulting in a desired increase in L-ascorbic acid production with virtually no or no increase in cell density. Continuing to add carbon source.

That is, the improved utilization of carbon source is L-
Observed for ascorbic acid (L-AA) production.
Proved by an increase in total L-AA expressed as mg of L-AA per liter of solution, preferably also an increase in the specific production of L-AA expressed as mg of L-AA per gram dry weight of cells. Increased yields are obtained, as described.

The present invention is further directed to novel high L-AA producing mutated microalgae, more specifically C. pyrenoido.
sa (Chlorella pyrenoidosa) isolate UTEX 1663
Chlorella pyrenoidosa UV 101-158; C. derivatized from U.S.A. and deposited on June 27, 1985 with the American Type Culture Collection (ATCC) under Deposit No. 53170.
Chlorella regularis UV 5-280 derived from regularis UTEX 1808;
Prototheca zopfii UV 3-132 derived from P. zopfii UTEX 1438; Ank
istrodesmus braunii Ankistrodesmu derived from 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 process of the invention are detailed below. Description of the Preferred Embodiments In general, the method of the present invention comprises three main steps: (1) L-AA.
A microorganism capable of heterotrophically producing, preferably a microalgae, containing an available carbon source and dissolved O ₂ at a first concentration in an amount sufficient to grow the algae to a high cell density, respectively. An early cell growth stage in which heterotrophic growth in the tank is achieved, while at the same time intracellular L-AA production and carbon source are substantially completely exhausted; (2) cell growth is substantially stopped. A substantially completely depleted carbon source stage in which the microbial cells are thus left intact in the depleted carbon source; and (3) the carbon source is supplied, and then a substantial increase in cell density is achieved. A step of conditioned carbon source addition that is effective in producing additional amounts of L-AA in the presence of dissolved O ₂ without effecting and is maintained in the fermentor at a second concentration lower than the first concentration Consists of.

Addition of a carbon source at relatively low concentrations, where L-AA production is prioritized over cell growth, results in L-AA
Can be continued until the ability of the microorganism to produce 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 invention include harvesting cells and separating and recovering L-AA substantially free of cellular material by techniques known in the art.

The L-AA product (biomass) recovered from the cells can be used as it is. Alternatively, L
The AA-containing biomass itself can be used as a vitamin C enriched animal feed composition or feed supplement, eg for use in aquaculture of fish.

The microorganisms used in the present invention are L-
AA-producing strains, particularly microorganisms capable of producing intracellular L-AA heterotrophically, can be widely modified. Preferred microorganisms are L-AA-producing green microalgae, and especially for economic reasons, so-called L-AA high-producing strains and sometimes also called overproducing strains. Potential organisms as high producers of L-AA can be identified using standard fermentation procedures for cell growth involving L-AA production. They also include physical or chemical mutagenesis tools such as UV radiation, X-
It is preferably mutated with Silicus, N-methyl-N'-nitro-N-nitrosoguanidine, dimethylsulfate 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, it is also possible to select microorganisms capable of maintaining or increasing L-AA production in the presence of chemical interference, by using analogues of metabolic intermediates to ascorbic acid or inhibitors of ascorbic acid synthesis.

A preferred progeny of the above procedure is a microorganism which improves the specific production of L-AA (dry weight basis), measured by mg L-AA per gram of cells. These progeny can then be further separated into individual clones and further subjected to the above manipulations.

One preferred microorganism is the genus Chlorella of the green microalgae Chlorella pyrenoidosa U
V 101-158 strain, high LA from UTEX 1663 strain by mutagenizing UV light
A productive mutant. C.pyrenoidosa UV 10
1-158 has been deposited with ATCC and has been deposited No. 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
Strain; and C. regularis, a UV-generated, high L-AA-producing mutant of the UTEX 1808 strain.
UV 5-280. Furthermore, another suitable L-AA-producing microalgae capable of growing heterotrophically is Protot.
It belongs to the genus heca and the genus Ankistrodesmus. Representative Prototheca species are UV-generated high L of P. zopfii UTEX 1438 strain and UTEX 1438.
-AA productive mutant. Representative Ankistrode
smus species are A. braunii ATC 12744 and high LA
A productive, UV produced, A. braunii UV 2-370, a mutant of ATC 12744.

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

In the practice of the method, the nutrient medium is generally accompanied by a first, generally low concentration of L-AA, but the active growth of the microorganism in an amount sufficient to result in a high cell density after a moderate growth period. Plant a sex culture. Typical initial cell densities are generally about 0.1 based on the dry weight of the cells.
It is 5 to 0.4 g / L. The medium contains a carbon source, various salts and generally also trace amounts of metals. It also contains a growth-promoting amount of carbon source as well as a molecular O ₂ source, generally air, provided in a growth-promoting amount. That is, both an effective carbon source and O ₂ must be available to the microorganism to achieve growth to high cell densities.

The carbon source is usually L-galactose or D
-Derived from glucose, 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 within wide limits depending on the individual organism and the desired result. Usually, in the case of high L-AA producing organisms, such as C. pyrenoidosa UV 232-1, the total amount of glucose source used is about 65-90 g / liter, more usually about 60, calculated as glucose, if not metabolized. 75-85 g / liter, preferably about 80 g
/ L concentration is provided. Usually about 15-30% of the total glucose is added first, more usually about 20-25%. Glucose is usually added initially and during fermentation with the other additives listed below. During the initial glucose addition and fermentation period, there is a period of unrestricted cell growth in which cells grow to a relatively high cell density, while at the same time L-AA is usually at a relatively low concentration. Is generated.

The amount of glucose source in the fermentor should be a non-inhibitory / non-limiting amount. That is, the amount should optimally promote cell growth and not prevent or unduly limit growth. The optimal non-growth limiting concentration of glucose source may vary from organism to organism, but is readily determined by testing on any individual organism. For example, C. pyrenoidosa UV 101-15
The glucose source concentration in the case of 8 is maintained by adding appropriately 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 glucose source with continuous addition of the glucose source. To be done. The ratio of the concentration of these additives to the concentration of the glucose source may be the same or different during fermentation.

Additives that can have different proportions from 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 dibasic sodium phosphate and monobasic potassium phosphate. The total amount of dibasic sodium phosphate is typically about 1-2 total g / l, usually about 1-1.5 total g / l, preferably about 1.3 total g / l. The amount of dibasic sodium phosphate initially present in the fermentor is typically about 35-50%, more usually about 40-45% of the total dibasic sodium phosphate added. The total amount of monobasic potassium phosphate is usually about 1.5-
It is 3 g / liter, more usually about 2-2.5 g / liter. The amount initially present is generally about 40 of the total amount.
-50%, more usually about 45-50%.

In addition to the above additives, a biologically acceptable chelating agent, such as sodium citrate, may be added to about 0.8-1.2.
It is advantageous to add it in a total amount of g / l, usually about 1 g / l. The monobasic sodium phosphate is generally about 0.8-1 g / liter, preferably about 0.95-1.
Present at g / liter. The addition of a biologically acceptable mineral acid keeps the trace metals in solution and also neutralizes the ammonia normally used as the 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 in metal
/ Liter is preferably present at 0.1 to 0.15 g / liter, but is particularly preferably present as a physiologically acceptable salt such as sulfate. The amount of iron and copper used is limited because these metals suppress the production of ascorbic acid. Iron (first) is initially present at about 5-7 mg / liter, preferably about 5.5-6 mg / liter and is not included in any subsequent addition. Copper is present in relatively small amounts, generally about 1 to 1 g of glucose.
It is 50 μg.

The following trace metal solutions are used in a total volume of about 10-15 ml / liter, more usually about 12-14 ml / liter. Based on glucose, 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 anhydrous ammonia.
It is also used as a pH adjuster. Parenchymal N levels in the medium are measured by the acid number of the medium and the buffer capacity of the medium.

The trace metal mixtures and solutions have the following compositions.

[Table 2]

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

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

In carrying out the fermentation, dibasic sodium phosphate and monobasic sodium phosphate were added to about 75
~ 90%, usually about 80-90% dissolved in and added to the fermentor.

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

The nutrient medium is then brought to the desired temperature. This temperature can be modified by the microorganism, but is generally about 3
The temperature is 0 to 40 ° C, preferably about 35 ° C. Inoculation of the fermenter generally yields an initial cell density of about 0.1-0.4 g / liter. A small amount of anti-foaming agent can be added during the fermentation process.

In the first stage fermentation, the cells are grown to high cell densities to build the basis for essentially high total product L-AA, and until the carbon source is substantially completely depleted, ie Cell growth is continued until the glucose equivalent concentration is usually below 0.1 g per liter of solution and a cell free supernatant is reached. The time required can vary from organism to organism, but usually is about 35
~ 50 hours, usually about 40-45 hours, growth ratio 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 it 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 aeration rate is generally about 0.2-air per minute
It is 0.6 liter. However, this may vary depending on the organism and other fermentation conditions. Although molecular oxygen in the culture medium by aeration (O ₂₎ is provided, 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. The O ₂ content of the nutrient medium can be conveniently monitored by standard techniques with an O ₂ probe electrode.

The glucose available to the microorganisms during this initial growth stage can be monitored in the supernatant, ie in the cell-free components of the medium, by any convenient method such as the glucose oxidase enzyme test, HPLC or other known methods. If the glucose concentration falls, it can be supplemented 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 / liter as described above. Below, but ensure that they are present below the growth suppression level. Glucose availability is maintained until the desired high cell density is obtained. For example, C. Pyrenoidosa UV 101-158 has a cell density of about 35 to about 35 calculated on a dry cell weight basis.
45 g / liter, preferably about 40 g / liter. At this point the glucose content of the medium is essentially 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 glucose depleted state is maintained. That is, the organism is starved during that time, cell growth is substantially arrested and cell density reaches a maximum. With the arrest of cell proliferation, L-AA production may also be substantially arrested. However, cells can generally utilize stored 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 re-fed with a glucose source, but at a regulated amount, with little or no increase in cell density. Start production of additional L-AA. The starvation period can range from minutes or less to hours or more, but typically about 1 to 4 hours, more usually 2 to 4 hours.
It's time. The optimal starvation time as well as the optimal amount or rate of glucose source supply to the fermentor should be added to each individual organism in small test doses and the effect on time on L-AA content and cell density monitored. Can be measured by The ratio of the increase in L-AA to the increase in cell density, if present, was L- during growth.
When the ratio of AA to cell density is greater than the maximum, such increased L-AA production status is generally associated with glucose sources at equal time intervals, with L-AA production predominating over increasing cell density. It is maintained by feeding in the amount or proportion that is given. In this way the optimum amount and feed rate for any individual microorganism can easily be 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 of the cells treated as the dry weight of the cells, and the glucose content is below the growth promoting concentration, for example, about 1 liter of the solution. 0.
It is maintained below 1 g.

The method of the present invention provides intracellular L-ascorbic acid in high yields, far higher than those obtained from other natural sources such as rosehips. Levels above 3.5% of biomass material can be achieved and levels above 4.0% are also achievable. The concentration of L-ascorbic acid can exceed 1.45 g / l and 3.3 g
/ 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 A 1-liter fermenter was charged with 0.6 liter of distilled water, 0.23 g of dibasic sodium phosphate and 0.27 g of monobasic potassium phosphate and sterilized. Then, the phosphoric acid solution was added to ferrous sulfate (heptahydrate) 1 in 5 ml of distilled water.
Groups of nutrients that were individually sterilized as follows at 1.2 mg and after cooling Group 1 56 g glucose, food grade, monohydrate (anhydrous basis) (80 g / l) dissolved in 80 ml water Group 2 0.7 g trisodium citrate dihydrate (1.0 g /
Liter) Magnesium sulfate, anhydrous (0.66 g / liter) and 1 ml sulfuric acid (1.4 ml / liter in 10 ml of water) Group 3 0.65 g monobasic sodium phosphate (0.97 g / liter) 1.3 g monobasic Potassium phosphate (1.9 g / l) 0.6 g Sodium dibasic phosphate (0.97 g / l) Dissolved in 10 ml water Group 4 9.4 ml Sterile glucose-salt prepared using trace metal solution 20 ml of concentrated liquid
Was added aseptically.

The temperature is raised to 35 ° C. and stirring is continued for about 200
Started at rpm. Air was passed through the medium at a rate of 0.2 liters per minute (lpm) to obtain 50 m of Chlorella pyrenoidosa UV 101-158 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, set the stirring speed to 5
The speed was increased to 00 rpm and the air flow was adjusted to 0.6 lpm. The agitation rate was then increased to 700 rpm and then 800 rpm as described in Table 3, while the airflow was constant at 0.6 lpm for the rest of the experiment.

The following table describes the conditions for fermentation and the analytical results.

[0042]

[Table 3]

The method used to measure L-ascorbic acid is
Grun and Loewus, Anelytical Biochemistry (1983) 13
0: 191-198. The method is 7.8 × 300
mm Organic Acid Analytical 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, L-ascorbic acid and isoascorbic acid can be resolved.

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

Transfer the biomass sample (5 ml) to one weighing pan and 5 ml of the supernatant liquid to the second weighing pan. The supernatant is centrifuged. Dry each pan in a convection oven (105 ° C for 3 hours). Weigh the content of each pan after cooling in a dessicator. The number of grams of cells per liter is determined as (sample weight-supernatant weight) x 200.

Based on the above results, a ratio generation based on grams of ascorbic acid per gram of cells is achieved 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, the ascorbic acid concentration can be increased to at least about 1.5 g / liter.

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

In these examples, three genus of microalgae selected to illustrate the invention, namely Chlorella, Proto
Theca and Ankistrodesmus are taxonomically broadly divergent and appear to be representative genera of L-ascorbic acid-producing heterotrophic microalgae.

Each of the above species was grown in a fed batch 1 liter stirred jar fermentor to give a cell density (dry weight basis) of about 40 g / liter. The nutrient nitrogen was replenished by the addition of ammonia and the pH was also adjusted by the addition of ammonia. The cells went into the absence of additional glucose for 1-3 hours after they had exhausted glucose-based nutrients. Thereafter, the cells were given 0.3 g of glucose per gram of the dry weight of the cells every 3 hours until it was pointed out that the ascorbic acid synthesis peaked and then declined by analysis twice a day (see below). The table shows post-proliferation glucose pulsing (pulsin
g)). In the table below, the results of these experiments are indicated by "Yes" in the subheading "Glucose pulsing after growth".

Control experiments were performed with the same strain under the same fed-batch conditions up to glucose depletion. However, no additional nutrients were added after glucose depletion. This growth condition is indicated by "No" in the "Glucose pulsing after growth" column.

[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 derived high ascorbic acid-producing strains compared to simple fed-batch conditions, i.e. growth after growth without any glucose addition, in mg / mg
It shows that it produced an increased yield of ascorbic acid, expressed as the highest concentration of ascorbic acid in liters. These results also show that C. pyrenoidosa UTEX
1663 and UV101-of it high L-AA productivity
158 mutant, C. regularis UTEX 1808 and its high L-AA producing UV 5-280 mutant and P. zopfii UTEX 1438 and its high L-AA producing UV 3-132 mutant. Are all under the conditions of the invention an enhanced specific production of L-AA (mg / g
Cells), which point to an improved utilization of the carbon source for ascorbic acid production as compared to that obtained without the addition of glucose after the initial growth and glucose depletion steps. is doing.

The C. pyrenoidosa 343 strain is a strain of other C.
Did not increase ascorbic acid yield under any combination of conditions compared to pyrenoidosa strains;
It should be noted that each of the istrodesmus braunii strains produced higher concentrations of ascorbic acid under the conditions of the present invention, but none of them improved the specific production of the acid at all. However, in this regard, the particular combination of conditions used after the initial growth and glucose depletion steps was optimized for the C. pyrenoidosa UTEX 1663 strain, whereas the 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 enhance ascorbic acid production for any of 2-370. It is believed that similar improved results may be obtained with these organisms as well.

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

The temperature is raised to 35 ° C. and the stirring is 350 rp
Starting at m, air was passed through the medium at 0.2 l / min (lpm) and pH was 6. with anhydrous ammonia (NH ₃ ) added to the air stream.
Adjusted to 9 and UV 232-1 strain was added to the medium. NH ₃ was supplied as a nitrogen source and pH adjusting agents nutrients during the experiment. After 6.2 hours, the air flow was increased to 0.6 lpm and the agitation was 400 rpm. After 11.8 hours, the air flow was increased to 0.6 lpm and kept it for the rest of the experiment, stirring speed was increased to 650 rpm. After 12.4 hours, add more than 40 ml of glucose-containing nutrient solution to the fermentor,
After 24.4 hours more than 20 ml and after 26.3 hours more than 15 ml were added. Meanwhile, the stirring speed is 22.
It was increased to 750 rpm after 8 hours, then adjusted to 900 rpm after 34.8 hours and maintained at that speed for the rest of the experiment.

The glucose content of the medium became exhausted after 31.7 hours, at which time the cell density (CD) was 19.5 and the L-AA concentration was 322 mg / l. 41.7 g of glucose (2% of 10% solution)
After the hour and every 4 hours between 41.7 hours and 94.8 hours, the fermentor was fed. Cell density and L-AA concentration
The time was shown in the table below with the calculated L-AA content of the biomass.

[0057]

[Table 5]

The above procedure was repeated four more times, essentially as described. The% L-AA was averaged at 5.2% of the dry weight of biomass in 5 experiments.

Although the present invention has been described in detail above, the present invention can be summarized and shown 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. Growing the cells of the microorganism heterotrophically, (b) growing the organism to a high cell density at an early stage,
Concomitantly producing a first amount of ascorbic acid, which then depletes the carbon source substantially completely, (c) (i) cell growth is substantially stopped, and (ii)
Subsequent addition of a controlled amount of carbon source resulted in the production of additional amounts of L-ascorbic acid in the cells in the presence of dissolved oxygen and a substantial amount of carbon source substantially without any increase in cell density. L), which is maintained in the complete absence of L-ascorbic acid, and (d) continues to regulated carbon source addition until the desired increase in L-ascorbic acid production is achieved with substantially no increase in cell density. -A method for producing ascorbic acid.

2. Produced L- in step (d)
The method according to the preceding paragraph 1, wherein the ratio of the total amount of ascorbic acid to the total cell density is larger than the ratio in the step (c) when the cell growth is substantially stopped and the increase of the cell density is stopped. 3. The method according to item 1 above, wherein ascorbic acid is recovered from cells substantially free of cellular substances.

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

8. The preceding paragraph 1 in which the microorganism is a green microalgae
The method described. 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 item 9 above, wherein the seed is pyrenoidosa. 11. The organism is a mutant strain of C. pyrenoidosa UTEX 1663 C. pyrenoidos designated UV 101-158 with ATCC Accession No. 53170.
11. The method according to 10 above, which is a strain of a.

12. A strain of Chlorella pyrenoidosa having ATCC Accession No. 53170. 13. C. py from Chlorella pyrenoidosa by UV irradiation
renoidosa UV 232-1 strain.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Internal reference number FI technical display location C12R 1:89) (72) Inventor Jeffrey A. Lanning 54220. Manitouotsk. North Fifth Street 834 (72) Inventor Thomas G. Skatztrad, Wisconsin, USA 53051. Menomonie Falls. Countryside Drive N 76 Doublet 15873

Claims (5)

[Claims] 1. The following steps: (a) heterotrophic addition of L-ascorbic acid in a growth promoting nutrient medium containing an appropriate carbon source and dissolved oxygen in respective amounts sufficient for cell growth and L-ascorbic acid production. Heterotrophically growing cells of microorganisms that can be produced physically, (b) growing the organism to a high cell density at an early stage,
Concomitantly producing a first amount of ascorbic acid and then depleting the carbon source substantially completely, (c) (i) cell growth being substantially arrested and (ii)
Subsequent addition of a controlled amount of carbon source resulted in the production of additional amounts of L-ascorbic acid in the cells in the presence of dissolved oxygen and a substantial amount of carbon source substantially without any increase in cell density. L), which is maintained in the complete absence of L-ascorbic acid, and (d) continues to regulated carbon source addition until the desired increase in L-ascorbic acid production is achieved with substantially no increase in cell density. -A method for producing ascorbic acid. 2. The ratio of the total amount of L-ascorbic acid produced to the total cell density in the step (d) is the step (c) when the cell growth substantially stops and the increase in the cell density stops. The method of claim 1, wherein the ratio is greater than 3. The microorganism is a green microalgae.
The method described. 4. The A.T.C.C. A Chlorella pyrenoidosa strain having the deposit No. 53170. 5. Chlorella pyrenoidosa by UV irradiation
C. pyrenoidosa UV 232-1 strain derived from.