Fermentation production of ascorbic acid from L-galactonic substrate

Processes for the fermentation production of L-ascorbic acid (Vitamin C), and to microorganisms (e.g., Candida Norvegensis MF-56, ATCC 20686) and fermentation media which are specifically adapted for such fermentation.

The present invention relates to methods for the production of L-ascorbic 
acid (Vitamin C) by fermentation, and to microorganisms and fermentation 
media which are particularly adapted for such fermentation. 
BACKGROUND OF THE INVENTION 
L-ascorbic acid is an essential dietary component for man, and is naturally 
present in citrus fruits and plants. It is conventionally synthesized by a 
variety of known methods such as that described in U.S. Pat. No. 2,265,121 
to T. Reichstein using D-glucose as the starting material. Various other 
chemical and biological methods are known for synthesis and manufacture of 
L-ascorbic acid, such as those described in U.S. Pat. Nos. 2,702,808, 
2,847,421 and 3,721,663, which are generally variations of the Reichstein 
process. However, as indicated, these are relatively complex processes 
which utilize glucose as starting material. Novel commercial-scale 
processes which utilize other starting materials would be desirable. 
As described in British Pat. No. 763,055, chemical-biological processes in 
which dehydrogenase (EC 1.3.2.3) present in enzyme animal or vegetable 
tissues is utilized to carry out terminal oxidation of the gamma lactones 
to provide L-ascorbic acid. A similar process is described in U.S. Pat. 
No. 4,259,433 in which hydrolyzed sugars of lactose and plant 
dehydrogenase enzyme (EC 1.3.2.3) derived from pea seedlings are utilized 
to produce L-ascorbic acid. The efficiency of the process was not 
disclosed but application at a commercial scale would appear to be 
restricted. 
It has been recognized that bakers and/or brewers yeast contain 
L-galactono-lactone oxidase(s), an enzyme(s) believed to catalyze the 
terminal oxidation step in L-ascorbic acid biosynthesis in which the 
enzyme(s) catalyzes the oxidation of L-galactono-gamma lactone to produce 
L-ascorbic acid and hydrogen peroxide [Enzymologia; 31 #2 (1966), Eur. J. 
Biochem.; 127, 391 (1982) and others, M. Nishikimi, et al., Arch. Biochem. 
Biphys., 191, 479 (1978)]. Studies of the ability of yeasts grown in a 
nutrient medium containing D-glucose (10%) as the carbon energy source to 
produce ascorbic acid analogs of the enediol class have also been carried 
out [Heick, et al., Can. J. Microbiol., 18, 597 (1972)]. In a similar 
study, Candida yeast strains have also been grown on sucrose, hexose or 
pentose to produce an ascorbic acid analog (D-erythroascorbic acid) [S. 
Murakawa, et al., Agric, Biol. Chem., 40 (6), 1255 (1976), 41 (9) 1799 
(1977)]. When the yeasts were grown in the added presence of L-galactono 
gamma lactone, L-ascorbic acid was also identified. Although 
D-erythroascorbic acid was formed from a variety of carbon sources, 
L-ascorbic acid was only formed when the L-sugar-lactone was also present 
in the fermentation medium. 
It is also known that a vast amount of lactose is available as a byproduct 
from cheese manufacture, in the form of whey, whey permeate or milk 
permeate. Utilization of these byproducts has long been a source of 
concern to cheese manufacturers. 
It has also long been known that lactose obtained from whey or other fluid 
milk derived byproducts may be hydrolyzed to provide glucose and galactose 
(e.g., U.S. Pat. Nos. 2,826,502, 2,826,503, 2,749,242, 2,681,858) and it 
is known that whey may be fermented to provide ethanol (e.g., Food 
engineering, November, 1977 pp. 74-75; British Pat. No. 1,524,618). A new 
process for the manufacture of L-ascorbic acid which could be adapted to 
utilize dairy byproduct lactose would be particularly desirable. 
Accordingly, a principal object of the present invention is to provide 
novel fermentation processes for producing L-ascorbic acid which may be 
carried out on a commercial scale. Another object of the present invention 
is to provide processes which may be adapted to utilize a dairy byproduct 
lactose source, such as whey, whey permeate or milk permeate in the 
manufacture of L-ascorbic acid. A still further object of the present 
invention is to provide microorganisms which are capable of producing 
L-ascorbic acid by aerobic fermentation of ethanol in the presence of 
various D- and L-galactose derivatives such as L-galactono-gamma-lactone. 
A further object is provision of fermentation media which are particularly 
adapted for the microbiological manufacture of L-ascorbic acid. These and 
other objects will become more apparent from the accompanying drawings and 
the following detailed description.

DESCRIPTION OF THE INVENTION 
Generally in accordance with the present invention, methods are provided 
for manufacture of L-ascorbic acid by the aqueous phase aerobic 
fermentation of an L-galactonic substrate selected from the group 
consisting of L-galactono-gamma-lactone, lower alkyl esters of 
L-galactonic acid, L-galactonic acid, and mixtures thereof. As will be 
discussed in more detail hereinafter, the L-galactonic substrate may be 
provided in any suitable manner, such as by oxidation of D-galactose and 
by hydrolysis of pectinaceous materials such as citrus pectin. 
L-galactono-gamma-lactone is the particularly preferred L-galactonic 
substrate. Further in accordance with such methods, a short chain carbon 
fermentation energy source which may be selected from the group consisting 
of ethanol, glycerol and mixtures thereof is utilized in the fermentation. 
Ethanol is the particularly preferred carbon source. 
The selection and utilization of an appropriate microorganism for the 
aerobic bioconversion fermentation is an important feature of the present 
methods. In this regard, microorganisms are desirably provided in the 
fermentation medium which are overproductive in L-ascorbic acid synthesis 
and which accumulate L-ascorbic acid from an L-galactonic substrate. By an 
organism which is "overproductive in L-ascorbic acid bio-synthesis" is 
meant an organism which either through natural mutation or genetic 
manipulation is capable of enhanced production of L-ascorbic acid as a 
metabolite at levels of at least about 0.3 grams per liter of fermentation 
medium based on the total volume of fermentation broth. 
As indicated, the production of L-ascorbic acid by fermenting ethanol in 
the presence of L-galactonic substrate by particular microorganisms is an 
important part of the present disclosure. Yeasts, and particularly 
selected yeasts of the genus Candida which are over productive in 
L-ascorbic acid formation from a L-galactonic substrate and which can 
utilize short chain carbon sources are particularly preferred. However, 
other suitable microorganisms (particularly including appropriately 
genetically modified microorganisms) such as yeast of other genera, such 
as Hansenula, Saccharomyces, Klyuveromyces, Debaromyces, Nadsonia, 
Lipomyces, Torulopsis, Kloeckera, Pichia, Schizosaccharomyces, 
Trigonopsis, Brettanomyces or Schwanniomyces may also be employed in some 
circumstances. 
In accordance with various aspects of the present invention, the 
microorganisms utilized should be capable of utilizing ethanol as the 
principal carbon source in oxidative fermentation to carry out 
bioconversion of L-galactono-gamma-lactone to produce L-ascorbic acid in 
yields of at least about 1 gram per liter. However, the preferred 
microorganisms are mutants which belong to the genus Candida and which 
have the characteristics necessary for the production of L-ascorbic acid, 
further including characteristics such as active transport of product into 
the fermentation broth and enhanced ability to metabolize alcohol under 
aerobic conditions. However, in some instances, strains which accumulate 
significant amounts within the cell may be of value. Further, while 
ethanol is the particularly preferred carbon source, it is recognized that 
glycerol may serve as a fermentation carbon source for growth and 
production of L-ascorbic acid or other enediol compounds. The critical 
determinant for the selection of a carbon source is that it not be 
converted to an isomer of L-ascorbic acid. The yeast may be naturally 
occurring, artificially mutated or genetically engineered strains, 
particularly including those belonging to the genus Candida, provided they 
have the ability to produce and accumulate L-ascorbic acid. 
Suitable mutants may be induced by conventional mutation procedures, such 
as exposure to ultraviolet (UV) rays, and/or chemical mutagens, such as 
N-methyl-N'-nitro-N-nitrosoguanidine, ethyl methane sulfonate, nitrous 
acid, acriflavin and caffeine. Hybridization of over-producing yeast 
strains using protoplast fusion or electrofusion to produce improved 
recombinants can be employed as can recombinant DNA technology. Further it 
is recognized that many genera of yeast can be induced to produce 
L-ascorbic acid or analogs under appropriate conditions. Such strains 
which are over-productive in ascorbic acid production may accumulate the 
bulk of the ascorbic acid within the cells so that it is not released 
until cellular autolysis occurs. However, for fermentation processes in 
which the L-ascorbic acid is most easily recovered from the fermentation 
medium, it is desirable for the microorganisms to transport the product 
into the medium. 
Accordingly, a particular feature of the present disclosure is the 
provision of specific microorganism strains which do not retain the 
L-ascorbic acid product within the cell, but rather permit transport from 
the site of formation into the fermentation medium. It is believed that 
the enzymatic system which carries out the manufacture of L-ascorbic acid 
from the L-galactono-gamma-lactone substrate is associated with the yeast 
mitochondria. Accordingly, the L-galactono-gamma-lactone substrate must 
cross the cell wall from the fermentation medium, and further must be 
transported across the mitochondrial membrane. The L-ascorbic acid 
reaction product must similarly be transported across the mitochondrial 
membrane and the cell wall to enter the fermentation medium. To provide 
for accumulation of L-ascorbic acid in the fermentation medium, 
microorganism strains which have such desirable transport properties 
across the cell and mitochondrial membranes are provided. In this regard, 
particularly preferred embodiments of the present invention utilize 
selected yeast species and strains such as the Candida mutants described 
hereinafter, that actively transport the bulk of the L-ascorbic acid or 
analogs they produce into the fermentation medium during their tropophase 
and idiophase growth sequences. In accordance with the present invention, 
novel microorganisms are provided which are particularly adapted to 
aerobically oxidize an L-galactonic acid substrate, and particularly 
L-galactono-gamma-lactone, to produce therefrom and accumulate 
substantially only L-ascorbic acid. Particularly preferred are such 
microorganisms which have enhanced ability to metabolize ethanol under 
aerobic conditions and which have transport of L-ascorbic acid across cell 
and mitochondrial membranes so that the L-ascorbic acid is provided in the 
aqueous fermentation medium. 
An example of a particularly preferred, artificially mutated yeast which 
produces and accumulates L-ascorbic acid when grown on an 
ethanol-containing standard fermentation medium ("SM-1") and a special 
glycine-containing medium ("glycine medium") each also containing 0.5% by 
weight L-galactono-gamma lactone, is Candida norvegensis Kraft, Inc. 
MF-56. This strain has been deposited with the American Type Culture 
Collection Rockville, Md. and has received culture identification ATCC 
20686. This yeast is a mutant strain derived and isolated through a series 
of mutagenic processes from Candida norvegensis CBS 2145. The genealogy of 
this L-ascorbic acid over-producer, and L-ascorbic acid yields in both 
standard and glycine fermentation media, is provided in the following 
table. 
TABLE I 
______________________________________ 
Geneaology of L-Ascorbic Acid Over-Producers 
from Candida norvegensis 
L-Ascorbic Acid 
Produced (grams/per liter) 
SM-1 Glycine Medium 
______________________________________ 
Candida norvegensis 
CBS 2145 (EMS) 0.09 0.30 
MF-27 (UV) 0.015 0.60 
MF-34 (UV/CAF) 0.020 0.72 
MF-39 (UV) 0.30 0.75 
MF-42 (NTG) 0.30 0.69 
MF-54 (NA) 0.33 0.75 
MF-55 (Ni.sup.+2) 0.34 0.80 
MF-56 0.34 1.07 
______________________________________ 
Fermentations were carried out at 30.degree. C. for 48 hours in low actinic 
500 ml Erlenmeyer flasks containing 50 ml of the respective fermentation 
medium containing ethanol 1.5% w/v and L-galactono-gamma-lactone 0.5% (400 
RPM). The mutation inducing agent from the previous strain is shown in 
parentheses, in accordance with the following abbreviations: 
UV=ultraviolet radiation; EMS=ethyl methane sulfonate; 
NTG=N-methyl-N'-nitro-N-nitrosoguanidine; NA=nitrous acid; CAF=caffeine; 
Ni.sup.+2 =Nickel L-galactonate. 
The process of screening selection of over producing mutants for L-ascorbic 
acid may be carried out by applying mutagenic treatment to a large number 
of yeast cells, and subsequently selecting yeast colonies based on the 
level of ascorbic acid production. The level of ascorbic acid production 
may be monitored by culturing isolated cells of the mutagenicly treated 
yeast in a culture medium which is sensitive to acid production. For 
example, a culture medium may be opacified with an acid-sensitive material 
such as powdered calcium carbonate. Acid production by a growing yeast 
colony will dissolve the calcium carbonate, thereby providing a clarified 
zone surrounding the colony, the diameter of which increases as a function 
of increased yeast colony acid production. 
Data for mutagenic treatment and screening in respect to one of the 
cultures in the geneology of Table I, designated as C. norvegensis MF-39 
is set forth in the following Table II. 
TABLE II 
______________________________________ 
STRAIN SELECTION FOR 
L-ASCORBIC ACID PRODUCTION 
UV Exposure 
Acid Units 
Time 0-1.0 1.0-1.5 1.5-2.0 
2.0-2.5 
&lt;2.5 
______________________________________ 
0 seconds 
464/468 4/468 -- 
(100% sur.) 
99.14% .85% -- -- 
15 seconds 
300/313 8/313 5/313 -- 
(13.66% sur.) 
95.84% 2.5% 1.59% -- 
30 seconds 
4433/4480 27/4480 18/4480 
2/4480 
(4.8% sur.) 
83% 7% 9% .88% 
45 seconds 
343/500 30/500 108/500 
18/500 
1/500 
(.52% sur.) 
68.6% 6% 21.6% 3.6% .20% 
60 seconds 
30/37 -- 6/37 1/37 -- 
(.03% sur.) 
81.08% -- 16.21% 2.7% -- 
75 seconds 
277/288 7/288 2/288 1/288 1/288 
(0.1% sur.) 
96.18% 2.43% .69% .34% .34% 
______________________________________ 
In the work described by Table II, superior producing mutants were screened 
on the basis of their acid unitage (AU). In this regard, following 
mutagenic treatment the parent strain and the survivors were plated on an 
acid indicating medium, which was SM-1 culture medium containing Agar, 
ethanol 1.5% w/v, 0.5 L-galactono-gamma-lactone and mono-sodium glutamate 
0.2%, further containing 0.3% Ca CO.sub.3 as an opacifying agent, and 
incubated for 96 hours at 30 C. to determine their AU values. By AU values 
is meant the diameter of clear zone (mm)/diamer of colony zone. In Table 
Ii, the mutagenic U.V. exposure time is given in the first column of the 
table for exposure times of 0, 15, 30, 45, 60 and 75 seconds with the 
total survival percentage culture at that exposure time being shown 
thereunder. For each exposure time, the number fraction and percentage of 
surviving colonies are shown for each of the five different zone sizes of 
acid unitage, in respective columns of the table. Shake flask testing as 
described in Table I is used for productivity evaluations. The MF- 42 
strain was selected from among the mutated strains having highest acid 
unitage. 
The morphological, culturaal and physiological characteristics of the 
mutant strain C. norvegensis Kraft, Inc. MF-56 and the parent strain C. 
norvegensis CBS 2145 are consistent with the yeast description provided in 
The Yeasts, a taxononomic study (J. Lodder (Ed.) 1970, North Holland 
Publishing Co., Amsterdam) and A New Key to the Yeasts (J. A. Barnett and 
R. J. Parkhurst (Ed) 1974, North Holland Publishing Co., Amsterdam). 
Morphological and identification tests are presented in Table III. 
When grown on malt extract at 25.degree. C., cells are cylindrical to ovoid 
(2-8).times.(5-13) microns. Colonies are cream colored, glistening, soft 
and smooth. Ascospores are not formed on Folwells acetate agar. 
TABLE III 
______________________________________ 
Assimilation of carbon 
compounds: 
Glucose + Ethanol + 
Galactose - Methanol - 
L-Sorbose - Glycerol + 
Sucrose - Erythritol - 
Maltose - Ribitol - 
Cellobiose + Galactinol - 
Trehalose - D-Mannitol - 
Lactose - D-Glucitol - 
Melibiose 
Methyl-D- - 
glucoside 
Raffinose - Salicin +/- 
Melezitose - Arbutin +/- 
Insulin - DL-Lactic Acid + 
Soluble Starch 
- Succinic acid + 
D-Xylose + latent or 
- Citric acid + 
L-Arabinose - Inositol * 
D-Arabinose - Glucono-delta-lactone 
- 
D-Ribose - 2-Keto-gluconate 
- 
L-Rhamnose - 5-Keto gluconate 
- 
- D-Glucosamine + 
Assimilation of KNO.sub.3 : negative 
Growth without added vitamins: negative; thiamine, 
biotin and pyridoxone are required 
Maximum temperature for growth: 41-45.degree. C. 
______________________________________ 
*sometimes weak 
Another feature of the present invention is provision of aqueous 
fermentation media and fermentation conditions under which metabolic 
processes of gluconeogenesis from ethanol may be repressed and formation 
of D-erythroascorbic acid may be minimized. Particularly preferred aqueous 
fermentation media may be provided which facilitate the recovery of 
L-ascorbic acid and which enhance the microbiological production of 
L-ascorbic acid. The provision of an appropriate aqueous fermentation 
medium is a further important feature of methods in accordance with the 
present invention, and selection and provision of a desired fermentation 
medium is in part a function of the particular microorganism utilized in 
the fermentation. The provision of an appropriate fermentation medium may 
also provide for more effective L-ascorbic acid recovery procedures by 
separation techniques including ion exchange resin separation methods, as 
will be discussed in more detail hereinafter. 
Ethanol is used as the carbon source in the fermentation medium and the 
initial concentration is preferably in the range of about 0.01-2.0% weight 
(grams)/volume (milliliters) herein "w/v" depending upon the particular 
strain employed. As ethanol is consumed during the fermentation it may be 
intermittently supplemented to give an optimum concentration (about 
0.01%-2.0% w/v) which can be tolerated by the yeast and does not inhibit 
growth or L-ascorbic acid production. 
In accordance with various aspects of such methods, an aqueous fermentation 
medium is provided comprising a carbon fermentation energy source having 
less than four carbon atoms selected from the group comprising ethanol, 
glycerol and mixtures thereof, an L-galactonic substrate selected from the 
group consisting of L-galactono-gamma-lactone, L-galactonic acid, and 
mixtures thereof. The fermentation medium will generally further contain 
nutrients necesssary for growth of the selected microorganism and will 
preferably have a pH in the range of from about 2.5 to about 6.5. 
Generally, at least about 0.01 weight percent, and preferably from about 
0.1 to about 2.0 weight percent of the carbon source will be provided in 
the aqueous fermentation medium, based on the total weight of the 
fermentation medium. The carbon source is consumed during the fermentation 
and may be periodically or continously added during the course of the 
fermentation. Similarly, at least about 0.1 weight percent of the 
fermentation substrate will desirably be provided in the fermentation 
medium based on the total weight of the medium. For yeast fermentations, 
the fermentation medium will generally include a nitrogen source, various 
organic nutrients, and various minerals. 
The nitrogen source (which may typically be utilized in an amount of about 
0.1 to about 0.5 weight percent based on the total weight of the aqueous 
fermentation medium) may be selected from the group metabolizable nitrogen 
compounds comprising ammonium sulfate, ammonium nitrate, ammonium chloride 
or ammonium phosphate, urea or ammonium ion in the form of ammonium 
hydroxide, etc. and mixtures thereof, depending on the ability of the 
particular strain to best utilize the nitrogen source. Further, various 
amounts of organic nutrients such as amino acids (e.g., monosodium 
glutamate, glutamine, aspartic acid, etc.) or purines (adenine, thymine) 
corn steep liquor, yeast extract, protein hydrolysates etc., inorganic 
salts, such as, sulfates or hydrochlorides of Ca, Mg, Na, K, Fe, Ni, Co., 
Cu, Mn, Mo, Zn; vitamins (e.g., water soluble B vitamins) may be added to 
prepare a culture or fermentation medium. One such medium which 
effectively carries out this function is the previously referred to SM-1 
ethanol medium. The compositional characteristics of this medium are 
listed as follows: 
______________________________________ 
SM-1 MEDIUM 
Amount 
G.L..sup.-1 
______________________________________ 
A. carbon - ethanol (weight/volume) 
15.0 
B. nitrogen - urea 2.0 
C. Supp mix - corn steep liquor 
5.0 
D. Inorganic 
Salts - 
K.sub.2 HPO.sub.4.3H.sub.2 O 
1.0 
KH.sub.2 PO.sub.4 3.0 
MgSO.sub.4.7H.sub.2 O 
0.5 
NaCl 0.1 
KCl 0.1 
H.sub.3 BO.sub.3 .0005 
FeCl.sub.3.6H.sub.2 O 
.0002 
MnSO.sub.4 H.sub.2 O 
.0004 
ZuSo.sub.4.5H.sub.2 O 
.0004 
CUSO.sub.4.5H.sub.2 O 
.0004 
KI .0001 
(NH.sub.4).sub.6 MO.sub.7 O.sub.24.4H.sub.2 O 
.0002 
E. Vitamins - 
Thiamine HCl .004 
Biotin .00002 
F. Bioconversion Compd. L-galactono-.gamma.-Lactone 
5.0 
G. Adjust to pH 4.0 
______________________________________ 
When selected mutant yeast are grown in this medium under appropriate 
cultural conditions essentially only L-ascorbic acid is produced as the 
bioconversion product of L-galactono-gamma-lactone provided in the culture 
medium. Modifications of this medium or other media (as will be more fully 
discussed hereinafter) may prove to be more beneficial. The conditions for 
culturing are typically a temperature in the range of from about 
20.degree. C. to about 37.degree. C. and preferably about 30.degree. C. 
The fermentation is desirably carried out at a pH in the range of from 
about 6.5 to about 2.6 and preferably about 4.0. The optimum conditions 
will depend on the particular yeast strain employed. The fermentation 
process may take from 1 to 7 days and is operated under aerobic 
conditions. When a high density yeast cell biomass (range 25-240G/WTCWT) 
is employed in the bioconversion process to produce L-ascorbic acid, the 
additional supplementation of pure oxygen or an oxygen enriched atmosphere 
to the aeration process may be required to prevent the development of 
anaerobic conditions or may be desirable to enhance yields. It is 
desirable that at least about 2.5 ppm oxygen be maintained in the aqueous 
culture medium, and preferably the oxygen content should not decrease 
below a predetermined level in the range of from about 3 to about 5 ppm. 
While standard culture media such as the SM-1 culture medium previously 
described may advantageously be utilized in the manufacture of L-ascorbic 
acid from a L-galactono-gamma-lactone substrate in accordance with the 
present invention, it is particularly preferred that the fermentation be 
carried out utilizing a culture medium of at least about 0.5 weight 
percent and preferably in the range of from about 0.6 weight percent to 
about 0.8 weight percent glycine, based on the total weight of the culture 
medium. It has been found that about 0.7 weight percent glycine in the 
medium is particularly effective in respect to yield enhancement. In this 
regard, it has been found that such high glycine culture media appear to 
enhance the yield productivity of L-ascorbic acid by factors of about 3 or 
more. The components of a glycine fermentation medium which has proven to 
be particularly effective in the fermentations described herein, and which 
are identified herein as "glycine medium" are listed as follows: 
______________________________________ 
GLYCINE MEDIUM 
______________________________________ 
component amount (grams per liter) 
______________________________________ 
Ethanol 20.0 
Glycine 7.0 
CSL w,v 5.0 
mono sodium glutamate 
2.0 
NH.sub.4 Cl 1.0 
MgSO.sub.4 0.5 
mineral mix 2.0 ml 
The mineral mix consists of: 
EDTA (2Na) 5.0 grams per liter 
ZnSO.sub.4.7H.sub.2 O 
0.22 grams per liter 
CaCl.sub.2.2H.sub.2 O 
0.735 grams per liter 
MnSO.sub.4.H.sub.2 O 
0.6725 grams per liter 
FeSO.sub.4.7H.sub.2 O 
0.915 grams per liter 
(NH.sub.4).sub.6 Mo.sub.7 O.sub.24.4H.sub.2 O 
0.10 grams per liter 
CuSO.sub.4.5H.sub.2 O 
0.25 grams per liter 
CoCl.sub.2.6H.sub.2 O 
0.293 grams per liter 
______________________________________ 
As will be more fully described, the L-galactonic substrate may desirably 
be manufactured from cheese or dairy byproduct lactose by hydrolyzing the 
lactose to produce glucose and D-galactose and oxidizing the D-galactose 
to produce D-galacturonic acid: 
##STR1## 
D-galacturonic acid may also conveniently be provided by hydrolysis, such 
as enzymatic hydrolysis, of pectinaceous materials such as citrus pectin. 
The D-galacturonic acid may be reduced to provide L-galactonic acid, which 
may be dehydrated to form L-galactano-gamma-lactone: 
##STR2## 
VArious derivatives of L-galactonic acid particularly including the lower 
alkyl esters may be manufactured by conventional esterification reaction 
of L-galactonic acid: 
##STR3## 
In accordance with the method, aerobic fermentative conditions are 
maintained under which the substrate is oxidized by the microorganism to 
substantially only L-ascorbic acid produced by the microbiological 
oxidation. The L-ascorbic acid produced by the aerobic fermentation may be 
subsequently recovered in an appropriate manner, as will be more fully 
described. 
It is necessary to maintain aerobic conditions during the fermentation, and 
in this regard, it is desirable that at least 20% (e.g., 20-30% 
oxygen-saturation (e.g., 2-3 ppm of oxygen) be maintained in the aqueous 
fermentation medium during the fermentation. Maintaining aerobic 
conditions may be carried out by introducing oxygen enriched gas into a 
fermentation medium, and by utilizing fermentation equipment, such as air 
lift reactors, which effectively disperse oxygen into the fermentation 
medium. 
As indicated, ethanol may be utilized as the principal carbon energy source 
under fermentation conditions in which L-galactono-gamma-lactone is 
substantially wholly converted to L-ascorbic acid. For example, when 
Candida yeast are grown on ethanol (a 2-carbon energy source), employing 
SM-1 -medium and 0.5 weight percent L-galactano-gamma-lactone rather than 
on hexose sugar (6-carbon energy source) as the energy source in SM-1 
medium with 0.5% L-galactono gamma-lactone essentially only L-ascorbic 
acid is formed and production of other ascorbic acid analogs, e.g., 
D-erythroascorbic acid is minimized. This is an important factor in the 
development of a practical process to produce L-ascorbic acid at levels of 
industrial interest. 
The fermentation process is a bioconversion process in which 
L-galactono-gamma-lactone is converted to a structurally related product 
by one or a small number of enzymes within a cell. The process may be 
carried out using growing cells, resting vegetative cells, dried cells or 
cells immobilized in various organic polymers, such as K-carrageenan, 
polyacrylamide, gelatin, or agar, or other macroporous resins or inorganic 
compounds, such as cordierite and silica. 
The L-ascorbic acid bioconversion process may be operated in conventional 
aerobic fermentation modes, e.g., batch, continuous, semi-continuous. Also 
cultivation methods usable to obtain high-density of biomass; dialysis 
culture, semi-batch with cell separator and fed-batch processes may be 
employed in which oxygen supplements to air may be required. 
Having generally described the present invention, various aspects thereof 
will now be more particularly described with respect to the process 
embodiment illustrated in block diagram by FIG. 1. 
FIG. 1 is a schematic illustration of an embodiment of a process for 
manufacturing L-ascorbic acid from a dairy by product lactose solution 
substrate 10 such as whey, whey permeate or milk permeate. The dairy fluid 
lactose solution may typically comprise from about 4.5 to about 5.0 weight 
percent lactose, which is hydrolyzed to provide its constituent sugars, 
glucose and galactose in the form of glucose-galactose solution 12. The 
hydrolysis may be accomplished in a conventional manner using a lactase 
enzyme derived from a yeast (K. fragilis or K. lactis) or a mold enzyme 
derived from A. niger or A. oryzae. The enzyme may be employed either in a 
free, entrapped or immobilized form. 
The glucose-galactose solution 12 or the lactose solution 10 may be 
deproteinized or demineralized in accordance with conventional procedures 
if necessary or desirable in carrying out the alcohol fermentation. The 
glucose-galactose solution provided by the hydrolysis treatment step may 
be concentrated to provide a solution comprising for example in the range 
of from about 15 percent to about 30 percent by weight, based on the total 
weight in the solids solutions. The non-lactose solids content may 
typically be in the range of from about 0.5 to about 1.0 weight percent of 
the total solids, such that the total glucose and galactose content of the 
solution may desirably be in the range of from about 20.0 to about 22.5 
percent by weight based on the total weight of the solution. 
After supplementation with suitable nutrients such corn steep liquor or 
yeast extract in accordance with conventional yeast fermentation 
procedures, the glucose-galactose solution 12 may be fermented under 
anaerobic conditions using an appropriate yeast strain for fermentation of 
glucose to ethanol, such as a selected yeast strain of S. cerevisiae, in 
order to convert the glucose to ethanol and carbon dioxide without 
substantially consuming the galactose component of the fermentation 
medium. In this manner an ethanol-galactose solution 14 is provided. 
The ethanol-galactose fermentate may typically comprise at least about 5 
percent ethanol on a weight to volume basis, and at least about 10 weight 
percent D-galactose based on the volume and weight of the fermentation 14 
respectively. The fermentate 14 is distilled to remove the alcohol 16 
which may subsequently be rectified to provide 190 proof grain alcohol, if 
desired. This alcohol 16 may then be utilized in the L-ascorbic acid 
fermentation process to serve as carbon energy for the selected 
microorganism utilized in the fermentation. 
In this regard, the stillage containing D-galactose recovered after removal 
of ethanol may be concentrated further by appropriate methods to provide a 
galactose solution having a total solids content in the range of from 
about 20 to about 75 weight percent based on the total weight of the 
solution, and which may desirably contain from about 16 to about 62 weight 
percent of galactose based on total solution weight. This galactose 
solution may be crystallized to obtain purified D-galactose 18. The 
mineral salts 20 may be utilized in the L-ascorbic acid fermentation, if 
desired; as a supply of inorganic nutrients for the fermentation medium. 
Alternatively the galactose may be separated from the rest of the 
fermentation components via ion exclusion. Such galactose can serve 
directly as the feedstock for reaction step 18. 
The D-galactose 18 is converted by catalytic oxidation to provide 
D-galacturonic acid 22. Processes to carry out this oxidative step for 
D-sugar acids are well known in the art and have been described in U.S. 
Pat. No. 2,265,121 by T. Reichstein. A variety of catalytic agents, e.g., 
platinum or palladium catalysts may be employed to convert blocked 
D-galactose (acetone) to the D-galacturonic acid product. The unblocked 
D-galacturonic acid may then be reduced by an appropriate reduction step 
such as reduction with gaseous hydrogen in the presence of a suitable 
hydrogenation catalyst such as Raney nickel, or palladium, to produce 
L-galactonic acid 24. Processes to carry out this chemical reduction are 
well known in the art such as described by H. Isbell, J. Res. Nat. Bur. 
Stds., 33, 45-60 (1944) Removal of water by distillation and condensation 
of the desalted L-sugar acid induces the formation of 
L-galactono-gamma-lactone 26 which is utilized in the microbiological 
conversion process of the present invention to form L-ascorbic acid. Other 
galactose derivatives such as L-galactonic acid esters and L-galactonic 
acid may be utilized although yields of L-ascorbic acid are reduced due to 
less efficient utilization. Keto-derivatives such as 5-Keto-L-Galactonic 
acid, are not utilized by the preferred yeast strains particularly 
described herein. The L-galactono-gamma-lactone 26, ethanol 16, and 
suitable organic and inorganic nutrients are combined to provide a 
fermentation medium 28 for a selected L-ascorbic acid overproducer. 
The L-ascorbic acid fermentation process may be carried out in conventional 
stirred aerated fermentors such as a 30 liter New Brunswick Scientific 
fermenter. Monitoring of product formation and control of the cellular 
environment using physical and chemical sensors linked to a microcomputer 
may be carried out by means of detectors for measuring ethanol, pressure, 
an input flow, exhaust gas, carbon dioxide, exhaust gas oxygen, pH, and 
dissolved oxygen. 
After cultivation, L-ascorbic acid 30 produced by the fermentation may be 
recovered from the clarified fermentation broth by a variety of methods 
such as by using ion exchange resins, absorption or ion retardation 
resins, activated carbon, concentration-crystallization, etc. 
The course of the fermentation may be monitored by appropriate analytical 
procedure. Quantitative assay of L-ascorbic acid and analogs may be 
carried out using redox-titration with 2,6 dichloroindophenol [N. G. 
Burton, et al., J. Assoc. Pub. Analysts, 17, 105 (1979)] and 
high-performance liquid chromatography using anion exchange [J. Chrom., 
196, 163 (1980)] and electro-redox procedures. [L. A. Pachia, Anal. Chem., 
48, 364 (1976)]. Enzymatic procedures involving the use of ascorbic acid 
oxidase (Boehringer-Mannheim) may also be employed. 
The fermentation may be terminated when maximal production of L-ascorbic 
acid has been attained in the fermentation broth. Unconverted portions of 
L-galactono-gamma-lactone may be recycled. 
Various aspects of the present invention will be further described with 
respect to the following specific examples, which are not intended to 
limit the scope of the invention. 
EXAMPLE I 
A stirred batch fermentation to produce L-ascorbic acid was carried out in 
a 30-Liter New Brunswick stirred fermentor. Fifteen liters of glycine 
medium composed of 0.25% corn steep liquor, 0.1% ammonium chloride, 0.7% 
glycine, 0.05% magnesium sulfate. 7H.sub.2 O, 0.2% monosodium glutamate, 
1.5% w/v ethanol and 0.30 ml of trace mineral mixture, was adjusted to pH 
4.2 and sterilized for 30 minutes at 121.degree. C. (Unless otherwise 
indicated values herein are in weight percentages). After cooling, 0.5% of 
cold-sterilized L-galactono-gamma-lactone was added to the sterile 
fermentation broth. The fermentor was inoculated with 500 ml of a 24 hour 
SM-1 broth culture of C. norvegensis KCC MF42 (Table I) grown in a 2-liter 
Erlenmyer flask on a rotary shaker at 30.degree. C., 200 RPM. 
The fermentor was operated at 30.degree. C., 250 RPM and an aeration rate 
of 0.25 vol/vol/min. with the pH initially maintained at 4.0. After 24 
hours, the supernatant broth contained 0.084 GL.sup.-1 of L-ascorbic acid. 
An additional 27.0 mg was present in the yeast cells. After 48 hours the 
clarified broth contained 0.43 GL.sup.-1 of L-ascorbic acid and the cells 
contained 29.6 mg. L.sup.-1. The product could be recovered using 
conventional ion exchange resin absorption and elution followed by 
decolorization, evaporation and crystallization. 
EXAMPLE II 
A system of process intensification using high density biomass and product 
recovery was developed for the production of L-ascorbic acid using Candida 
yeast and mutants. In this procedure KCC MF-42 yeast cells were cultivated 
in SM-1 medium (ETOH 1.5% w/v) (L-Galactono-gamma-lactone 0.1%) for 18 
hours in a stirred fermentor and centrifuged under sterile conditions. The 
cell paste provided by centrifugation was then aseptically reconstituted 
at 37.5 g L.sup.-1 wet cell weight in sterile fresh SM-1 medium, pH 4.0 
(Ethanol 1.5%--monosodium glutamate 0.2%--L-galactono-gamma-lactone 0.5%) 
and aerated with oxygen at a dissolved oxygen level of 65% of saturation. 
Production of L-ascorbic acid rose to 0.470 GL.sup.-1 in 24 hours and 
increased to 0.580 GL.sup.-1 in 45 hours. The pH of the medium dropped to 
2.6 during the fermentation. 
Four liters of chilled, clarified fermentation broth were passed through a 
500 ml column of IR120 (H.sup.+) resin, an ion exchange resin manufactured 
by Rohm and Haas. The effluent and washwater were collected and evaporated 
to 100 ml volume at 37.degree. C. under vacuum. One hundred milliliters of 
cold ethanol was added and the precipitate (protein) was removed by 
centrifugation at 5000 RPM at 5.degree. C. The product was again 
evaporated to 25 ml volume and stored at 0.degree. C. for five days until 
crystallization was complete. The filtered crystals were washed 3 times 
with acetone, redissolved in warm alcohol and recrystallized. About 1.4 g 
of crude L-ascorbic acid crystals (HPLC) were recovered in the first crop. 
Recovery and purification can also be carried out by absorption of 
L-ascorbic acid from broth on anion retardation resin (Dowex 1 type), 
acetic form and elution with 0.1M H.sub.2 SO.sub.4. 
EXAMPLE III 
A process was developed in which resting cells of Candida yeast and mutants 
were used to produce L-ascorbic acid from ethanol and 
L-galactono-gamma-lactone in buffered salt solution. Both ethanol and 
L-sugar lactone are required by the yeast. The resulting cells may be used 
in a free state or immobilized in various polymeric gels or attached to 
polymeric resins, or inorganic mineral compounds. 
In this example, yeast cells Candida norvegensis CBS #1911 cultivated 18 
hours in SM-1 medium were centrifuged, washed in phosphate buffer (pH 4.5) 
and resuspended at a level of 3.0 grams wet cell weight/50 ml of 0.03% 
phosphate buffer solution (pH 4.5) containing 0.8% ethanol and 0.5% 
L-galactono-gamma-lactone. The 50 ml mixture in a 500 milliliter 
low-actinic, borosilicate Erlenmeyer flask was placed on a rotary shaker 
and aerated at 300 RPM at 30.degree. C. Broth samples were taken 
periodically in order to monitor ethanol utilization and L-ascorbic acid 
production. Additions of ethanol were made periodically to maintain the 
alcohol concentration at about 0.3% w/v concentration. Results of the 
L-ascorbic acid accumuation by the yeast after 96 hours are shown in TABLE 
IV: 
TABLE IV 
______________________________________ 
Microorganism used 
L-ascorbic acid 
C. Norvegensis accumulated Time 
CBS - #1911 (mg L.sup.-1) 
Hours 
______________________________________ 
90 33 
130 48 
200 73 
260 96 
______________________________________ 
EXAMPLE IV 
A screening program was initiated for the selection of microorganisms 
capable of converting galactose derivatives and preferably 
L-galactono-gamma-lactone to L-ascorbic acid. Microorganisms of the genus 
Candida were selected from the varieties of yeast reported capable of 
producing enediol compounds. 
A large number of Candida species readily available in various culture 
collections, e.g., American Type Culture Collection, Rockville, Md., 
Central-Bureau voor Schimmelculture, Delft, Institute Pasteur, Paris and 
Northern Region Research Lab, Peoria, Ill., were accessed and purified 
prior to screening studies. The cultures were maintained on G-agar slant 
tubes or other nutrient media. 
A saline suspension of a 24 hour slant of yeast grown on G-agar was 
employed as the inoculum. A 0.5 ml cell suspension was aseptically added 
to 50 ml of sterile SM-1 medium (ethanol 1.5% w/v, urea 0.2%) in a 500 ml 
low actinic Erlenmeyer flask. L-galactano-gamma-lactone (0.5%) was cold 
sterilized and added to the cooled flasks. The flasks were placed on a 
rotary shaker and aerated at 200 rpm, 30.degree. C. for 48 hours. The 
clarified broths were examined for L-ascorbic acid production. The 
centrifuged, washed cell pastes were treated with 3.0 ml of 10% 
trichloracetic acid and titrated with 2,6, dichloroindophenol to establish 
the level of reducing compounds present within the cell. Conversion of 
L-galactono-gamma-lactone to L-ascorbic acid was observed in the following 
species, as shown by Table V. 
TABLE V 
______________________________________ 
Production of L-Ascorbic Acid (L-AAH.sub.2) 
Micrograms 
per deciliter 
Organism Source No # Broth Cells 
Total 
______________________________________ 
C. ingens CBS 4603 350 3717 4067 
C. truncata CBS 1899 2730 5691 8421 
C. lusitaniae 
CBS 4413 1820 2100 3920 
C. berthetii 
ATCC 18808 1330 2394 3724 
C. maltusa ATCC 20184 630 1470 2100 
C. langeronii 
ATCC 22972 910 1386 2296 
C. parapsilosis 
ATCC 22019 140 462 602 
C. maltosa ATCC 28140 560 1764 2324 
C. silvae ATCC 22685 70 168 238 
C. reukaufii 
CBS 611 0 147 147 
RDICC 5506 9660 7581 17241 
C. utiliis NRRL Y-900 
H. anomala ATCC 20029 1610 2961 4571 
C. utilis ATCC 15239 11970 3402 15372 
Y. lipolytica 
ATCC 20390 280 3696 3976 
Y. lipolytica 
ATCC 8661 70 3465 3535 
C. guilliermondii 
IP 47 1820 798 2618 
C. zeylanoides 
IP 207 0 168 168 
C. pseudotropicalis 
IP 513 70 168 238 
C. pelliculosa 
IP 606 1750 420 2170 
C. pulcherrima 
IP 622 420 756 1176 
C. robusta IP 826 0 231 231 
T. candida ATCC 10539 280 546 826 
C. sloofii ATCC 22978 0 147 147 
C. norvegensis 
CBS 1911 10780 1113 11893 
C. amyloanta 
NRRL Y-7784 140 168 308 
C. buinensis 
NRRL Y-11706 0 147 147 
C. cacaoi NRRL Y-7302 1960 1428 3388 
C. conglobata 
NRRL Y-1504 0 147 147 
C. deformans 
NRRL Y-321 70 168 238 
F. fluviotilis 
NRRL Y-7711 420 546 966 
C. vinii NRRL Y-94 4130 2520 6650 
______________________________________ 
EXAMPLE V 
Airlift fermentors have several distinct advantages over conventional 
agitator-driven shaft fermentors. Among these are improved mass transfer 
of oxygen, reduced power requirements, and a more gentle environment for 
the cultivation of organisms compared to the high degree of shear present 
in mechanically agitated fermentors. Because of these features, airlift 
fermentors are desirably employed on an industrial scale. The following 
examples illustrate the use of an airlift tower fermentor for the 
production of Vitamin C using Candida yeast. 
A 4.0 liter laboratory scale airlift fermentor was filled with sterile 
glycine medium, pH 4.1, containing 2.75% w/v ethanol, 0.7% glycine and 
0.5% L-galactono-gamma-lactone. The fermentor was inoculated with a 24 
hour suspension of the C. norvegensis KCC MF-42 cells washed from G-Agar 
(2.5%) flasks. The viable cell count at 0 hour was 5.5.times.10.sup.6. 
Aeration of the fermentor was adjusted to 1.9 V/V/m which provided a cycle 
rate of 5.0 m.sup.-1. After 24 hours at 30.degree. C., the viable cell 
count rose to 1.1.times.10.sup.8, and at 48 hours was 3.0.times.10.sup.8, 
at 72 hours the viable cell count was 2.8.times.10.sup.8. The count 
declined to 1.7.times.10.sup.8 after 91 hours of cultivation. A level of 
0.72 GL.sup.-1 of L-ascorbic acid was produced. 
EXAMPLE VI 
In a similar airlift tower experiment a high cell density fermentation was 
performed. In this instance a 24 hour wet cell paste of C. norvegensis KCC 
MF-42 was dispersed in the 4.0 liter tower at a level of 100 GL.sup.-1 in 
SM-1 medium containing 0.7% glycine and 0.7% L-Galactono-gamma-lactone. 
Ethanol was supplied continuously at a level (0.1-0.3%) neither limiting 
or inhibitory to yeast growth or productivity. Oxygen-enriched aeration 
was supplied to the fermentor at 1:1 oxygen-air ratio. Total mixed gas 
volume was 1.7 V/V/min. Under these conditions a dissolved oxygen 
saturation level of 30% was maintained in the upper section of the tower. 
After 20 hours of fermentation, L-ascorbic acid was produced at a level of 
1.44 GL.sup.-1. 
It will be appreciated from the previous description that in accordance 
with the present invention, useful new methods, organisms and culture 
media have been provided for the manufacture of ascorbic acid. While 
various aspects of the invention have been specifically described with 
respect to certain specific embodiments, it will be appreciated that 
various modifications and adaptations will become apparent from the 
present disclosure, which are within the spirit and scope of the present 
invention and are intended to be within the scope of the following claims.