The present invention provides a process for the cell fusion of strains of Phaffia rhodozyma thereby providing novel strains of Phaffia rhodozyma.

FIELD OF THE INVENTION 
The present invention relates to a process for the fusion of Phaffia 
rhodozyma spheroplasts and novel strains of Phaffia rhodozyma. 
BACKGROUND OF THE INVENTION 
Astaxanthin (trans 3,3'-dihydroxy-4,4'-diketo-.beta..beta.'-carotene also 
known as trans 3,3'-dihydroxy-.beta.,.beta.'-carotene-4,4'-dione) is an 
oxycarotenoid pigment widely distributed in plants and animals. It is a 
predominant oxycarotenoid pigment in crustaceans, and salmonids. 
Astaxanthin is also found in algae, yeast (such as Phaffia rhodozyma), 
bacteria and birds. 
In commercial aquaculture it is desirable to add astaxanthin to the diet of 
salmonids and crustaceans to impart the distinctive pink coloration found 
in indigenous salmonids, crustaceans and birds. Imparting this distinctive 
pink coloration to salmonids and crustaceans produced by commercial 
aquaculture is believed to be important in encouraging consumer acceptance 
of salmonids and crustaceans produced through aquaculture. Currently no 
economical source for astaxanthin exists. 
One potential source of aztaxanthin for aquacultural purposes is the yeast 
Phaffia rhodozyma. Phaffia rhodozyma has been recognized, since its 
classification as a yeast species having a high astaxanthin content 
(.about.85% of its carotenoid pigment is astaxanthin, N. W. Miller, et al. 
Int. J. Syst. Bacteriol., Vol. 26, p. 286 (1976). Use of this yeast as a 
dietary supplement in salmonid and crustacean diets has also been explored 
by Eric A. Johnson and other researchers since the early 1980's. 
The development of Phaffia rhodozyma as a commercial source of astaxanthin 
has been hampered by the absence of strains of Phaffia rhodozyma which 
produce high levels of astaxanthin. The strains of Phaffia rhodozyma 
currently available generally produce from 30 to 2000 micrograms per gram 
of cell mass. Unfortunately the strains of Phaffia rhodozyma which are 
high astaxanthin producer exhibit extremely slow growth rates which render 
them unsuitable for commercial fermentation. Thus, it would be very 
advantageous to develop strains of Phaffia rhodozyma which produce high 
levels of astaxanthin and desirable growth rates (thereby providing higher 
overall yields). 
Unfortunately the only method currently available for improving Phaffia 
rhodozyma strains is through repeated rounds of mutagenesis. However, 
repeated rounds of mutagenesis produces Phaffia rhodozyma strains with 
numerous mutations deleterious to the commercial fermentation of these 
strains. Thus improving Phaffia rhodozyma strains becomes increasingly 
difficult with each successive round of mutagenesis. This problem cannot 
be solved by utilizing classical mating techniques because sexual 
reproduction is unknown in Phaffia rhodozyma. Spheroplast fusion 
techniques could offer an alternative to classical mating techniques as a 
method for improving Phaffia strains, if a technique could be developed to 
generate Phaffia rhodozyma spheroplasts. However, Phaffia rhodozyma has an 
incredibly tough cell wall which has prevented researchers from being able 
to produce Phaffia spheroplasts suitable for cell fusions. 
Thus, it would be advantageous to develop new strains of Phaffia rhodozyma 
which produce higher yields of astaxanthin. 
It would also be advantageous to develop a process to produce Phaffia 
rhodozyma spheroplasts suitable for use in cell fusions. 
It would further be useful to develop a process for fusing spheroplasts of 
Phaffia rhodozyma. 
Thus it is an object of the present invention to provide strains of Phaffia 
rhodozyma which produce high yields of astaxanthin. 
It is a further object of the present invention to provide a process for 
producing Phaffia rhodozyma spheroplasts suitable for use in cell fusions. 
It is yet another object of the present invention to provide a process for 
fusing spheroplasts of Phaffia rhodozyma. 
Other aspects, objects and several advantages of this invention will be 
apparent from the instant specification. 
SUMMARY OF THE INVENTION 
In accordance with the present invention I have discovered a stable fusion 
strain of Phaffia rhodozyma which produces in the range of from about 1430 
.mu.g/g to about 1660 .mu.g/g of astaxanthin, and from about 2350 .mu.g/g 
to about 2950 .mu.g/g of total carotenoids and provides a yield of at 
least 24 percent on a dry weight basis when cultivated under suitable 
growth conditions in a shake flask with a productivity of in the range of 
from about 4500 .mu.g/l to about 8600 .mu.g/l based on a 5 day shake flask 
assay wherein the strain is cultivated under suitable conditions to 
facilitate near optimum growth of said strain. 
In accordance with the present invention, also I have discovered a process 
for producing spheroplasts of Phaffia rhodozyma suitable for use in cell 
fusions comprising contacting a viable Phaffia rhodozyma cell having a 
cell wall, under suitable conditions with an effective amount of a 
suitable digestive enzyme preparation obtained from Trichoderma 
harzianium, to facilitate the removal of said cell wall and the formation 
of a viable Phaffia rhodozyma spheroplast. 
In another embodiment of the present invention, I have also discovered a 
process for the fusion of Phaffia rhodozyma cells comprising 
(a) contacting viable Phaffia rhodozyma cells having cell walls, under 
suitable conditions with an effective amount of a suitable digestive 
enzyme preparation obtained from Trichoderma harzianium, to facilitate the 
removal of said cell wall and the formation of viable Phaffia rhodozyma 
spheroplasts; 
(b) treating said spheroplasts in a manner which facilitates the fusion of 
the cell walls of one or more spheroplasts.

DETAILED DESCRIPTION OF THE INVENTION 
Cell fusion of Phaffia rhodozyma strains provides a process for restoring 
vigorous growth characteristics to high astaxanthin producing strains of 
Phaffia rhodozyma with poor growth characteristics. The discovery of a 
process to form Phaffia cell fusions thus provides a easier way to 
continuously improve Phaffia cell strains. The fusion of Phaffia rhodozyma 
cell strains was not previously possible because of the inability of 
researchers to remove the tough cell walls characteristic of Phaffia 
rhodozyma cells. I have discovered that spheroplasts of Phaffia rhodozyma 
can effectively be formed utilizing an enzyme preparations obtained from 
the fungus Trichoderma harzianium. Utilizing these preparations it is 
possible for the first time to form Phaffia rhodozyma spheroplasts and 
perform cell fusions with these spheroplasts. 
Suitable strains of Trichoderma harzianium are publicly available from the 
American Type Culture Collection, 12301 Parklawn Drive, Rockville, Md. 
(such as ATCC 64986). These strains may be cultured and stimulated to 
produce digestive enzymes by submerged culture fermentation as is known by 
those skilled in the art. A suitable source of Trichoderma harzianium 
digest enzyme preparations is Novo Enzymes SP-299-Mutanase.TM. and 
Novozyme.TM. SP-234 a purified form of Mutanese.TM.. 
Viable Phaffia rhodozyma cells should be treated with an effective amount 
of Trichoderma harzianium digestive enzyme preparation to result in the 
substantial removal of the Phaffia rhodozyma cell walls while retaining 
the viability of a portion of the spheroplasts formed by this process. 
Generally cell wall removal of the Phaffia rhodozyma can be determined 
utilizing suitable techniques known to those skilled in the art including 
microscopic examination, or by photometric monitoring of turbidity, or by 
plating. Presently it is preferred to follow removal of the cell wall by 
photometrically monitoring turbidity. Typically a sample of Phaffia 
rhodozyma cells will be placed in an aqueous solution with the digestive 
enzyme preparation and the turbidity of the solution monitored until a 
significant drop in the turbidity is observed. The drop in turbidity will 
generally correspond to a portion of the cells being lysed by the 
digestive enzyme preparation. Generally the amount of digestive enzyme 
preparation per 100 grams/liter of aqueous Phaffia rhodozyma will be 
dependent on the temperature, pH and condition of the Phaffia rhodozyma 
cells employed. As a guideline it is recommended that the amount of 
Trichoderma harzianium utilized range from about 0.5 units to about 5.0 
units of Trichoderma harzianium digestive enzyme preparation per 100 
grams/liter of Phaffia rhodozyma cells. Currently, it is preferred that in 
the range of from 1 to 2 units of Trichoderma harzianium digestive enzyme 
preparation per 100 grams/liter of Phaffia rhodozyma cells be utilized. A 
unit is defined as the amount of Trichoderma harzianium digestive enzyme 
which will provide the equivalent amount of released astaxanthin as the 
acetone extraction described in Example V, on a sample of aqueous Phaffia 
rhodozyma with a density of 100 grams/liter, removed while in a 
logarithmic growth phase, when the digestive enzyme is contacted with the 
Phaffia rhodozyma cells at 22.degree. C., and pH 4.5 and allowed to 
incubate for 24 hours. 
Temperature at which Phaffia rhodozyma cells are contacted with the 
digestive enzyme preparation may be any temperature which allows the 
digestive enzyme preparation to digest Phaffia rhodozyma cell walls. 
Generally temperatures should range from about 0.degree. C. to about 
60.degree. C. Preferred for the practice of this invention are 
temperatures in the range of from about 20.degree. C. to about 30.degree. 
C. 
The pH at which Phaffia rhodozyma cells are contacted with the digestive 
enzyme preparation may be any suitable pH which permits the digestive 
enzyme preparation to digest Phaffia rhodozyma cell walls. Generally the 
pH at which Phaffia rhodozyma cells are contacted with the digest enzyme 
preparation should range of from about pH 4.0 to about pH 5.5 and 
preferably be in the range of from about pH 4.5 to about pH 5.0. 
Phaffia rhodozyma cells may be contacted with the digestive enzyme 
preparation derived from Trichoderma harzianium at any time during the 
life cycle of Phaffia rhodozyma. However, it is preferred that the Phaffia 
rhodozyma cells be contacted with the digestive enzyme preparation after 
the Phaffia rhodozyma cells have reached late logarithmic growth phase or 
early stationary phase, preferably in the range of from about 1 generation 
to about 10 generations after a logarithmic growth phase and most 
preferably in the range of from about 2 generations to about 4 
generations. 
The mixing of an aqueous suspension of Phaffia rhodozyma cells and the 
Trichoderma harzianium digestive enzyme preparation may be accomplished by 
any suitable means. Mixing is generally accomplished by contacting a dried 
digestive enzyme preparation with an aqueous Phaffia rhodozyma 
fermentation broth or aqueous cell suspension and admixing said dry 
digestive enzyme preparation into solution. 
The digestive enzyme preparation derived from Trichoderma harzianium may be 
contacted with viable Phaffia rhodozyma cells for an amount of time 
effective to result in the substantial removal of cell walls of the 
Phaffia rhodozyma cells. The amount of time depends on the cell 
concentration, pH, temperature and units of digestive enzyme preparation 
utilized. Generally the time of contacting the Phaffia rhodozyma cells 
with the digestive enzyme preparation derived from Trichoderma harzianium 
should be in the range of about 1 hour to about 4 hours and preferably the 
time of contacting will be about 2 hours. 
Phaffia rhodozyma Spheroplast Fusion Techniques 
Once the spheroplasts are formed, standard yeast fusion techniques may be 
used to fuse the Phaffia rhodozyma spheroplasts. Yeasts fusion techniques 
are well know to those skilled in the art. One suitable technique is 
described in Example I. The following discussion of Phaffia rhodozyma 
fusion is illustrative of techniques which may be utilized in Phaffia 
rhodozyma spheroplast fusion. 
After the cell walls have been substantially removed, the viable 
spheroplasts so formed should be collected and removed from contact with 
the digestive enzyme preparation. Suitable separatory techniques for 
removing the cell from the digestive enzyme preparation include but are 
not limited to centrifugation or filtration. The suitable separatory 
techniques used to separate the spheroplasts from contact with the 
digestive enzyme preparation should facilitate the continued viability of 
the spheroplasts. The Phaffia rhodozyma spheroplasts may be optionally 
washed in an isotonic solution to facilitate the complete removal of the 
digestive enzyme preparation. The Phaffia rhodozyma spheroplasts will then 
be recovered from the isotonic wash solution by suitable separator 
techniques. 
Following the removal of the Phaffia rhodozyma cell wall, the spheroplasts 
so formed must be treated with care to avoid rupturing the cell membrane. 
To avoid rupturing the spheroplast, it is advisable that the spheroplasts 
be maintained in a substantially isotonic solution until after the fusion 
of the spheroplasts is complete and the cell wall has regenerated. 
Isotonic solution can be formed with a variety of buffers and water 
soluble nontoxic agents including but not limited to glucose, D-mannitol, 
D-sorbitol, sucrose, or potassium chloride. It is currently preferred that 
the isotonic solution have a concentration of solutes (which includes at 
least the buffer and the soluble nontoxic agents) in the range of from 
about 0.5M to about 3M, preferred is a concentration of about 1M. 
To induce spheroplast fusion, the spheroplasts should be treated with an 
agent which promotes cell membrane to cell membrane adhesion and fusion 
such as polyethylene glycol. The spheroplasts should be placed in contact 
with the polyethylene glycol for a limited period of time because of its 
toxicity to the spheroplasts. Generally the concentration of polyethylene 
glycol contacted with the spheroplasts should be sufficient to induce 
fusion within a reasonable time but of a low enough concentration to avoid 
excessive spheroplast mortality. It is currently preferred that the 
polyethylene glycol be provided in a substantially isotonic solution at a 
concentration from in the range of about 100 grams/liter to about 300 
grams/liter, it is most preferred that the concentration of polyethylene 
glycol be about 200 grams/liter of the isotonic solution contacted with 
the spheroplasts. The length of time for which the polyethylene glycol 
should be contacted with the spheroplasts will depend on the number of 
cell per unit volume and the degree of fusion desired (the longer the 
period of contacting and the higher the cell concentration per unit 
volume, the greater the likelihood that multiple fusions of three or more 
cells will occur). For the practice of the present invention it is 
currently preferred that the polyethylene glycol be contacted with the 
spheroplasts in the range of from about 10 minutes to about 20 minutes and 
most preferably for about 15 minutes. The fused spheroplasts should then 
be washed with an isotonic solution and recovered by centrifugation. 
After the fused spheroplasts have been recovered the fused cells will need 
to be placed under suitable conditions to facilitate the regeneration of 
the fused Phaffia rhodozyma cell wall. One suitable technique for 
facilitating the regeneration of the Phaffia rhodozyma cell wall is to 
plate the fused spheroplasts on plates in an isotonic top agar. Plating 
Phaffia rhodozyma spheroplasts requires that a few precautions be taking. 
Since Phaffia rhodozyma cells are temperature sensitive, the top agar 
should have either a low melting point or be applied in a very thin layer 
which will cool quickly. Currently it is preferred that the top agar be 
applied in a very thin layer because it is desirable that the colonies 
which form in the agar penetrate the top agar and come in contact with 
air. Only those colonies which penetrate the top agar and contact the air 
will exhibit the carotenoid coloration characteristic of Phaffia rhodozyma 
cells (this facilitates easy identification of the colonies of interest). 
It also preferred for the practice of the present invention that the 
bottom or support layer of agar on to which the top agar is poured be 
isotonic. The colonies formed after fusion may be isolated and further 
screened for astaxanthin production, strain stability and growth 
characteristics using standard microbiological techniques. 
The following table demonstrates the strain improvements possible utilizing 
spheroplast fusion between selected strains of Phaffia rhodozyma to 
provide significantly improved fusion strains of Phaffia rhodozyma. These 
fusion strains exhibit significantly improved astaxanthin production and 
improved growth characteristics compared to the parent strain, PC 8055. 
TABLE I 
______________________________________ 
Phillips Culture Astaxanthin.sup.1 
Collection No. NRRL No. .mu.g/l 
______________________________________ 
PC 8055* Y-10291 1260 
PC 8166.sup.2 Y-18730 8200 
PC 8168.sup.2 Y-18731 7636 
PC 8170.sup.2 Y-18732 7706 
PC 8239.sup.2 Y-18733 8568 
PC 8243.sup.2 Y-18734 7661 
______________________________________ 
*The parent strain was 67210, also known as PC 8055, which is deposited 
and accessible to the public from the United States Department of 
Agriculture, Agricultural Research Service, Northern Regional Research 
Center located in Peoria, Illinois under accession number NRRL Y10291. 
.sup.1 Astaxanthin content was determined by the method described in 
Example II. The strains were grown under the conditions described in 
Example II. 
.sup.2 The isolated substantially pure strains of Phaffia rhodozyma with 
corresponding NRRL numbers have been deposited with the United States 
Department of Agriculture, Agricultural Research Service, Northern 
Regional Research Center, 1815 North University Street, Peoria, Illinois 
61604, under the terms of the Budapest Treaty. 
With the inventive Phaffia rhodozyma strains PC8166, PC8168, PC8170, 
PC8239, and PC8243, the increase in astaxanthin productivity is due to 
increased levels of astaxanthin (trans 
3,3'-dihydroxy-4,4'-diketo-.beta.,.beta.'-carotene also know as 
trans-3,3'-dihydroxy-.beta.,.beta.-carotene-4,4'-dione) production. The 
increased astaxanthin productivities of these strains under suitable 
growth conditions is in the range of from about 4500 .mu.g/l to about 8600 
.mu.g/l of astaxanthin when cultivated in a shake flask. Preferably the 
astaxanthin productivity of these strains under the conditions described 
above will be in the range of from about 5600 .mu.g/l to about 8600 
.mu.g/l and most preferably will range from about 7600 .mu.g/l to about 
8600 .mu.g/l of astaxanthin. The increased astaxanthin productivities of 
these strains also will result in an increased level of astaxanthin of 
from in the range of from about 1430 .mu.g/g to about 1660 .mu.g/g of 
astaxanthin on a dry weight basis when cultivated in shake flask. 
Preferably the level of astaxanthin produced will be in the range of from 
about 1515 .mu.g/g to about 1660 .mu.g/g of astaxanthin on a dry weight 
basis when cultivated in shake flask. The increased productivity observed 
will also translate into increased total carotenoid productivity in the 
range of from about 2350 .mu.g/g to about 3000 .mu.g/g of carotenoid on a 
dry weight basis. Preferably the amount of carotenoid produced will be in 
the range of from about 2460 .mu.g/g to about 2950 .mu.g/g of carotenoid 
on a dry weight basis. 
Suitable growth conditions in a shake flask are defined as the conditions 
necessary to provide the maximum specific growth rate for the Phaffia 
rhodozyma strain being cultivated in a shake flask which is being 
vigorously agitated after 5 days of growth. Suitable growth conditions for 
the Phaffia rhodozyma strains of the present invention in a shake flask 
include utilizing Shake Flask Assay Growth Medium as defined in the 
Examples of this application and cultivating the strain at between 
20.degree. C. to 22.degree. C. with vigorous shaking (as set forth in 
Example II). 
Fermentation 
Phaffia rhodozyma is a relatively new organism for use in industrial 
fermentation. Several workers in the area of Phaffia rhodozyma 
fermentation have observed that alcohol or aldehydes will accumulate in 
levels toxic to Phaffia if an excess carbon-energy source in the form of 
sugar is provided. This has led these workers to suggest growing Phaffia 
rhodozyma cells under conditions where the amount of carbon-energy source 
provided limits growth conditions. However, Phaffia rhodozyma responds to 
carbon-energy source limitation by producing lower astaxanthin yields and 
releasing compounds which cause excessive foaming in the fermentation 
vessel. The presence of these foam-causing compounds necessitates the use 
of antifoamants to avoid fermentation vessel overflow. Unfortunately the 
utilization of antifoamants can reduce the per cell astaxanthin yields. 
We, however, have discovered that by maintaining a measurable excess of at 
least one suitable carbon-energy source in the fermentation broth 
containing the aqueous Phaffia rhodozyma cells and nutrients that alcohol 
and aldehyde production can be easily controlled and foaming avoided. 
Additionally the presence of a measurable excess of carbon-energy source 
also results in increased cell growth rates and astaxanthin yields. 
Particularly important in improving astaxanthin and cell yields is the 
maintenance of a measurable excess of at least one suitable carbon-energy 
source while the Phaffia rhodozyma cells are in the transition phase 
between inoculation and the logarithmic growth phase. Preferably the 
Phaffia rhodozyma cells will be contacted with a measurable excess of at 
least one suitable carbon-energy source from the transition phase after 
inoculation through a substantial portion of the logarithmic growth phase. 
The measurable excess of at least one suitable carbon-energy source 
provided should be an effective amount to avoid excessive foam formation 
during the fermentation of Phaffia rhodozyma and also not result in the 
generation of growth repressing or toxic levels of alcohol or aldehyde. 
Preferably the measurable excess of at least one carbon-energy source 
detectable in the fermentation broth consisting of the aqueous Phaffia 
rhodozyma cells and nutrients, will range from about 1.0 gram/liter to 
about 20 grams/liter and most preferably it will range from about 1.0 
grams/liter to about 5.0 grams/liter. The amount of measurable excess of 
at least one suitable carbon-energy source in the fermentation broth 
should be controlled to avoid excess alcohol or aldehyde production. 
Preferably the amount of alcohol in the fermentation broth should range 
from about 0.0 grams/liter to about 3.0 grams/liter. Preferably the amount 
of aldehyde present in the fermentation broth will range from about 0.0 
grams/liter to about 0.1 grams/liter. 
The fermentation of Phaffia rhodozyma can be conducted in a aqueous 
continuous or batch-fed manner, utilizing a variety of carbon-energy 
sources and/or nutrient sources. Suitable carbon-energy sources for 
growing Phaffia rhodozyma include but are not limited to the carbon-energy 
source selected from the group consisting of succinate, furamate, malate, 
pyruvate, glucose, sucrose, fructose, maltose, corn syrup, hydrolyzed 
starch and combinations of any two or more thereof. Preferred 
carbon-energy sources for growing Phaffia rhodozyma are carbon-energy 
sources selected from the group consisting of succinate, glucose, and 
combinations thereof. A suitable nutrient or media source for Phaffia 
rhodozyma would include at least one nitrogen source, at least one 
phosphate source, at least one source of minerals such as iron, copper, 
zinc, magnesium, manganese, calcium, and other trace elements, and 
vitamins (such as biotin, pantothenic acid and thiamine) as required. 
Suitable sources of at least one carbon-energy source and nutrients can be 
obtained from a variety of sources or may consist of a single source such 
as cane molasses. However, preferred are at least one carbon-energy source 
and/or nutrient sources which have a defined character. At least one 
carbon-energy source and nutrient composition which has proven 
particularly effective is set forth in Table 2. 
TABLE 2 
______________________________________ 
Carbon-Energy Source and Nutrients 
Component per Liter of Water 
______________________________________ 
Glucose 10-100 (g/l) 
H.sub.3 PO.sub.4 (85%) 
0.16-2.7 (ml/l) 
CaSO.sub.4.2H.sub.2 O 
0.011-0.8 (g/l) 
K.sub.2 SO.sub.4 0.171-1.3 (g/l) 
MgSO.sub.4.7H.sub.2 O 
0.140-1.56 
(g/l) 
KOH 0.047-0.35 
(g/l) 
Biotin 0.006-0.044 
(mg/l) 
Thiamine 0.12-9.8 (mg/l) 
.sup.1 Yeast extracts 
1.2-6.0 (g/l) 
.sup.2 Minerals and Trace metals 
0.118-9.8 (ml/l) 
______________________________________ 
.sup.1 Yeast extract is Amberex 1003 which is available from and a 
trademark of Universal Foods Corporation, Milwaukee, Wisconsin. 
.sup.2 Minerals and trace metals are FeSO.sub.4.7H.sub.2 O 65.0 g/l, 
CuSO.sub.4.5H.sub.2 O 6.0 g/l, ZnSO.sub.4.7H.sub.2 O 20 g/l, MnSO.sub.4 
3.0 g/l and H.sub.2 SO.sub.4 5.0 ml/l 
The yeast extracts utilized in the present invention include but are not 
limited to yeast extracts selected from the group consisting of 
Amberex.TM. 1003 (Universal Foods Corporation) and Bacto.TM. Yeast Extract 
(Difco Laboratories Incorporated). 
Trace metals utilized in the present invention are those trace metals 
generally utilized in yeast growth provided in an amount sufficient to not 
limit the growth rate or astaxanthin production of Phaffia rhodozyma which 
include but are not limited to trace metals selected from the group 
consisting of cobalt and molybdenum. 
The fermentation temperature should generally range from about 18.degree. 
C. to about 22.degree. C. and preferably should be about 20.degree. C. 
The dissolved oxygen content in the fermentation vessel where the 
fermentation is conducted in a batch-fed manner may range from about 10% 
to about 80% of saturation and preferably will range from about 30% to 
about 60% of saturation. The dissolved oxygen content in a continuous 
fermentation should range from about 70% to about 100% of saturation and 
preferably be in the range of from about 70% to about 80% of saturation. 
The pH at which the Phaffia rhodozyma cells are cultivated should range 
from about 3.0 to about 5.5 and preferably the pH will range from about 
4.5 to about 5.4. 
After the fermentation broth containing the Phaffia rhodozyma cells has 
reached a desired cell density or astaxanthin content, the cell mass may 
be harvested. It is preferred that the Phaffia rhodozyma culture be held 
in a stationary phase for from the range of from about 4 to about 24 hours 
and most preferably in the range of from about 8 to about 12 hours to 
increase the astaxanthin yield. 
However, Phaffia rhodozyma should not be maintained for extended periods of 
time in a stationary phase because the Phaffia rhodozyma cells will form 
tough cell walls which will be detrimental to cell breakage. 
Cell Breakage 
Salmonids, crustaceans and birds cannot utilize astaxanthin from unbroken 
Phaffia rhodozyma cells. To utilize Phaffia rhodozyma as a dietary source 
of astaxanthin, the cell walls of Phaffia rhodozyma must be disrupted by 
physical, chemical, mechanical, or enzymatic means. Phaffia rhodozyma cell 
walls are very resistant to normal lysis protocols. For example, bead 
milling will only release .about.40% of the astaxanthin present in Phaffia 
rhodozyma cells after three passes through a bead mill (more passes 
through a bead mill will not substantially increase the release of 
astaxanthin). A Gaulin Press will release .about.95% of the astaxanthin 
present in Phaffia rhodozyma but only after three passes through the 
Gaulin Press (which is time consuming and requires a significant capital 
expenditure). Enzymatic lysis of Phaffia rhodozyma also had not proven to 
be economical or effective in releasing astaxanthin from Phaffia rhodozyma 
until the discovery of the present invention. 
Applicants have discovered that an effective amount of a digestive enzyme 
preparation from the fungus Trichoderma harzianium is capable of digesting 
the cell wall of Phaffia rhodozyma. This results in an almost complete 
availability of the astaxanthin present in Phaffia rhodozyma as determined 
by comparison to acetone extraction which is described in Example V. 
Suitable strains of Trichoderma harzianium are publicly available from the 
American Type Culture Collection, 12301 Parklawn Drive, Rockville, Md. 
(such as ATCC 64986). These strains may be cultured and stimulated to 
produce digestive enzymes by submerged culture fermentation as is known by 
those skilled in the art. One suitable source of Trichoderma harzianium 
digest enzyme preparations is Novo Enzymes SP-299-Mutanase. 
Phaffia rhodozyma cells containing astaxanthin should be treated with an 
effective amount of Trichoderma harzianium digestive enzyme preparation to 
result in the availability of substantially all the astaxanthin contained 
therein. Generally the amount of digestive enzyme preparation per 100 
grams/liter of aqueous Phaffia rhodozyma will be dependent on the 
temperature, pH and condition of the Phaffia rhodozyma cells employed. as 
a guideline it is recommended that the amount of Trichoderma harzianium 
utilized range from about 0.2 units to about 10.0 units of Trichoderma 
harzianium digestive enzyme preparation per 100 grams/liter of Phaffia 
rhodozyma cells. A unit is defined as the amount of Trichoderma harzianium 
digestive enzyme which will provide the equivalent amount of released 
astaxanthin as the acetone extraction described in Example V, on a sample 
of aqueous Phaffia rhodozyma with a density of 100 grams/liter, removed 
while in a logarithmic growth phase, when the digestive enzyme is 
contacted with the Phaffia rhodozyma cells at 22.degree. C., and pH 4.5 
and allowed to incubate for 24 hours. 
Temperature at which Phaffia rhodozyma cells are contacted with the 
digestive enzyme preparation may be any temperature which allows the 
digestive enzyme preparation to digest Phaffia rhodozyma cell walls. 
Generally temperatures should range from about 0.degree. C. to about 
60.degree. C. Preferred for the practice of this invention are 
temperatures in the range of from about 20.degree. C. to about 30.degree. 
C. 
The pH at which Phaffia rhodozyma cells are contacted with the digestive 
enzyme preparation may be any suitable pH which permits the digestive 
enzyme preparation to digest Phaffia rhodozyma cell walls. Generally the 
pH at which Phaffia rhodozyma cells are contacted with the digest enzyme 
preparation should be in be in the range of from about pH 4.0 to about pH 
5.5 and preferably be in the range of from about pH 4.5 to about pH 5.0. 
Phaffia rhodozyma cells containing astaxanthin may be contacted with the 
digestive enzyme preparation derived from Trichoderma harzianium at any 
time during the life cycle of Phaffia rhodozyma. However, it is preferred 
that the Phaffia rhodozyma cells be contacted with the digestive enzyme 
preparation as soon as possible after the Phaffia rhodozyma cells have 
been in a logarithmic growth phase, preferably in the range of from about 
0 hours to about 72 hours after a logarithmic growth phase and most 
preferably in the range of from about 0 hours to about 24 hours. 
The mixing of an aqueous suspension of Phaffia rhodozyma cells and the 
Trichoderma harzianium digestive enzyme preparation may be accomplished by 
any suitable means. Mixing is generally accomplished by contacting a dried 
digestive enzyme preparation with an aqueous Phaffia rhodozyma 
fermentation broth or aqueous cell suspension and admixing said dry 
digestive enzyme preparation into solution. 
The digestive enzyme preparation derived from Trichoderma harzianium may be 
contacted with Phaffia rhodozyma cells which contain astaxanthin for an 
amount of time effective to result in the substantial release of 
astaxanthin present in the Phaffia rhodozyma cells as compared to acetone 
extraction described in Example V.A. The amount of time depends on the 
cell concentration, pH, temperature and units of digestive enzyme 
preparation utilized. Generally the time of contacting the Phaffia 
rhodozyma cells with the digestive enzyme preparation derived from 
Trichoderma harzianium should be in the range of about 12 hours to about 
24 hours and preferably the time of contacting will be about 24 hours. 
Drying of Phaffia rhodozyma Cells 
The Phaffia rhodozyma cells after having been broken or digested in a 
manner which renders the astaxanthin contained therein available for use 
as a dietary pigment supplement can be dried. Drying may be performed 
using a fluidized bed drier, drum drier, or spray drier. Spray drying is 
presently preferred because of the short exposure time to high 
temperatures which could possibly degrade the astaxanthin present. 
After drying, the resultant product will be a powdery yeast material which 
may be recovered by any suitable means such as a cyclone, and further 
handled for use in feed, storage, or shipping. 
EXAMPLES 
______________________________________ 
Strains 
______________________________________ 
Phaffia rhodozyma PC 8055 
NRRL Y-10921 
Phaffia rhodozyma PC 8166 
NRRL Y-18730 
Phaffia rhodozyma PC 8168 
NRRL Y-18731 
Phaffia rhodozyma PC 8170 
NRRL Y-18732 
Phaffia rhodozyma PC 8239 
NRRL Y-18733 
Phaffia rhodozyma PC 8243 
NRRL Y-18734 
Shake Flask Assay Growth Medium 
glucose 20.0 g/L 
KH.sub.2 HPO.sub.4 10.0 g/L 
K.sub.2 HPO.sub.4 5.0 g/L 
(NH.sub.4).sub.2 SO.sub.4 
1.0 g/L 
calcium pantothenante 0.0200 g/L 
pyridoxine.HCl 0.0125 g/L 
thiamine.HCl 0.0100 g/L 
nicotinic acid 0.0100 g/L 
CaCl.sub.2.2H.sub.2 O 0.01 g/l 
ZnSO.sub.4.7H.sub.2 O 0.0070 g/L 
Hemin 0.005 g/l 
CuSO.sub.4.5H.sub.2 O 0.0006 g/L 
MnSO.sub.4.2H.sub.2 O 0.0002 g/L 
biotin 0.00015 g/L 
Mazu DF 37C antifoam 10.0 drops/L 
Modified YMA Medium 
Bacto Yeast Extract 3.0 g/l 
Difco Malt Extract 3.0 g/l 
Dextrose 20.0 g/l 
Agar 20.0 g/l 
Water 1.0 L 
(per liter of water) 
Bio Lafitte Media 
H.sub.3 PO.sub.4 (85%) 14.5 ml 
CaSO.sub.4.2H.sub.2 O 0.60 g 
K.sub.2 SO.sub.4 9.12 g 
MgSO.sub.4.7H.sub.2 O 7.60 g 
KOH 2.60 g 
glucose 40.0 g 
yeast extract 20.0 g 
trace metals.sup.1 4.0 ml 
biotin 8 mg 
thiamine 8 mg 
MAZU DF 37C antifoam 12 drops 
.sup.1 trace metals contain (YTM-4): 
FeSO.sub.4.7H.sub.2 O 16.25 g/250 ml 
CuSO.sub.4.5H.sub.2 O 1.50 g/250 ml 
ZnSO.sub.4.7H.sub.2 O 5.00 g/250 ml 
MnSO.sub.4.H.sub.2 O 0.75 g/250 ml 
H.sub.2 SO.sub.4 1.25 g/250 ml 
______________________________________ 
EXAMPLE I 
Fusion of Spheroplasts 
Cultures of Phaffia rhodozyma were prepared in 100 ml of modified YMA broth 
and allowed to incubate for five days at 20.degree. C. 2.5 ml (total 
volume) of equivalent Klett units were mixed for each culture in a sterile 
centrifuge tube and pelleted at 12,000 g at 20.degree. C. for 10 min. The 
pellet was washed 2.times. with sterile 100 mM phosphate buffer (pH 4.0) 
containing 1M sorbitol and then resuspended in 25 ml of the same buffer 
with sorbitol. The resulting solution was split into two 10 ml aliquots. 
To one aliquot was added 0.5 ml of 10 mg/ml Mutanase (SP299) and both 
aliquots were incubated at room temperature for 2 hours followed by 
chilling on ice. Cell wall removal was monitored by adding 0.1 ml of each 
aliquot to 4.9 ml of 5% SDS and measuring the absorbance at 600 nm 
(A.sub.600), followed by observing a portion of each aliquot under a light 
microscope. 
Spheroplasts were pelleted from each aliquot by centrifugation at 120 g at 
20.degree. C. for 10 min and washed 2.times. with 10 ml of sterile 100 mM 
phosphate buffer (pH 4.0) containing 1M sorbitol. Each aliquot was gently 
resuspended in 1.0 ml of 100 mM phosphate buffer (pH 4.0) containing 1M 
sorbitol. 9 ml of sterile 20% polyethylene glycol-3350 in 100 mM phosphate 
buffer (pH 4.0) was added and incubated at room temperature for 15 min. 
The spheroplasts were then pelleted at 120 g for 10 min at 20.degree. C., 
and the PEG was removed using sterile pasteur pipetes. The spheroplasts 
were resuspended in 10 ml of modified YM broth containing 1M sorbitol and 
incubated for 30 min at room temperature. 0.1 ml of each aliquot was 
pipetted onto the surface of 20 modified YMA plates containing 1M 
sorbitol. 10 ml of modified YM containing 1M sorbitol and 1% agar (which 
had been held at 42.degree. C.) was added to the surface of the plates. 
The cells were mixed into the top agar by gentle swirling, and the plates 
were then incubated at room temperature for 5 to 10 days, single colonies 
wee picked and plated on modified YMA plates, incubated at 20.degree. C. 
for 5 days and assayed for astaxanthin content. 
EXAMPLE II 
Astaxanthin Production in Fusion Strains 
The following Table denotes the fusion strains generated in Example I and 
the levels of astaxanthin produced by each strain, when grown for 5 days 
in a shake flask. Each strain was grown in a 100 ml modified YM shake 
flask, which was innoculataed with a loopful of Phaffia rhodozyma culture 
from a 5-10 day old modified YMA plate. The shake flask was incubated 5 
days on an orbital shaker (10 cm strokes at 200 rpm) at 20.degree. C. 10 
ml of the shake flask culture was innoculated into 1000 ml of Shake Flask 
Assay Medium in a 2.8 L tripple-baffled Fernbach flask. Samples were 
analyzed for washed cell dry weight, astaxanthin content (HPLC), and total 
carotenoid content (HPLC) after 5 days on an orbital shaker (10 cm strokes 
at 200 rpm) at 20.degree. C. Cell yield (based on total glucose 
concentration) is calculated using the following formula: 
##EQU1## 
Volumetric astaxanthin productivity was calculated using the following 
formula: 
EQU Washed cell dry weight (g/L).times.Astaxanthin (.mu.g/g of cells) 
TABLE I 
__________________________________________________________________________ 
Phaffia Fusion Strains 
ASTAXANTHIN.sup.2 
CAROTENOIDS.sup.2 
PRODUCTIVITY.sub.3 
PC # 
DESCRIPTION % YIELD.sup.1 
(PPM) (PPM) .mu.g/K 
__________________________________________________________________________ 
8166 
8055 .times. 8059 .times. 8117 #27 (stable) 
24.7 1660 2775 8200 
8055 
ENT 35.0 180 495 1260 
8059 
ENT 35.0 535 915 3745 
8117 
ENT (Unstable) 
19.5 1960 3245 7644 
8168 
8055 .times. 8059 .times. 8117 #7 (Stable) 
25.2 1515 2460 7636 
8055 
ENT 35.0 180 495 1260 
8059 
ENT 35.0 535 915 3745 
8117 
ENT (Unstable) 
19.5 1960 3245 7644 
8170 
8055 .times. 8117 #23 (Stable) 
25.1 1535 2490 7706 
8055 
ENT 35.0 180 495 1260 
8117 
ENT (Unstable) 
19.5 1960 3245 7644 
8239 
8055 .times. 8059 .times. 8146 .times. 8147 .times. 
28.0 1530 2950 8568 
8148 #60 (Stable) 
8055 
ENT 35.00 
180 495 1260 
8059 
ENT 35.0 535 915 3745 
8146 
ENT 22.1 1725 2715 7625 
8147 
ENT (Unstable) 
22.8 1070 1675 4879 
8148 
ENT (Unstable) 
21.1 1275 1965 5381 
8243 
8216 .times. 8059 #41 (Stable) 
28.5 1430 2350 7661 
8059 
ENT 35.0 535 915 1260 
8216 
ENT (Unstable) 
32.5 1245 2435 8013 
__________________________________________________________________________ 
.sup.1 Shake Flask Average of 3 Data Points (WCDW/Wt. Glucose) (WCDW = 
Washed Cell Dry Weight) represents percent of a theoretical yield based o 
20 grams of carbon source available. Thus 4.94 grams of cell mass provide 
a yield of 24.7% (i.e. 4.94/20 .times. 100). 
.sup.2 Data is From HPLC (see Example III) for only trans 
3,3dihydroxy-.beta.,carotene-4,4dione 
.sup.3 .mu.g of astaxanthin/liter calculated by multiplying the ppm by th 
cell concentration after 5 days of growth in Shake flask as described in 
Example II. 
As demonstrated by PC 8166, PC 8239 and PC 8243 some of the fusion strains 
substantially out preform their parent strains in the production of 
astaxanthin. However, strain such as PC 8168, and PC 8170 also represent 
significant improvements over their parental strains. The parental strains 
PC 8117 and PC 8216 which were used in some of the fusion experiments 
above were unsuitable for large scale fermentation production because of 
their tendency to generate daughter cells which were unpigmented or 
lightly pigmented. The fusion strains developed from cell fusions 
utilizing PC 8117 and PC 8216, however, do not generate unpigmented or 
light colored daughter cells. Thus the fusion strains generated from 
unstable from highly pigmented strains provide a method of continuing 
strain development on highly productive Phaffia rhodozma. 
EXAMPLE III 
HPLC Pigment Analysis of Phaffia 
The carotenoid pigments in Phaffia rhodozyma were analyzed using a normal 
phase HPLC system. This system uses an extraction step into n-heptane, 
which can be loaded directly onto the normal phase column. 
0.5 ml of Phaffia culture broth was pipetted into a 2.0 ml microcentrifuge. 
The cells were pelleted by centrifugation in the microfuge for 5 minutes. 
The supernatant was decanted and glass beads (Sigma, 0.4-0.5 mm) were 
added to cover the remaining pellet. 300 .mu.l of glacial acetic acid was 
added to the pellet, and the cells were broken by vibrating them in a 
mechanical shaker for 15 minutes. 1.0 ml of deionized water was added, 
followed by 0.5 ml of n-heptane. The carotenoids were extracted into the 
upper heptane phase by putting the tubes on a mechanical shaker for 10 
minutes. The solvent phases were separated by centrifugution in the 
microfuge for 5 minutes. The tubes were stored in the cold until they were 
ready to be analyzed. The injection volume was 30 .mu.l. 
The HPLC system used for the quantitative analysis of astaxanthin was a 
Waters HPLC system, which consisted of two Model 510 pumps, a U6K manual 
injector, a Model 490E Programmable Multiwavelength UV/Visible Detector, a 
Model 680 Automated Gradient Controller, and a Model 740 
Recorder/Integrator. The stationary phase used was Waters microPorosil 
with 10 .mu.m packing, in a 3.9 mm.times.300 mm column. The stationary 
phase was pretreated after initial installation by pumping a 1.0% (w/v) 
solution of phosphoric acid in methanol through the column for one hour at 
a rate of 1 ml per minute. The mobile phase was n-hexane and acetone. 
Separation of all of the components of Phaffia extracts that absorb at 470 
nm required the use of a gradient program. The chromatogram took 30 
minutes to run, and 5 minutes to reestablish the initial solvent 
concentration ratio. This quantition of astaxanthin can also be 
accomplished using an isocratic hexane:acetone (87:13) system which takes 
20 minutes to run without the need for equilibration time before the next 
sample is injected. 
EXAMPLE IV 
Astaxanthin Assays 
A. Plate Assay of Astaxanthin 
Modified YMA plates were streaked with cultures to be tested and incubated 
at 20.degree.-22.degree. C. aerobically. After four days, using a sterile 
applicator stick or loop, a patch of culture was scraped off (0.1-0.2 
grams net weight) from each Modified YMA culture plate. The cells were 
resuspended in 1.0 ml of deionized, distilled water and placed in a 2.0 ml 
conical bottom microcentrifuge tube. 
0.1 ml of each cell suspension was pipetted into 9.9 ml of deionized, 
distilled water, and the absorption was measured at 600 nm, to determine 
the cell concentration (in grams of washed cell dry weight per liter). 
The original cell suspension (0.9 ml volume) was then pelleted by 
centrifugation at 14,000 g and 4.degree. C. for 5 minutes and the 
supernatant was decanted. Dimethylformamide was added to bring the volume 
to exactly 10 ml. 0.25 grams of 450-500 micron glass beads were added and 
vortexed for at least 10 min. total time (with periodic cooling intervals 
on ice). The resulting cell debris and glass beads were pelleted by 
centrifugation at 14,000 g and 4.degree. C. 0.5 ml of supernatant was 
removed and 1.0 ml of dimethylformamide was added. The absorption was then 
measured at 478 nm. 
Astaxanthin concentration was calculated as follows: 
assume 1 .mu.g/l of pure astaxanthin 
has an A.sub.478 =176 * 10.sup.-6 with a 1 cm light path 
Total astaxanthin concentration of the original 1.0 ml cell suspension: 
##EQU2## 
where b=length of light path in cm (usually 1) and 
##EQU3## 
Cellular astaxanthin concentration is then calculated as follows: 
##EQU4## 
EXAMPLE V 
Astaxanthin Assays 
A. Spectrophotometric Method of Determination of Cell Breakage in Treated 
Broth 
Cells from duplicate 0.2 ml treated broth samples (approximately 100 grams 
of cells per liter of broth) were pelleted in a table top microcentrifuge 
(Hermle Z 230 M, National Labnet Co, Woodbridge, N.J.) at 14,000 g for 5 
min at 4.degree. C., resuspended in 1.0 ml of deionized, distilled water 
and transferred to 2.0 ml conical bottom, polypropylene microcentrifuge 
tubes. The cells were repelleted in the polypropylene tubes by 
centrifugation at 14,000 g for 5 min at 4.degree. C. and the supernatants 
were decanted. Acetone was added to each sample to give a volume of 
exactly 1.0 ml in the tube. 0.25 grams of glass beads (450-500 microns) 
were added to one sample and both tubes were vortexed on a Vortex, Jr. 
Mixer (Scientific Products, Inc) for a total of 10 min at 4.degree. C. 
Glass beads and cell debris were pelleted by centrifugation at 14,000 g 
for 5 min at 4.degree. C. A 0.5 ml aliquot of the supernatant was removed 
and added to 1.0 ml of acetone. Absorbance was measured at 478 nm. Percent 
breakage was determined by calculating the ratio: 
##EQU5## 
B. Batch-Fed Growth of Phaffia rhodozyma in BioLafitte 
A fresh culture of Phaffia rhodozyma PC 8115 was inoculated into the 
fermenter. This is preferably done in the following manner: A colony was 
picked from a plate and transferred to a 100 ml shake flask for 72 hrs. 
The contents of the 100 ml shake flask was then transferred to a 1000 ml 
shake flask for 24 hours. The contents of the 1000 ml shake flask was then 
transferred to the fermenter. The shake flask medium utilized comprised 
0.6% yeast extract, 0.6% malt extract, 2.0% glucose. The initial fermenter 
volume was 10 liters with pH=5.2 and 20.degree. C. 
During the 4 day run, pH was controlled between 4.5 and 5.2 (using ammonia 
and phosphoric acid), the temperature was controlled between 19.degree. C. 
and 21.degree. C. (using a cooling water bath), and the % dissolved oxygen 
(DO) was maintained between 50% and 100% (by increasing agitation to a 
maximum of 1000 rpm and increasing the airflow to a maximum of 2 
volumes/min. if necessary). 
The fermentation was run so as to maintain a slight positive glucose (0.1% 
to 0.3%) for the first 100 hours. After depletion of the original glucose 
in the fermenter, the feed was started from a 50% glucose reservoir (1750 
g glucose+1750 g H.sub.2 O, sterile). The initial feed rates were 
generally slow, but increased rapidly thereafter. Glucose concentration 
was monitored by LC during this process. The fermentation was then allowed 
to run glucose limited (glucose not detectable). The fermentation was 
complete 24 hours after 1750 grams of glucose had been used, provided 
there is no detectable glucose in the broth. 
NOTE: to control foaming 3-4 drops of Mazu DF 37C was added per liter of 
working volume as needed.