World
Journal
of Microbiology
and Biotechnology
9, 514-520
Selection and evaluation astaxanthin-overproducing of Phaffia rhodozyma
of mutants
P.S. Meyer, J.C. du Preez* and S.G. Kilian Mutagenesis of PhaBa rhodozyma with NTG yielded a mutant with an astaxanthin content of 1688 pg (g dry biomass)-‘, a cell yield coefficient of 0.47 on glucose and a maximum specific growth rate of 0.12 h-l. Re-mutation of the mutant decreased the cell yield and maximum specific growth rate but increased the astaxanthm content. The use of mannitol or succinate as carbon sources enhanced pigmentation, yielding astaxanthin contents of 1973 pg g-1 and 1926 pg g-‘, respectively. The use of valine as sole nitrogen source also increased astaxanthin production, but severely decreased the maximum specific growth rate and cell yield coefficient. The optiium pH for growth of P. rhodoqmza was between pH 4.5 and 5.5, whereas the astaxanthin content remained constant above pH 3. Key words: Astaxanthin, mannitol, mutants, NTG, ~~~
r~~~zy~~, succinate, valine, yeast.
Astaxanthin (3,3’-dihydroxy-j$fl-carotene-4,4’-dione) is an ubiq~tous carotenoid pigment in the marine enviro~ent (Weedon I971), probably originating from certain algae, fungi and small crustacea and leading to the pigmentation of larger animals, including salmonids, via the food chain (Johnson & Lewis 1979; Johnson et al. 1980). An important factor affecting the consumer acceptance of salmonids is the colour of the flesh and, in the caseof commercial production in captivity, carotenoid pigments are included in the feed to obtain the desirable pigmentation (Johnson ef al. 1977). According to Johnson & An (1991), approximately 460,000 tonnes of farmed salmon will be produced by the end of the decade, while more than 250,000 tonnes of farmed trout were produced worldwide during 1987. It is estimated that more than 100,000 kg of carotenoid pigments might be required by the year 2000 for inclusion in fish feed (Johnson & An 1991). Only a few microorganisms synthesize astaxanthin (Nelis & De Leenheer 1989) of which Pha#a rhodozyma is a possible candidate for industrial-scale production becauseof its high astaxanthin content (Johnson & Lewis 1979).
P.S. Meyer, J.C. du Preer and S.G. Kilian are with the Department of Microbiology and Biochemistry, University of the Orange Free State, P.O. 60x 33% Bloemfontein 9300, Republic of South Africa; fax: (27) 51 482004. * Corresponding author. @ 7993 Rapid
514
Communications
World Ioumal
of Oxford
ofMicrobi&gy
Lfd
and Biotcchnolqy,
Vol 9, 1993
Hoffman-La Roche, Switzerland, markets a synthetic astaxanthin (‘Carophyll pink’) selling at approximately US $25oo/kg dry wt in beadlets containing 8% (w/w) astaxanthin, but it has not yet been approved by the United StatesFood and Drug Administration (FDA) (Johnson & An 1991). Astaxanthin is, therefore, the most costly component of salmonid diets (An ef al. ~991). Because of the high efficiency of astaxanthin transfer to salmonid flesh and the fact that a preparation of P. yh~~zy~ could also supply nutrients required for growth of the fish, the inclusion of the yeast in the fish feed might improve both the colour and flavour of pen-reared salmon more effectively than a synthetic feed additive (johnson et al. 1980). The astaxanthin content typical of the naturally-occurring P. rhodozyma strains would have to be increased to facilitate economic extraction (Johnson & An 1991). In this study, ~-methyl-~-nitro-~-nitroso~a~d~e (NTG) was chosen as mutagen because An et al. (1989) reported that UV light yielded no highly pigmented mutants and that ethyl meth~es~phonate (EMS) yielded mutants which reverted at a high frequency. The objective of this study was to obtain P. rhadozyma mutants with increased astaxanthin content and to determine the influence of various culture conditions, especially the effect of a wide variety of carbon and nitrogen sources, on astaxanthin production.
Astaxanthin-overproducing
Materials
and Methods
Yeast strains Phafia rhodozyma strains CBS 5905T
and IGC 4172 were from the culture collection of the Deparhnent of Microbiology and Biochemistry, University of the Orange Free State. All the other P. rhodozyma strains, designated VKPM, were from the National Collection of Industrial Microorganisms, Moscow. The strains were maintained on agar slants of YPG medium containing (g I-‘): glucose, 20; peptone, 10; yeast extract, 10; and agar, 20.
Media and Cultivation The strains and mutants were evaluated in 500-ml Erlenmeyer shake-flasks, equipped with side-arms to facilitate turbidimetric measurements, containing 50 ml YM medium (g 1-l: glucose, 10; peptone, 5: malt extract, 3; and yeast extract, 3). The yeasts were grown at 22 “C and pH 6 for 120 h, unless stated differently in the text, on a rotary shaker at 180 rev/min. Each 500 ml shake-flask received 1 ml inoculum from a 250 ml Erlenmeyer shake-flask containing 25 ml YM medium which had been incubated at 22 “C for 36 h. Yeast carbon base (Difco), with 100 mM MES and supplemented with various nitrogen sources to yield 75 mM nitrogen, was used to evaluate the influence of different nitrogen sources. The effect of various carbon sources was determined in YM medium in which the usual glucose was replaced with 10 g of the alternative carbon source 1-l. The effect of pH was determined by supplementing YM medium with 15 g KH,PO, I-’ and adjusting the pH prior to autoclaving.
Mutagenesis Mutagenesis was performed on freshly grown cells using NTG and antimycin A (Sigma) according to An et al. (1989). After exposure to NTG, cells were cultivated in YM medium on a rotary shaker (180 rev/min) overnight in the dark at 22 “C before plating on selective agar. Astaxanthin-overproducing mutants were selected by using b-ionone to suppress pigmentation (Lewis et al. 1990). B-Ionone was dissolved in ethanol and added to YM agar immediately before After 7 to 10 days of pouring plates to give 10e4 M p-ionone. incubation at 22 “C, the colonies which appeared the darkest were replica plated to YM plates as well as P-ionone-containing plates and incubated for a further 7 days. These plates were visually screened and the darkest pigmented colonies brought into pure cultures and evaluated in shake-flasks.
Analytical Methods The DMSO method, as described by Sedmak et al. (1990), was used to rupture cells prior to extraction of the carotenoids into either methanol or acetone. The total carotenoids were calculated using E:20M1 = 2100 (An et al. 1989). Astaxanthin wag determined by HPLC using a stainless steel Nova-Pak Cl8 (Waters Associates), reversed phase column (3.9 mm x 150 mm). The mobile phase was an isocratic solvent of methanol:acetonitrile (9:l v/v) at 1 ml mir-’ (Ben-Amotz et al. 1988). Authentic astaxanthin (Roche Products, Isando, Republic of South Africa) was used as external standard. A Beckman System Gold diode array detector module 168 with a programmable solvent module 126 or a Waters Liquid Chromatograph equipped with a Waters 501 HPLC pump, Waters WISP 7lOB autosampler and a Waters Lambda-Max Model 480 detector were used. Growth was monitored with a Klett-Summerson calorimeter (Klett, New York) using a No. 64 red filter. The side-arms on the
mutants
P. rhodozyma
flasks facilitated turbidimetric measurements and the determination of maximum specific growth rate. Dry cell mass was determined gravimetrically by drying centrifuged and washed samples to a constant mass at 105 “C. Sugars were analysed by HPLC according to Van Zyl et al. (1988). Individual carotenoids were analysed by TLC and identified by & values and cochromatography with b-carotene (Sigma) and trans-astaxanthin (Roche Products) standards (An et al. 1989).
Results
and Discussion
The naturally-occurring less
than
330
pg
Phafia rhodozyma strains astaxanthin
(g
dry
contained
cells)-’
and
the
specific growth rate did not exceed 0.2 h-’ (Table I). The total carotenoid content of 287 pg g-’ obtained with P. rhodozyma CBS 5905T (Table I) was similar to the total carotenoid content of 29.5 pg g-’ of P. rhodozyma UCF-FST 67-210 (also subcultured from strain CBS 5905T) reported by An et al. (1989). The low astaxanthin content of these strains is typical of the values reported by Fleno et al. (1988) and An et al. (1989) for wild strains. Strains CBS 5905T (which was the best strain initially available), VKPM Y1657, Y1660 and Y1673 were selected for mutagenesis. Phafia rhodozyma CBS 5905T yielded a mutant (M4) after the first mutation cycle with an astaxanthin content of 567 pg g-’ (Table 2). By successively selecting the mutant producing the most astaxanthin and subjecting it to further mutation cycles, the astaxanthin content of a mutant &I-3) derived from strain CBS 5905T was increased to 1392 pg g-‘. Mutant N9, which was the strain with the highest pigmentation, namely 2289 pg total pigment (g dry cells)-’ and 1834 lug astaxanthin (g dry cells)-‘, was isolated later (Table 2). /?-Ionone proved to be a powerful agent for the indication of over-producing mutants on agar maximum
plates
during
the
first
three
mutation
cycles,
but
failed
to
discriminate between mutants during the successive mutations. Mutant N9 was the highest astaxanthin producer obtained and further attempts to produce better mutants failed, probably because of the lack of a more selective screening procedure. The use of antimycin A did not yield higher astaxanthin-producing mutants (data not shown) despite the presence of highly-pigmented papillae arising after approximately 1 month of incubation, as described by An et al. (1989). With each mutation cycle, the growth rate and cell yield of the higher astaxanthin-containing mutants decreased with increasing astaxanthin content (Table 2). The decrease in cell yield might be a consequence of the higher astaxanthin content, since in these mutants less carbon and energy would be available for cell biosynthesis. A lower growth rate of such mutants was also observed by Johnson & An (1991) and might indicate that carotenoid production retards growth. The selected VKPM strains also yielded mutants with higher astaxanthin levels. However, these were
World Journal
ofMicrobiology
and Biotechnology, Vol 9, 1993
515
P.S.Meyer, JC. du Freezand S.G. K&m Table t s The eV8fUatfOIl of verfous totei earotenoid and 8staxanthbI Strain
maximum Spectfk growth rate @-‘I
CBS 6QO5T IGC 4172 VKPM YQO2T VKPM Y989 VKPM Y1562 VKPM Y1651 VKPM Yf653 VKPM Y1664 VKPM Y1655 VKPM Y1656 VKPM Y1657 VKPM Y1658 VKPM Y1669 VKPM Yl660 VKPM Y1661 VKPM Y1662 VKPM Y1663 VKPM Y1864 VKPM Yl665 VKPM Y1666 VKPM Y1667 VKPM Y1668 VKPM Y1669 VKPM Y1670 VKPM Y1671 VKPM Y1672 VKPM Yf673
#%?iiia Content.*
ri#of#oz$~3&3
Cell yield [g ceik
The strains were cultivated 22°C for120 h. The values are
Maximum speclttc growth rat% W-‘1
of growth,
Total carotenolds
(g SubStr8te
Ml
0.55 0.53 0.48 0.44 0.80 0.61 0.65 0.59 0.60 0.48 0.48 0.60 0.59 0.61 0.63 0.61 0.56 0.56 0.58 0.64 0.56 0.63 0.56 0.61 0.42 0.49 0.50
cus 93
e-3 287 330 353 318 348 338 277 276 334 386 464 268 304 402 316 294 330 341 281 292 291 295 306 297 360 311 412
204 254 261 222 217 253 227 206 232 214 327 202 228 300 225 245 222 267 232 256 218 219 218 252 256 277 320 I-’
at pW 6.0 and
~~~ rhtaioxyma Cgg 59osT totei carotenotd and astaxanthin
Cell yield [g cells (g substrate utlliz%d)-‘I
Totat
pfgment
@g ml-‘)
CBS 5905T M4 83-34 E3-29 Hl-27 J4-3 NQ
0.18 0.16 0.15 0.15 0.16 0.12 0.12
0.65 0.59 0.53 0.49 0.51 0.47 0.46
* The strains/mutan~ for 120 h. The vatues
were cultivated on YM medium containing are means of duplicate determinations.
cell ytefd,
Astaxanthin
in YM medium containing 10 g glucose means of duplicate determinations.
Tabfe 2. The evaluation of mutants derfved from mu~Uons with NT& in terms of growth, cell ytetd, ~r8in/m~t
in terms
utilized)-‘]
0.18 0.17 0.16 0.16 0.16 0.17 0.20 0.16 0.15 0.13 0.15 0.18 0.17 0.15 0.17 0.17 0.17 0.15 0.18 0.17 0.15 0.19 0.17 0.16 0.16 0.16 0.20
l
strains
1.58 4.49 4.66 5.92 7.75 7.93 10.12 10 g glucose
during soccesske content.* Astaxanthln
w B-7 @II s-9
287 746 889 1213 1523 1688 2289 I-’
204 667 773 1097 1176 1392 1834 at pH 6.0 and 22%
AsfAxanfkin-aveq7roduci,?g Table 3. The effect of dlfferent carbon content of Phaffla fhodozyma J4-3.* Carbon source (at 10 g I-‘)
L-Arabinose Cerlobiose Fructose Glucose Maltose M~nnOS~
Fiaffinose Starch Sucrose a-Trehalose Xylose Ethanol GfyCX3r0t
Mannitol o-Sorbitol Fumaric acid Glyoxylic acid cc-Ketoglutaric acid oL-Malic acid a-~ethyf-oft-f-giucoside Pyruvic acid Succinic acid ‘Growth occurred
was measured on acetic. citric
sources
Maximum specific growth rate (h-‘1 0.08 0.07 0.14 0.14 0.10 0.16 0.10 0.07 0.11 0.08 0.08 0.11 0.13 0.12 0.13 a.11 0.13 0.05 0.12 O.DT 0.07 0.05 in YM-based or oxaloacetic
on growth,
Biomass f.g 1-‘1
3.63 5.01 4.75 4.71 4.85 4.60 3.62 2.52 4.90 4.76 4.11 3.97 3&r& 4.71 5.25 1.17 1.26 2.03 1.23 1.53 1.15 2.37 medium acids.
unsuitable for further mutation due to a dramatic decrease in the cell yields and/or growth rates (data not shown). The total pigment contents of 1688 ,ug g-’ and 2289 @gg-’ of mutants J4-3 and N9, respectively, compared favourably with the values of 1050 pg g-” (An & Johnson 1990}, xooo gg 8-l (Lewis et al. 1990) and 1250 pg g-’ (An pi tll. 1991) for other mutants. An et al. (1989) obtained a mutant strain by successive mutagenesis claimed to consistently produce more than 2000 /~g astaxanthin (g yeast biomass)-‘. When P. &odozyma J4-3 was grown on different carbon sources (Table 31, the highest astaxanthin contents of 1973 pg g-’ and 1926 ,ug g -’ were obtained with mannitol and succinate as sole carbon sources, respectively. On succinate, however, the maximum specific growth rate and final biomass concentration were severely decreased. Significant increases in astaxanthin content were also observed with raffinose, L-arabinose, cellobiose, cc-ketoglutaric acid, maltose, sucrose, starch and a-trehalose as the respective carbon sources (Table 3). Johnson & Lewis (1979) atso reported higher astaxanthin contents when P, rkodozyma UCD 67-210 was grown on cellobiose, maltose, mannitol, succinate and sucrose. They suggested that the higher astaxanthin content obtained on succinate was due
after
total
pigment
and
Total pigment w g-7
astaxanthin
Astaxantbln (119 e-‘I
1814 1983 1485 1688 1940 1815 1795 2030 2007 2122 1623 1818 1688 2426 1463 1902 1716 2093 1459 1858 1831 2336 120 h at pH 5.5 and
P. rhodozyma mafan#s
1573 1607 1311 1392 1652 1300 1569 1653 1539 1775 1339 1285 1101 1973 1003 1004 1009 1652 668 1137 657 1926 22°C.
NO growth
to its direct assimilation into the tricarboxylic acid cycle, whereas cellobiose stimulated carotenoid production because it can be utilized only aerobically. The maximum specific growth rate of P. rkaduzyma J4-3 was decreased by 50% or more, relative to growth on glucose, when grown on ceIlobiose* &etogIutaric acid, ~-methyl-D-glucoside, pyruvic acid, succinate and starch (Table 3). Only on mannose did the maximum specific growth rate exceed that on glucose. In ~aemafocuc~ p&a/is, several intermediates of the TCA-cycle (succinate, fumarate, malate and oxalacetate) inhibited astaxanthin production (Kobayashi et aI. 1991), as was found here. These compounds, however, stimulated p-carotene production in Blakeslea brispora (Bj;irk & Neujahr 19693. Carotenoid synthesis was, however, stimulated by pyruvate and acetate in H. plttvialis (Kobayashi et al. 1991), whereas pyruvate did not enhance astaxanthin production by P. rkodozyma (Table 3). According to Goodwin f198trfs carbohydrates affect carotenoid production differently in different fungi. Johnson & Lewis (1979) and Nelis & De Leenheer (1989) reported that changing nitrogen source had no significant influence on pigment production. However, in the alga Dunaliella ferfioleda the nitrogen source exerted a significant effect on b-carotene production (Abalde & Eabregas 1991f.
P.S. Meyer, JC. du Preez and S.G. Kilian Table 4. The Influence of various Phaflle rhodozyma 54-L* Nltrogen source (at 75 mM nitrogen)
Peptone NH&l PJH&SOI NH,NO, V-M&O Ammonium Ammonium Ammonium Arginine Aspartate Glutamate Glycine lsoleucine Leucine Histidine Methionine Phenylalanine Valine
Maximum specific growth rate W’)
oxalate tartrate
Growth was measured at 22°C and pf-l 6.0
sources
after
120 h in yeast
on the growth,
Cell yield [g cells (g substrate utilized)-‘]
0.13 0.13 0.12 0.13 0.14 0.10 0.06 0.11 0.08 0.05 0.10 0.04 0.07 0.08 0.04 0.06 0.06 0.06
citrate
l
nitrogen
0.40 0.35 0.35 0.36 0.38 0.52 0.36 0.39 0.38 0.37 0.38 0.38 0.33 0.24 0.34 0.31 0.32 0.27 carbon
base
A major problem, when comparing the effect of different nitrogen sources on the growth of microorganisms, is the different patterns of pH change during cultivation (Lin & Demain 1991). According to Lin & Demain (1991), MOPS buffer stabilized the pH without affecting cell growth or pigment production in Monascus. However, MOPS is only active in the pH range of 6.5 to 8 and therefore MES, active in the pH range of 5.8 to 6.5, was used in this study. The use of 100 mM MES minimized the change in culture pH during growth; except when urea and ammonium citrate were used, the pH remained relatively constant during shake-flask cultivations (Table 4). Phafia rhodozyma J4-3 grown on yeast carbon base (Difco) with MES and peptone as nitrogen source yielded a total pigment content of 1549 pg g-i (Table 4). which compared well with the value of 1688 pg g-’ obtained with the same mutant on YM medium (Table 2). However, the astaxanthin content was lower (Table 4), indicating that MES and/or yeast carbon base negatively influenced astaxanthin production. The growth rate, total pigment content and astaxanthin content of cells grown on the different ammonium salts (NH&l, (NH&SO,, NH,NO,) and urea were similar, whereas ammonium citrate, ammonium oxalate and ammonium tartrate gave lower values (Table 4). The cell yield coefficients on carbon substrate [g cells (g carbon supplied)-‘] in the presence of different nitrogen sources (75 mu nitrogen) were similar for all the nitrogen sources,
518
World Journal
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Microbmlogy and Bmfechnology, Vol 9, 1993
(11.7 g I-‘),
total
pigment
Total
pigment
and
astaxanthln
content
of
Astaxanthln
Final
@!a g-7
019 g-7
PH
1549 1270 1432 1468 1401 1082 858 1170 1396 1193 1264 1795 1780 1762 1243 1897 1818 2018
865 737 798 793 701 657 583 589 693 624 494 669 884 772 555 935 837 822
5.8 5.3 5.3 5.3 7.1 7.1 5.8 5.6 5.7 6.0 6.2 5.8 5.4 5.5 5.4 5.5 5.9 5.5
with 100 mM MES as buffer,
in shake-flasks
except for the lower values obtained with valine and leucine and higher value on ammonium citrate. In Aspergilltis nidulans, Neurospora crassa and Saccharomyces cerevisiae, ammonium, glutamate and glutamine were the favoured nitrogen sources and the maximal growth rates were obtained with these sources (Jennings 1989). Glucose was completely utilized, except when L-histidine was the sole nitrogen source and approximately 45% glucose remained after 5 days of incubation (data not shown). Phafia rhodozyma also failed to utilize thymine in auxanograms (data not shown), indicating P. rhodozyma is probably unable to utilize nitrogen in ring structures as sole nitrogen source. The maximum specific growth rate was markedly lower with amino acids (especially with glycine, isoleucine, histidine, methionine, phenylalanine and valine) as the sole nitrogen source (Table 4), indicating that nitrogen utilization from these compounds was rate-limiting. The total pigment content obtained with glycine, isoleucine, leucine, methionine, phenylalanine and valine was higher than that obtained on the ammonium salts (Table 4) and the highest total pigment content of 2018 pg g-l was obtained with valine. Nelis & De Leenheer (1989) reported leucine and valine stimulated carotenoid formation in fungi. However, the low specific growth rate of 0.06 hh’, the lower biomass concentration produced (Table 4) and the cost factor rendered the use of valine and other amino acids as sole nitrogen source unsuitable for commercial use. Valine and leucine are precursors of fl-hydroxy-fi-methylglutaryl-
Astaxanfhin-ovenwodttcing
P. rhodozyma
muf~nfs
approximately nine times by repeated NTG mutagenesis, without the antimycin A exposure used by An ef al. (1989). The maximum specific growth rate and cell yield coefficient of these mutants decreased, however. The use of mannitol or succinate as sole carbon source and valine as sole nitrogen source yielded the highest astaxanthin contents. Changing pH values between pH 3.5 and 6 exerted only a slight effect on astaxanthin production but growth rate was affected much more; the optimum maximum specific growth rate was reached at pH 5.
Acknowledgements 0.15
0.12
0.09
^
This research was supported by grants from Analysis, Management and Systems Pty. Ltd. (AMS), Stellenbosch and the Foundation for Research Development. We thank J.P. van der Walt for advice and PJ. Botes for technical assistance with the chromatographic analyses.
f 0.06
0.03
g z t u
0 2.5
3.5
4.5
5.5
6.5
pH Figure 1. The effect of initial pH on the maximum specific growth rate (0). ceil yield coefficient (a), final pH (A). total pigment (D;& and astaxanthin (0) content of Phaffia fhodozyma J4-3 during shake-flask cultivation at 22 “C for 5 days. Initial
CoA, a key intermediate in isoprenoid synthesis, and this might explain their stimulatory effect on carotenoid production (Nelis & De Leenheer 1989). In Monascus, glutamate stimulated pigment production significantly (Lin & Demain 1991), whereas this amino acid has no significant effect on carotenoid production in P. rhudozyma (Table 4). During cultivation of p. rhodozyma in shake-flasks at different initial pH values, no significant difference was observed between the initial and final pH (Figure I). The op~~urn pH in terms of ma~mum specific growth rate was pH 5, with a sharp decrease below pH 4 (Figure I). The optimum pH in terms of the volumetric production of total pigment (pg ml-.‘) was pH 5, with a sharp decrease at pH 3. The astaxanthin content (pg g-i) similarly decreased at pH 3 but remained constant between pH 3.5 and pH 6 (Figure I). The cell yield coefficient remained constant between pH 5 and pH 6, but decreased linearly below pH 5 (Figure I). In contrast, Monascus biomass production increased with decreasing pH, with a concomitant decrease in the specific production of pigment (Lin & Demain 1991). In conclusion, our results show that the astaxanthin content of I? rhodozyma CBS 5905T was increased
References Abalde, J. & Fabregas, J. 1991 P-Carotene, vitamin C and vitamin E content of the marine microalga Duneli& terfiolecta cultured with different nitrogen sources. Bioresource Technology 38, 121-125.
An, G.-H., Bielich, H., Auerbach, R. & Johnson, E.A. 1991 Isolation and characterization of carotenoid hyperproducing mutants of yeast by flow cytometry and ceff sorting. Biotechnology 9,%--73. An, G.-H. & Johnson, E.A. 1990 Influence of light on growth and pigmentation of the yeast Pk~~~ rkuduzymff. Anfonie vary leeu~en~oek; ~~~~~ af ~icrobioIogy and ~olffgy 57, 19X-203. An, G.-H., Schuman, D.B. & Johnson, E.A. 1989 Isolation of Pka~a rkodozyma mutants with increased astaxanthin content. Applied and Environmental Microbiology 55, 116-124. Ben-Amotz, A., Lers, A. & Avron, M. 1988 Stereoisomers of p-carotene and phytoene in the alga Dunaliellu bard&l. Plant Physiology
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Bjork, L. & Neujahr, H.Y. 1969 Stimulation of p-carotene synthesis in Blukesleu trisporu by pyruvate and intermediates of the tricarboxylic acid (TCA-) cycle. Acfa Ckemice Scandinauicu 23‘2908-2909.
Fleno, B., Christensen, 1.& Larsen, R. 1988 Astax~thin-producing yeast cells, methods for their preparation and their use. Patent application no. RCT/DK88/00068 filed by Danisco Bioteknologi A/S in Copenhagen, Denmark. Goodwin, T.W. 1980 Fungi. In The Biuckemisfry of fke Carofenoids, ed Goodwin, T.W. pp. 257-283. London and New York: Chapman and Hall. Jennings, D.H. 1989 Some perspectives on nitrogen and phosphorus metabolism in fungi. In Nitrogen, Phosphorus and Strlpkur Ufilizafion by Fungi, eds Boddy, L., Marchant, R. & Read, DJ. pp. l-32. Cambridge: Cambridge University Press. Johnson, E.A. & An, G.-H. 1991 Astaxanthin from microbial sources. Crificai Rev&us in ~~~feck~ff~~gy 11, 297-326. Johnson, E.A., Conklin, D.E. & Lewis, M.J. 1977 The yeast Plugs r~o~zy~a as a dietary pigment source for salmonids and crustaceans. $~urmai Fisheries Research Board of Canada 34, 24x7-2421.
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P.S. A4eyer, J.C. Au Preez ana’ 5.G. K&an Johnson, E.A. & Lewis, MJ. 1979 Astaxanthin formation by the yeast Pan rhodozyma. fc~urnul of General ~~ffubi~~ogy 115, 173-183. Johnson, E.A., Villa, T.G. & Lewis, M.J. 1980 Pita rh~dozy~a as an astaxanthin source in salmonid diets. Aqwculture 20, 123-134. Kobayashi, M., Kakizono, T. & Nagai, S. 1991 Astaxanthin production by a green alga, ~~~fococc~ pl~vial~ accompanied with morphological changes in acetate media. Journa/ of Fermentation and Bioengineering 71, 335-339. Lewis, M.J., Ragot, N., Berlant, M.C. & Miranda, M. 1990 Selection of astaxanthin-overproducing mutants of Phafia rhodozyma with @-ionone. Applied and Environ~~f~~ ~icr~biol5gy 56, 29442945. Lin, T.F. & Demain, A.L. 1991 Effect of nutrition of Munascus sp. on formation of red pigments. Applied Microbiology and Eio~echnu~Q~
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Weedon, B.C.L. 1971 Occurrence. In Curotenoids, 29-59. Basel: Birkhauser Verlag.
(Received ..% ~a~~~
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17,~
7-369.
ed Isler, 0. pp
1993)
Journal
of Microbiology
and Biotechnology
9, 514-520
Selection and evaluation astaxanthin-overproducing of Phaffia rhodozyma
of mutants
P.S. Meyer, J.C. du Preez* and S.G. Kilian Mutagenesis of PhaBa rhodozyma with NTG yielded a mutant with an astaxanthin content of 1688 pg (g dry biomass)-‘, a cell yield coefficient of 0.47 on glucose and a maximum specific growth rate of 0.12 h-l. Re-mutation of the mutant decreased the cell yield and maximum specific growth rate but increased the astaxanthm content. The use of mannitol or succinate as carbon sources enhanced pigmentation, yielding astaxanthin contents of 1973 pg g-1 and 1926 pg g-‘, respectively. The use of valine as sole nitrogen source also increased astaxanthin production, but severely decreased the maximum specific growth rate and cell yield coefficient. The optiium pH for growth of P. rhodoqmza was between pH 4.5 and 5.5, whereas the astaxanthin content remained constant above pH 3. Key words: Astaxanthin, mannitol, mutants, NTG, ~~~
r~~~zy~~, succinate, valine, yeast.
Astaxanthin (3,3’-dihydroxy-j$fl-carotene-4,4’-dione) is an ubiq~tous carotenoid pigment in the marine enviro~ent (Weedon I971), probably originating from certain algae, fungi and small crustacea and leading to the pigmentation of larger animals, including salmonids, via the food chain (Johnson & Lewis 1979; Johnson et al. 1980). An important factor affecting the consumer acceptance of salmonids is the colour of the flesh and, in the caseof commercial production in captivity, carotenoid pigments are included in the feed to obtain the desirable pigmentation (Johnson ef al. 1977). According to Johnson & An (1991), approximately 460,000 tonnes of farmed salmon will be produced by the end of the decade, while more than 250,000 tonnes of farmed trout were produced worldwide during 1987. It is estimated that more than 100,000 kg of carotenoid pigments might be required by the year 2000 for inclusion in fish feed (Johnson & An 1991). Only a few microorganisms synthesize astaxanthin (Nelis & De Leenheer 1989) of which Pha#a rhodozyma is a possible candidate for industrial-scale production becauseof its high astaxanthin content (Johnson & Lewis 1979).
P.S. Meyer, J.C. du Preer and S.G. Kilian are with the Department of Microbiology and Biochemistry, University of the Orange Free State, P.O. 60x 33% Bloemfontein 9300, Republic of South Africa; fax: (27) 51 482004. * Corresponding author. @ 7993 Rapid
514
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ofMicrobi&gy
Lfd
and Biotcchnolqy,
Vol 9, 1993
Hoffman-La Roche, Switzerland, markets a synthetic astaxanthin (‘Carophyll pink’) selling at approximately US $25oo/kg dry wt in beadlets containing 8% (w/w) astaxanthin, but it has not yet been approved by the United StatesFood and Drug Administration (FDA) (Johnson & An 1991). Astaxanthin is, therefore, the most costly component of salmonid diets (An ef al. ~991). Because of the high efficiency of astaxanthin transfer to salmonid flesh and the fact that a preparation of P. yh~~zy~ could also supply nutrients required for growth of the fish, the inclusion of the yeast in the fish feed might improve both the colour and flavour of pen-reared salmon more effectively than a synthetic feed additive (johnson et al. 1980). The astaxanthin content typical of the naturally-occurring P. rhodozyma strains would have to be increased to facilitate economic extraction (Johnson & An 1991). In this study, ~-methyl-~-nitro-~-nitroso~a~d~e (NTG) was chosen as mutagen because An et al. (1989) reported that UV light yielded no highly pigmented mutants and that ethyl meth~es~phonate (EMS) yielded mutants which reverted at a high frequency. The objective of this study was to obtain P. rhadozyma mutants with increased astaxanthin content and to determine the influence of various culture conditions, especially the effect of a wide variety of carbon and nitrogen sources, on astaxanthin production.
Astaxanthin-overproducing
Materials
and Methods
Yeast strains Phafia rhodozyma strains CBS 5905T
and IGC 4172 were from the culture collection of the Deparhnent of Microbiology and Biochemistry, University of the Orange Free State. All the other P. rhodozyma strains, designated VKPM, were from the National Collection of Industrial Microorganisms, Moscow. The strains were maintained on agar slants of YPG medium containing (g I-‘): glucose, 20; peptone, 10; yeast extract, 10; and agar, 20.
Media and Cultivation The strains and mutants were evaluated in 500-ml Erlenmeyer shake-flasks, equipped with side-arms to facilitate turbidimetric measurements, containing 50 ml YM medium (g 1-l: glucose, 10; peptone, 5: malt extract, 3; and yeast extract, 3). The yeasts were grown at 22 “C and pH 6 for 120 h, unless stated differently in the text, on a rotary shaker at 180 rev/min. Each 500 ml shake-flask received 1 ml inoculum from a 250 ml Erlenmeyer shake-flask containing 25 ml YM medium which had been incubated at 22 “C for 36 h. Yeast carbon base (Difco), with 100 mM MES and supplemented with various nitrogen sources to yield 75 mM nitrogen, was used to evaluate the influence of different nitrogen sources. The effect of various carbon sources was determined in YM medium in which the usual glucose was replaced with 10 g of the alternative carbon source 1-l. The effect of pH was determined by supplementing YM medium with 15 g KH,PO, I-’ and adjusting the pH prior to autoclaving.
Mutagenesis Mutagenesis was performed on freshly grown cells using NTG and antimycin A (Sigma) according to An et al. (1989). After exposure to NTG, cells were cultivated in YM medium on a rotary shaker (180 rev/min) overnight in the dark at 22 “C before plating on selective agar. Astaxanthin-overproducing mutants were selected by using b-ionone to suppress pigmentation (Lewis et al. 1990). B-Ionone was dissolved in ethanol and added to YM agar immediately before After 7 to 10 days of pouring plates to give 10e4 M p-ionone. incubation at 22 “C, the colonies which appeared the darkest were replica plated to YM plates as well as P-ionone-containing plates and incubated for a further 7 days. These plates were visually screened and the darkest pigmented colonies brought into pure cultures and evaluated in shake-flasks.
Analytical Methods The DMSO method, as described by Sedmak et al. (1990), was used to rupture cells prior to extraction of the carotenoids into either methanol or acetone. The total carotenoids were calculated using E:20M1 = 2100 (An et al. 1989). Astaxanthin wag determined by HPLC using a stainless steel Nova-Pak Cl8 (Waters Associates), reversed phase column (3.9 mm x 150 mm). The mobile phase was an isocratic solvent of methanol:acetonitrile (9:l v/v) at 1 ml mir-’ (Ben-Amotz et al. 1988). Authentic astaxanthin (Roche Products, Isando, Republic of South Africa) was used as external standard. A Beckman System Gold diode array detector module 168 with a programmable solvent module 126 or a Waters Liquid Chromatograph equipped with a Waters 501 HPLC pump, Waters WISP 7lOB autosampler and a Waters Lambda-Max Model 480 detector were used. Growth was monitored with a Klett-Summerson calorimeter (Klett, New York) using a No. 64 red filter. The side-arms on the
mutants
P. rhodozyma
flasks facilitated turbidimetric measurements and the determination of maximum specific growth rate. Dry cell mass was determined gravimetrically by drying centrifuged and washed samples to a constant mass at 105 “C. Sugars were analysed by HPLC according to Van Zyl et al. (1988). Individual carotenoids were analysed by TLC and identified by & values and cochromatography with b-carotene (Sigma) and trans-astaxanthin (Roche Products) standards (An et al. 1989).
Results
and Discussion
The naturally-occurring less
than
330
pg
Phafia rhodozyma strains astaxanthin
(g
dry
contained
cells)-’
and
the
specific growth rate did not exceed 0.2 h-’ (Table I). The total carotenoid content of 287 pg g-’ obtained with P. rhodozyma CBS 5905T (Table I) was similar to the total carotenoid content of 29.5 pg g-’ of P. rhodozyma UCF-FST 67-210 (also subcultured from strain CBS 5905T) reported by An et al. (1989). The low astaxanthin content of these strains is typical of the values reported by Fleno et al. (1988) and An et al. (1989) for wild strains. Strains CBS 5905T (which was the best strain initially available), VKPM Y1657, Y1660 and Y1673 were selected for mutagenesis. Phafia rhodozyma CBS 5905T yielded a mutant (M4) after the first mutation cycle with an astaxanthin content of 567 pg g-’ (Table 2). By successively selecting the mutant producing the most astaxanthin and subjecting it to further mutation cycles, the astaxanthin content of a mutant &I-3) derived from strain CBS 5905T was increased to 1392 pg g-‘. Mutant N9, which was the strain with the highest pigmentation, namely 2289 pg total pigment (g dry cells)-’ and 1834 lug astaxanthin (g dry cells)-‘, was isolated later (Table 2). /?-Ionone proved to be a powerful agent for the indication of over-producing mutants on agar maximum
plates
during
the
first
three
mutation
cycles,
but
failed
to
discriminate between mutants during the successive mutations. Mutant N9 was the highest astaxanthin producer obtained and further attempts to produce better mutants failed, probably because of the lack of a more selective screening procedure. The use of antimycin A did not yield higher astaxanthin-producing mutants (data not shown) despite the presence of highly-pigmented papillae arising after approximately 1 month of incubation, as described by An et al. (1989). With each mutation cycle, the growth rate and cell yield of the higher astaxanthin-containing mutants decreased with increasing astaxanthin content (Table 2). The decrease in cell yield might be a consequence of the higher astaxanthin content, since in these mutants less carbon and energy would be available for cell biosynthesis. A lower growth rate of such mutants was also observed by Johnson & An (1991) and might indicate that carotenoid production retards growth. The selected VKPM strains also yielded mutants with higher astaxanthin levels. However, these were
World Journal
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and Biotechnology, Vol 9, 1993
515
P.S.Meyer, JC. du Freezand S.G. K&m Table t s The eV8fUatfOIl of verfous totei earotenoid and 8staxanthbI Strain
maximum Spectfk growth rate @-‘I
CBS 6QO5T IGC 4172 VKPM YQO2T VKPM Y989 VKPM Y1562 VKPM Y1651 VKPM Yf653 VKPM Y1664 VKPM Y1655 VKPM Y1656 VKPM Y1657 VKPM Y1658 VKPM Y1669 VKPM Yl660 VKPM Y1661 VKPM Y1662 VKPM Y1663 VKPM Y1864 VKPM Yl665 VKPM Y1666 VKPM Y1667 VKPM Y1668 VKPM Y1669 VKPM Y1670 VKPM Y1671 VKPM Y1672 VKPM Yf673
#%?iiia Content.*
ri#of#oz$~3&3
Cell yield [g ceik
The strains were cultivated 22°C for120 h. The values are
Maximum speclttc growth rat% W-‘1
of growth,
Total carotenolds
(g SubStr8te
Ml
0.55 0.53 0.48 0.44 0.80 0.61 0.65 0.59 0.60 0.48 0.48 0.60 0.59 0.61 0.63 0.61 0.56 0.56 0.58 0.64 0.56 0.63 0.56 0.61 0.42 0.49 0.50
cus 93
e-3 287 330 353 318 348 338 277 276 334 386 464 268 304 402 316 294 330 341 281 292 291 295 306 297 360 311 412
204 254 261 222 217 253 227 206 232 214 327 202 228 300 225 245 222 267 232 256 218 219 218 252 256 277 320 I-’
at pW 6.0 and
~~~ rhtaioxyma Cgg 59osT totei carotenotd and astaxanthin
Cell yield [g cells (g substrate utlliz%d)-‘I
Totat
pfgment
@g ml-‘)
CBS 5905T M4 83-34 E3-29 Hl-27 J4-3 NQ
0.18 0.16 0.15 0.15 0.16 0.12 0.12
0.65 0.59 0.53 0.49 0.51 0.47 0.46
* The strains/mutan~ for 120 h. The vatues
were cultivated on YM medium containing are means of duplicate determinations.
cell ytefd,
Astaxanthin
in YM medium containing 10 g glucose means of duplicate determinations.
Tabfe 2. The evaluation of mutants derfved from mu~Uons with NT& in terms of growth, cell ytetd, ~r8in/m~t
in terms
utilized)-‘]
0.18 0.17 0.16 0.16 0.16 0.17 0.20 0.16 0.15 0.13 0.15 0.18 0.17 0.15 0.17 0.17 0.17 0.15 0.18 0.17 0.15 0.19 0.17 0.16 0.16 0.16 0.20
l
strains
1.58 4.49 4.66 5.92 7.75 7.93 10.12 10 g glucose
during soccesske content.* Astaxanthln
w B-7 @II s-9
287 746 889 1213 1523 1688 2289 I-’
204 667 773 1097 1176 1392 1834 at pH 6.0 and 22%
AsfAxanfkin-aveq7roduci,?g Table 3. The effect of dlfferent carbon content of Phaffla fhodozyma J4-3.* Carbon source (at 10 g I-‘)
L-Arabinose Cerlobiose Fructose Glucose Maltose M~nnOS~
Fiaffinose Starch Sucrose a-Trehalose Xylose Ethanol GfyCX3r0t
Mannitol o-Sorbitol Fumaric acid Glyoxylic acid cc-Ketoglutaric acid oL-Malic acid a-~ethyf-oft-f-giucoside Pyruvic acid Succinic acid ‘Growth occurred
was measured on acetic. citric
sources
Maximum specific growth rate (h-‘1 0.08 0.07 0.14 0.14 0.10 0.16 0.10 0.07 0.11 0.08 0.08 0.11 0.13 0.12 0.13 a.11 0.13 0.05 0.12 O.DT 0.07 0.05 in YM-based or oxaloacetic
on growth,
Biomass f.g 1-‘1
3.63 5.01 4.75 4.71 4.85 4.60 3.62 2.52 4.90 4.76 4.11 3.97 3&r& 4.71 5.25 1.17 1.26 2.03 1.23 1.53 1.15 2.37 medium acids.
unsuitable for further mutation due to a dramatic decrease in the cell yields and/or growth rates (data not shown). The total pigment contents of 1688 ,ug g-’ and 2289 @gg-’ of mutants J4-3 and N9, respectively, compared favourably with the values of 1050 pg g-” (An & Johnson 1990}, xooo gg 8-l (Lewis et al. 1990) and 1250 pg g-’ (An pi tll. 1991) for other mutants. An et al. (1989) obtained a mutant strain by successive mutagenesis claimed to consistently produce more than 2000 /~g astaxanthin (g yeast biomass)-‘. When P. &odozyma J4-3 was grown on different carbon sources (Table 31, the highest astaxanthin contents of 1973 pg g-’ and 1926 ,ug g -’ were obtained with mannitol and succinate as sole carbon sources, respectively. On succinate, however, the maximum specific growth rate and final biomass concentration were severely decreased. Significant increases in astaxanthin content were also observed with raffinose, L-arabinose, cellobiose, cc-ketoglutaric acid, maltose, sucrose, starch and a-trehalose as the respective carbon sources (Table 3). Johnson & Lewis (1979) atso reported higher astaxanthin contents when P, rkodozyma UCD 67-210 was grown on cellobiose, maltose, mannitol, succinate and sucrose. They suggested that the higher astaxanthin content obtained on succinate was due
after
total
pigment
and
Total pigment w g-7
astaxanthin
Astaxantbln (119 e-‘I
1814 1983 1485 1688 1940 1815 1795 2030 2007 2122 1623 1818 1688 2426 1463 1902 1716 2093 1459 1858 1831 2336 120 h at pH 5.5 and
P. rhodozyma mafan#s
1573 1607 1311 1392 1652 1300 1569 1653 1539 1775 1339 1285 1101 1973 1003 1004 1009 1652 668 1137 657 1926 22°C.
NO growth
to its direct assimilation into the tricarboxylic acid cycle, whereas cellobiose stimulated carotenoid production because it can be utilized only aerobically. The maximum specific growth rate of P. rkaduzyma J4-3 was decreased by 50% or more, relative to growth on glucose, when grown on ceIlobiose* &etogIutaric acid, ~-methyl-D-glucoside, pyruvic acid, succinate and starch (Table 3). Only on mannose did the maximum specific growth rate exceed that on glucose. In ~aemafocuc~ p&a/is, several intermediates of the TCA-cycle (succinate, fumarate, malate and oxalacetate) inhibited astaxanthin production (Kobayashi et aI. 1991), as was found here. These compounds, however, stimulated p-carotene production in Blakeslea brispora (Bj;irk & Neujahr 19693. Carotenoid synthesis was, however, stimulated by pyruvate and acetate in H. plttvialis (Kobayashi et al. 1991), whereas pyruvate did not enhance astaxanthin production by P. rkodozyma (Table 3). According to Goodwin f198trfs carbohydrates affect carotenoid production differently in different fungi. Johnson & Lewis (1979) and Nelis & De Leenheer (1989) reported that changing nitrogen source had no significant influence on pigment production. However, in the alga Dunaliella ferfioleda the nitrogen source exerted a significant effect on b-carotene production (Abalde & Eabregas 1991f.
P.S. Meyer, JC. du Preez and S.G. Kilian Table 4. The Influence of various Phaflle rhodozyma 54-L* Nltrogen source (at 75 mM nitrogen)
Peptone NH&l PJH&SOI NH,NO, V-M&O Ammonium Ammonium Ammonium Arginine Aspartate Glutamate Glycine lsoleucine Leucine Histidine Methionine Phenylalanine Valine
Maximum specific growth rate W’)
oxalate tartrate
Growth was measured at 22°C and pf-l 6.0
sources
after
120 h in yeast
on the growth,
Cell yield [g cells (g substrate utilized)-‘]
0.13 0.13 0.12 0.13 0.14 0.10 0.06 0.11 0.08 0.05 0.10 0.04 0.07 0.08 0.04 0.06 0.06 0.06
citrate
l
nitrogen
0.40 0.35 0.35 0.36 0.38 0.52 0.36 0.39 0.38 0.37 0.38 0.38 0.33 0.24 0.34 0.31 0.32 0.27 carbon
base
A major problem, when comparing the effect of different nitrogen sources on the growth of microorganisms, is the different patterns of pH change during cultivation (Lin & Demain 1991). According to Lin & Demain (1991), MOPS buffer stabilized the pH without affecting cell growth or pigment production in Monascus. However, MOPS is only active in the pH range of 6.5 to 8 and therefore MES, active in the pH range of 5.8 to 6.5, was used in this study. The use of 100 mM MES minimized the change in culture pH during growth; except when urea and ammonium citrate were used, the pH remained relatively constant during shake-flask cultivations (Table 4). Phafia rhodozyma J4-3 grown on yeast carbon base (Difco) with MES and peptone as nitrogen source yielded a total pigment content of 1549 pg g-i (Table 4). which compared well with the value of 1688 pg g-’ obtained with the same mutant on YM medium (Table 2). However, the astaxanthin content was lower (Table 4), indicating that MES and/or yeast carbon base negatively influenced astaxanthin production. The growth rate, total pigment content and astaxanthin content of cells grown on the different ammonium salts (NH&l, (NH&SO,, NH,NO,) and urea were similar, whereas ammonium citrate, ammonium oxalate and ammonium tartrate gave lower values (Table 4). The cell yield coefficients on carbon substrate [g cells (g carbon supplied)-‘] in the presence of different nitrogen sources (75 mu nitrogen) were similar for all the nitrogen sources,
518
World Journal
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Microbmlogy and Bmfechnology, Vol 9, 1993
(11.7 g I-‘),
total
pigment
Total
pigment
and
astaxanthln
content
of
Astaxanthln
Final
@!a g-7
019 g-7
PH
1549 1270 1432 1468 1401 1082 858 1170 1396 1193 1264 1795 1780 1762 1243 1897 1818 2018
865 737 798 793 701 657 583 589 693 624 494 669 884 772 555 935 837 822
5.8 5.3 5.3 5.3 7.1 7.1 5.8 5.6 5.7 6.0 6.2 5.8 5.4 5.5 5.4 5.5 5.9 5.5
with 100 mM MES as buffer,
in shake-flasks
except for the lower values obtained with valine and leucine and higher value on ammonium citrate. In Aspergilltis nidulans, Neurospora crassa and Saccharomyces cerevisiae, ammonium, glutamate and glutamine were the favoured nitrogen sources and the maximal growth rates were obtained with these sources (Jennings 1989). Glucose was completely utilized, except when L-histidine was the sole nitrogen source and approximately 45% glucose remained after 5 days of incubation (data not shown). Phafia rhodozyma also failed to utilize thymine in auxanograms (data not shown), indicating P. rhodozyma is probably unable to utilize nitrogen in ring structures as sole nitrogen source. The maximum specific growth rate was markedly lower with amino acids (especially with glycine, isoleucine, histidine, methionine, phenylalanine and valine) as the sole nitrogen source (Table 4), indicating that nitrogen utilization from these compounds was rate-limiting. The total pigment content obtained with glycine, isoleucine, leucine, methionine, phenylalanine and valine was higher than that obtained on the ammonium salts (Table 4) and the highest total pigment content of 2018 pg g-l was obtained with valine. Nelis & De Leenheer (1989) reported leucine and valine stimulated carotenoid formation in fungi. However, the low specific growth rate of 0.06 hh’, the lower biomass concentration produced (Table 4) and the cost factor rendered the use of valine and other amino acids as sole nitrogen source unsuitable for commercial use. Valine and leucine are precursors of fl-hydroxy-fi-methylglutaryl-
Astaxanfhin-ovenwodttcing
P. rhodozyma
muf~nfs
approximately nine times by repeated NTG mutagenesis, without the antimycin A exposure used by An ef al. (1989). The maximum specific growth rate and cell yield coefficient of these mutants decreased, however. The use of mannitol or succinate as sole carbon source and valine as sole nitrogen source yielded the highest astaxanthin contents. Changing pH values between pH 3.5 and 6 exerted only a slight effect on astaxanthin production but growth rate was affected much more; the optimum maximum specific growth rate was reached at pH 5.
Acknowledgements 0.15
0.12
0.09
^
This research was supported by grants from Analysis, Management and Systems Pty. Ltd. (AMS), Stellenbosch and the Foundation for Research Development. We thank J.P. van der Walt for advice and PJ. Botes for technical assistance with the chromatographic analyses.
f 0.06
0.03
g z t u
0 2.5
3.5
4.5
5.5
6.5
pH Figure 1. The effect of initial pH on the maximum specific growth rate (0). ceil yield coefficient (a), final pH (A). total pigment (D;& and astaxanthin (0) content of Phaffia fhodozyma J4-3 during shake-flask cultivation at 22 “C for 5 days. Initial
CoA, a key intermediate in isoprenoid synthesis, and this might explain their stimulatory effect on carotenoid production (Nelis & De Leenheer 1989). In Monascus, glutamate stimulated pigment production significantly (Lin & Demain 1991), whereas this amino acid has no significant effect on carotenoid production in P. rhudozyma (Table 4). During cultivation of p. rhodozyma in shake-flasks at different initial pH values, no significant difference was observed between the initial and final pH (Figure I). The op~~urn pH in terms of ma~mum specific growth rate was pH 5, with a sharp decrease below pH 4 (Figure I). The optimum pH in terms of the volumetric production of total pigment (pg ml-.‘) was pH 5, with a sharp decrease at pH 3. The astaxanthin content (pg g-i) similarly decreased at pH 3 but remained constant between pH 3.5 and pH 6 (Figure I). The cell yield coefficient remained constant between pH 5 and pH 6, but decreased linearly below pH 5 (Figure I). In contrast, Monascus biomass production increased with decreasing pH, with a concomitant decrease in the specific production of pigment (Lin & Demain 1991). In conclusion, our results show that the astaxanthin content of I? rhodozyma CBS 5905T was increased
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