Fed-batch and continuous culture ofPhaffia rhodozyma (Xanthophyllomyces dendrorhous)

The basidiomycetous yeastPhaffia rhodozyma was grown in batch, fed-batch and continuous culture, and some parameters governing growth and total carotenoid production were determined.     

Effect of squalene on astaxanthin production by a mutant of Phaffia rhodozyma

The effect of different sesquiterpenes on carotenoid synthesis in Phaffia rhodozyma was studied. Addition of squalene to the culture medium resulted in a decrease in the echinenone and trans-astaxanthin concentrations, whereas -carotene remained unchanged. The role of squalene as an inhibitor of ketocarotenoid synthesis in Ph. rhodozyma is discussed.     

High concentrations of biotechnologically produced astaxanthin by lowering pH in a <Emphasis Type="Italic">Phaffia rhodozyma</Emphasis> bioprocess

Astaxanthin additions to animal diets predominantly serve as colorization aid to satisfy consumer expectations and desire for a consistent product with familiar coloration, e.g. the characteristic pink colorization of the flesh of species being produced by aquaculture. The heterobasidiomycetous yeast Phaffia rhodozyma (Xanthophyllomyces dendrorhous) can be used as natural feed source of astaxanthin. However, currently, the majority of astaxanthin used for the feed market is produced by chemical synthesis. We present a further step in direction of a competitive production of natural astaxanthin in an optimized bioprocess with non-genetically modified Phaffia rhodozyma. After medium optimization AXJ-20, a mutant strain of P. rhodozyma wild-type strain ATCC 96594, was able to grow to a cell dry weight concentration of over 114 g per kg of culture broth in a fed-batch process. In this bioprocess, where pH was lowered from 5.5 to 3.5 during the maturation phase, AXJ-20 produced the highest value reported for astaxanthin production with P. rhodozyma up to now: 0.7 g astaxanthin per kg of culture broth with a space-time-yield of 3.3 mg astaxanthin per kg of culture broth per hour. Lowering the pH during the bioprocess and increasing trace element and vitamin concentrations prevented loss of cell dry weight concentration in the maturation phase and proved to be critical for astaxanthin concentration and purity.     

Astaxanthin production by a Phaffia rhodozyma mutant on grape juice

During fermenter cultivation of Phaffia rhodozyma on a grape juice medium, the presence of glucose initially delayed fructose utilization, although fructose was consumed before glucose depletion. Total pigment and astaxanthin production were growth associated and reached maximum values of 15.9 g/ml and 9.8 g/ml, respectively, after depletion of the carbon source. The total cellular pigment and astaxanthin content increased during the stationary growth phase due to a decrease in biomass, reaching final values of 2120 g/g and 1350 g/g, respectively, without the volumetric concentration in the culture changing. The final cell yield was 0.33 g/g sugar utilized. High sugar concentrations in shake-flasks as well as O₂ limitation decreased the astaxanthin content of the cells. Addition of yeast extract to a grape juice minimal medium markedly increased the maximum specific growth rate, total pigment and astaxanthin content of the cells. An excess of ammonia decreased the intracellular astaxanthin content, which reached a maximal value in cultures with no residual glucose or ammonia.The authors are with the Department of Microbiology and Biochemistry, University of the Orange Free State, P.O. Box 339, Bloemfontein 9300, South Africa;     

Mevalonic acid increases trans-astaxanthin and carotenoid biosynthesis in Phaffia rhodozyma

Summary Mevalonic acid has been tested as enhancer of pigment biosynthesis in wild-type Phaffia rhodozyma. The addition of 0.1% mevalonic acid to the culture media stimulated both trans-astaxanthin and total carotenoids biosynthesis, with average increases by ca 400%.     

A factorial approach for a sugarcane juice-based low cost culture medium: increasing the astaxanthin production by the red yeast Phaffia rhodozyma

A sugarcane juice-based low cost culture medium was previously explored to produce the carotenoid pigment astaxanthin in liquid culture by the red yeast Phaffia rhodozyma (1300?μg astaxanthin/g of dry yeast and 6500?μg/l whole culture medium). Two peculiar limitations in Phaffia are growth temperature (<26?°C) and lack of sugar osmotolerance. Two advantages are the wide biochemical ability for the assimilation and metabolization of disaccharides and the prompt utilization of simple nitrogen sources. For instance, the sucrolytic/ureolytic enzymatic activities deserves exploration. In order to improve the culture medium composition and the conditions of fermentation for highly oxygenated carotenoids (e.g., astaxanthin) a study was carried out with a factorial design in two steps. As a first step, the production of astaxanthin was studied as a function of the nutrient concentration levels and their interactions. The production increase (μg/l) obtained was 23.0% but at the expense of 16.0% pigment content decrease (μg/g). In the second step, the variables pH and agitation level (OTR, oxygen transfer rate) were optimized and then, both goals were attained: the increase of pigment content (418?μg astaxanthin/g of yeast) as well as the absolute pigment production enhancement (1987?μg/l).     

Production of carotenoids byPhaffia rhodozyma grown on media composed of corn wet-milling co-products

Summary Natural isolates of the carotenoid-producing yeastPhaffia rhodozyma were analyzed for their ability to grow and to produce carotenoids in culture media composed exclusively of co-products of corn wet-milling for fuel ethanol production. FiveP. rhodozyma strains were tested for biomass produced (dry weight) and carotenoid yield. Six co-products were examined, ranging in cost from approximately $0.02 per kg to $0.11 per kg, all less expensive than conventional or agricultural growth substrates previously tested. The three co-products allowing the greatest accumulation of biomass and carotenoids byP. rhodozyma were thin stillage (TS), corn condensed distiller's solubles (CCDS) and corn gluten feed (CGF). Of the medium compositions tested, 10–15% CGF, 70% TS and 6–8% CCDS generally allowed maximum carotenoid production. Cultures grown in these three media produced up to 65%. 148% and 104% of the carotenoid yield per ml of yeast extract/malt extract (YM) cultures, respectively. Under the conditions tested, this was at an approximate medium cost of $0.67 per g carotenoids for CCDS and $0.73 per g for CGF as compared to $385.00 per g for YM. These results indicate that certain co-products of corn wet-milling can serve, at the appropriate concentration, as efficient, economical substrates for growth and carotenoid production byPhaffia rhodozyma.The mention of firm names or trade products does not imply that they are endorsed or recommended by the US Department of Agriculture over other firms or similar products not mentioned.     

An industrial medium for improved production of carotenoids from a mutant strain of Phaffia rhodozyma

A medium based on less expensive nutrient sources, such as corn starch hydrolyzate (hydrol), corn steep liquor (CSL), urea and potassium phosphate was used for the growth of the yeast Phaffia rhodozyma 2A2N strain. A central composite experimental design has been employed to derive a statistical model on the effect of hydrol and CSL on carotenoid production. An initial concentration of sugars as glucose equivalent 73?g/l in hydrol and 43?g/l CSL were found optimal for the maximization of final carotenoid production in shake flask cultures. The carotenoid production was increased by adding urea and phosphate sources. Laboratory scale fermentation was performed with the optimized medium and total carotenoid production of 52.4?mg/l was obtained using constant fed-batch culture.     

Relationship between astaxanthin production and the intensity of anabolic processes in the yeast <Emphasis Type="Italic">Phaffia rhodozyma</Emphasis>

The astaxanthin synthesis in the yeast Phaffia rhodozyma was shown to depend on the rate of growth occurring in the first two days of cultivation. The growth rate of the yeast culture studied was preset by the cultivation conditions, among which the C : N ratio was decisive. The intense anabolic processes coupled with active culture growth during the first 24 h significantly inhibited the synthesis of the key enzymes involved in astaxanthin synthesis, which led to a marked decrease in the carotenoid production. It was demonstrated that, for the maximum yield of astaxanthin to be obtained from 1 l of nutrient medium, it is necessary to carry out cultivation, beginning with the first day, at a growth rate significantly lower than µ_max. The optimum budding rate of the mutant strain Ph. rhodozyma VKPM Y-2409 consistent with the maximum astaxanthin synthesis was determined. The specific astaxanthin productivity of the strain studied was about 7.0 mg/g of dry biomass at a budding rate of <0.5.Translated from Mikrobiologiya, Vol. 73, No. 6, 2004, pp. 751–757.Original Russian Text Copyright © 2004 by Vustin, Belykh, Kishilova.     

Effect of acetic acid on astaxanthin production by Phaffia rhodozyma

Summary Low concentrations of acetic acid decreased the growth rate of and astaxanthin production by Phaffia rhodozyma on glucose, with growth completely inhibited by 2 g acetic acid/l. Using H₂SO₄ for pH control after sugar depletion caused a decline in the biomass concentration, whereas using acetic acid as titrant resulted in an increase in the biomass with a high astaxanthin content of 1430 g/g cells. An extended culture with a continuous glucose feed failed to maintain a high astaxanthin content.     

Use of alfalfa residual juice as a substrate for propagation of the red yeast Phaffia rhodozyma

Summary Alfalfa residual juice (ARJ) supported good growth of the yeast Phaffia rhodozyma but formation of astaxanthin was inhibited. Supplementary nutrients did not reverse the inhibition, indicating that the the juice probably contained some inhibitor of astaxanthin biosynthesis. Six strains of P. rhodozyma were tested and found to be susceptible to the inhibitory effects of the juice. Concentrations of ARJ above 1.25% (v/v) were inhibitory to pigmentation of the yeast. Above approximately 3.7%, total inhibition of astaxanthin formation was observed but some chromogenic components of the juice were adsorbed on Phaffia cells and appeared as artefacts in astaxanthin analyses. Phaffia biomass produced in ARJ showed greater susceptibility to autolysis than that produced in a peptone-glucose-salts medium. Supplementation of ARJ with glucose enhanced yield of cell mass and minimised the autolytic phenomenon, and is potentially useful for producing Phaffia biomass for use as a source of single cell protein.Unsupplemented brewer's malt wort and molasses, separately and in a suitable combination, were compared with ARJ and were found suitable for growth and pigmentation of P. rhodozyma.     

Reduction of fatty acid flux results in enhancement of astaxanthin synthesis in a mutant strain of Phaffia rhodozyma

A moderate-temperature mutant strain of the yeast Phaffia rhodozyma, termed MK19, was selected by 1-methyl-3-nitro-1-nitrosoguanidine (NTG) and Co60 mutagenesis. MK19 displayed fast cell growth and elevated astaxanthin content at 25°C, whereas optimal temperature for growth and astaxanthin synthesis of wild-type P. rhodozyma was 17–21°C. Optimized astaxanthin yield for MK19 after 4 days culture in shaking flask at 25°C, determined by response surface methodology, was 25.8 mg/l, which was 17-fold higher than that of the wild-type. MK19 was tolerant of high initial concentration of glucose (>100 g/l) in optimized medium. Total fatty acid content of MK19 was much lower than that of the wild-type. Acetyl-CoA is a common precursor of fatty acid and terpenoid biosynthesis, and it is possible that decreased fatty acid synthesis results in transfer of acetyl-CoA to the carotenoid biosynthetic pathway. Our results indicate that astaxanthin content is negatively correlated with fatty acid content in P. rhodozyma. Nutrient analysis showed that MK19 cells are enriched in lysine, vitamin E, and other rare nutrients, and have potential application as fish food without nutritional supplementation. This moderate-temperature mutant strain is a promising candidate for economical industrial-scale production.     

Carbon-source assimilation pattern of the astaxanthin-producing yeast Phaffia rhodozyma

Eleven Phaffia rhodozyma strains were assayed for their ability to utilize 99 compounds as single carbon source. Some of them showed modified coloration compared to colonies of the same strain grown on glucose medium.     

Estimation of the concentrations of cells, astaxanthin and glucose in a culture of Phaffia rhodozyma by near infrared reflectance spectroscopy

Summary Near infrared reflectance spectroscopy (NIR) was employed to estimate the concentrations of cells, astaxanthin and glucose in the culture broth of Phaffia rhodozyma. The culture broth (119 samples) was directly subjected for NIR analysis without any pretreatment. When the data obtained by NIR were compared with those obtained by conventional methods, high correlation coefficients were obtained: 0.98 for cells, 0.99 for astaxanthin and 0.94 for glucose. These results suggest that NIR analysis, which is very simple and requires only 3 to 5 mm for a sample, is applicable to monitor P. rhodozyma cultures.     

Carotenoids of Phaffia rhodozyma, a red-pigmented fermenting yeast

The red-pigmented fermenting yeast Phaffia rhodozyma contains astaxanthin as the principal carotenoid pigment. Echinenone, 3-hydroxyechinenone and phoenicoxanthin were also isolated and identified; isocryptoxanthin and canthaxanthin were absent. Evidence is presented for a new carotenoid, 3-hydroxy-3′4′-didehydro-β,ψ-caroten-4-one. A possible biosynthetic scheme for the formation of astaxanthin in P. rhodozyma is suggested.     

Mixed culture ofBacillus circulans WL-12 andPhaffia Rhodozyma on different carbon sources: Yeast-wall lytic enzyme production and extractability of astaxanthin

Summary Lytic enzyme production byBacillus circulans WL-12 and modification of the red yeastPhaffia rhodozyma in mixed culture, is feasible on some simple sugars other than glucose. Advantages of the mixed culture are discussed.     

Phaffia rhodozyma is polyploid

The ploidy of the red yeast Phaffia rhodozyma was evaluated using flow cytometric analyses of propidium iodide- stained cells and mutagenic inactivation kinetics. Our findings suggest that Phaffia rhodozyma is not haploid. Auxotrophic strains were generated at a high frequency following treatment of mutagenized cells with a combination of benomyl and ethyl acetate. Studies of an auxotrophic mutant using flow cytometry and UV inactivation indicated possible chromosome loss to an aneuploid state. Received 21 June 1998/ Accepted in revised form 25 September 1998     

<Emphasis Type="Italic">Phaffia rhodozyma</Emphasis>: colorful odyssey

Phaffia rhodozyma was isolated by Herman Phaff in the 1960s, during his pioneering studies of yeast ecology. Initially, the yeast was isolated from limited geographical regions, but isolates were subsequently obtained from Russia, Chile, Finland, and the United States. The biological diversity of the yeast is more extensive than originally envisioned by Phaff and his collaborators, and at least two species appear to exist, including the anamorph Phaffia rhodozyma and the teleomorph Xanthophyllomyces dendrorhous. The yeast has attracted considerable biotechnological interest because of its ability to synthesize the economically important carotenoid astaxanthin (3,3-dihydroxy-, -carotene-4,4-dione) as its major pigment. This property has stimulated research on the biology of the yeast as well as development of the yeast as an industrial microorganism for astaxanthin production by fermentation. Our laboratory has isolated several mutants of the yeast affected in carotenogenesis, giving colonies a vivid array of pigmentation. We have found that nutritional and environmental conditions regulate astaxanthin biosynthesis in the yeast, and have demonstrated that astaxanthin protects P. rhodozyma from damage by reactive oxygen species. We proposed in the 1970s that P. rhodozyma could serve as an economically important pigment source in animal diets including salmonids, lobsters, and the egg yolks of chickens and quail, in order to impart characteristic and desirable colors. Although P. rhodozyma/Xanthomyces dendrorhous has been studied by various researchers for nearly 30 years, it still attracts interest from yeast biologists and biotechnologists. There is a bright and colorful outlook for P. rhodozyma/X. dendrorhous from fundamental and applied research perspectives.     

Astaxanthin from Jerusalem artichoke: Production by fed-batch fermentation using Phaffia rhodozyma and application in cosmetics

Jerusalem artichoke extract or powder was used for astaxanthin production using Phaffia rhodozyma without acidic or enzymatic inulin hydrolysis. The culture medium containing Jerusalem artichoke as carbon source was optimized, and feeding strategies, including constant, exponential, pH-stat, and substrate feedback fed-batch fermentations, were also compared for enhancing the cell biomass and astaxanthin synthesis by P. rhodozyma. Substrate-feedback fed-batch fermentation resulted in the highest dry cell weight of 83.60 g/L, with a carotenoid concentration and yield of 982.50 mg/L and 13.30 mg/g, respectively, under optimized medium components using Jerusalem artichoke extract as carbon source in a 3-L stirred-tank bioreactor. Moreover, 482.50 mg/L of carotenoids and 253.10 mg/L of astaxanthin were obtained by continuous feeding of Jerusalem artichoke powder, which was used as carbon source. Astaxanthin essence with high DPPH-scavenging activity was obtained from the extracted astaxanthin, and the DPPH free radical scavenging rate of 40 ppm astaxanthin essence reached 76.29%. When stored at 4 °C, astaxanthin essence showed the highest stability, with a minimum k value of 0.0099 week⁻¹ and maximum half-life (t_1/2) value of 70 weeks.     

Transport-limited sucrose utilization and neokestose production by Phaffia rhodozyma

Summary Growth of an astaxanthin hyper-producing strain of Phaffia rhodozyma on sucrose is accompanied by the accumulation of glucose and fructose in the medium due to the limited capacity of the corresponding monosaccharide transport system or systems. This is accompanied by the production of the trisaccharide neokestose by transglycosylation reactions.