See discussions, stats, and author profiles for this publication at: https://www.researchgate.

net/publication/325719659

Effect of Different Carbon and Nitrogen Sources on the Production of

Carotenoid Pigments by Phaffia rhodozyma

Article · December 2011

CITATIONS READS

0 28

2 authors:

Sivakumar Natesan Selvakumar Gopal

Madurai Kamaraj University Alagappa University
45 PUBLICATIONS   157 CITATIONS    29 PUBLICATIONS   130 CITATIONS   

SEE PROFILE SEE PROFILE

Some of the authors of this publication are also working on these related projects:

Bioactive pigments from estuarine and marine environments View project

Evoloution of Livestock-Associated S. aureus genomes View project

All content following this page was uploaded by Sivakumar Natesan on 09 August 2018.

The user has requested enhancement of the downloaded file.

Research & Reviews: A Journal of Life Sciences
Volume 1 Issue 2, December 2011, Pages 1–9.
___________________________________________________________________________

Effect of Different Carbon and Nitrogen Sources on the Production of

Carotenoid Pigments by Phaffia rhodozyma
Sivakumar Natesan
School of Biotechnology, Madurai Kamaraj University, Madurai, Tamil Nadu, India

Selvakumar Gopal*
Department of Microbiology, DDE, Alagappa University, Karaikudi, Tamil Nadu, India

Abstract

Carotenoids are natural compounds that are attractive in color and easy to extract from
microorganisms. Astaxanthin is the principal carotenoid pigment responsible for the
distinctive orange red pigmentation in marine invertebrates, fish and crustaceans. Phaffia
rhodozyma (red yeast) produces astaxanthin as the major carotenoid. In the present
investigation, improved astaxanthin production by the P. rhodozyma was performed with
different carbon and nitrogen sources. Maximum amount of carotenoid obtained in
sucrose-supplemented medium at 2% (W/V) was 6.80 mgL1. It was followed by maltose
(5.3 mgL1), and glucose (1.8 mgL1). Urea at 0.25% was the best nitrogen source for
higher carotenoid production (6.74 mgL1), followed by (NH4)2 SO4 (4.81 mgL1) and
yeast extract (4.48 mgL1).

Keywords: Astaxanthine, carotenoid, optimization, Phaffia rhodozyma

*Author for Correspondence E-mail: gselvakumar75@gmail.com

1. INTRODUCTION The red yeast, Phaffia rhodozyma, has

Astaxanthin (3, 3-dihydroxy-ß, ß-
carotene-4, 4-dione) is one of the most
abundant carotenoids in nature and occur
in certain microorganisms, crustaceans [1]
and some fishes [2], but animals cannot
synthesize carotenoids, and it must be
offered through their diets. Astaxanthin
has a strong antioxidant activity and some
vital biological functions [3–4]. The use of
astaxanthin as a colorant in the aquaculture
industry has been increasing in the recent
past [5]. Synthetic colorants become
undesirable due to their hazardous effects
to health and environment [6]. Presently,
the number of permitted artificial colors
has reduced considerably, and as a
consequence, the interest in natural
colorants has increased significantly [7–8].
been considered as a good source of
astaxanthin [9–10] as the primary
carotenoid pigment and can be grown in an
economically feasible process [11]. Many
published works report astaxanthin
fermentation by P. rhodozyma at lab scale
[11–12]. Commonly, astaxanthin
production by P. rhodozyma utilizes
inexpensive substrates and co-products
[13–15]. Because of the increasing
demand, the high cost of synthetic
astaxanthin, and the need of astaxanthin
obtained from natural sources at a low cost
with high productivity, the present study
aimed to optimize the carotenoid and
astaxanthin pigment production by P.
rhodozyma WG 07 isolated from Western
Ghats under different carbon and nitrogen
sources.

1
Research & Reviews: A Journal of Life Sciences
Volume 1 Issue 2, December 2011, Pages 1–9.
___________________________________________________________________________
2. MATERIALS AND METHODS
2.1. Microorganism and Culture Condin
The strain Phaffia rhodozyma WG 07 (red-
yeast) was isolated from the leaf samples
collected from Western Ghats, Tamil
Nadu, India. Identification of red yeast up
to species level was carried on the basis of
standard morphological and biochemical
protocols followed by Barnett et al. [16]
and Kurtzman and Fell [17]. P. rhodozyma
WG 07 was inoculated in to 25 mL of
yeast extract peptone (YEP) medium
containing yeast extract, 0.3%; bacto
peptone, 0.5%; glucose, 1%; and
manganous chloride, 0.075%, and
incubated for 5 days at 25°C. Yeast strain
WG 07 was maintained on YM broth
(yeast extract malt extract peptone
dextrose medium) (Himedia, India) agar
slants at 4°C [18, 19].
2.2. Inoculum Preparation
The inoculum was grown on a medium (L)
consisting of 20 g sucrose, 1 g yeast
extract, and 5 g peptone. The inoculum
was cultivated in 250 mL Erlenmeyer
flasks in a rotatory shaker at 150 rpm,
25°C for 48 h. This culture was used to
inoculate the batch processes in order to
produce an initial absorbance in the
fermentation medium of about 0.45
(650 nm).
2.3. Fermentation Condition
Batch fermentation process was performed
in 1000 mL Erlenmeyer flasks kept on a
rotatory shaker at 100 rpm, 24°C for 5
days. To the best of our knowledge, the
initial composition of the fermentation
media was prepared with different carbon
(glucose, maltose, sucrose, xylulose, and
manitol) (2% W/V) and nitrogen sources
(yeast extract, urea, (NH4)2SO4, and
ammonium nitrate) (0.25% w/v). The pH
was adjusted to pH 6.0 ± 0.2 with 0.1 M
NaOH or 0.1 M H2SO4. The temperature
was controlled at 24 ± 0.5°C. All
experiments were done in triplicate and the
average (mean) values of the results are
shown.
2.4. Analytical Methods
At the end of the processes, biomass was
measured by dry cell mass. The
astaxanthin content was determined by the
method described by Moriel et al. [20]. In
this method, the freeze-dried cells were
ruptured with 2 mL of dimethyl sulfoxide
(DMSO) for 30 min. Then, 6 mL of
acetone was added and agitated vigorously
in order to extract the astaxanthin. The
sample was centrifuged and the solvent
phase was separated. This process was
repeated twice with the precipitate in order
to ensure the extraction of all the
astaxanthin completely. The two solvent
phases obtained were pooled together and
10 mL of 20% NaCl and 10 mL of
petroleum ether were added. After the
agitation and separation of the phases, the
solvent phase was separated. Another
5 mL of petroleum ether was added to
ensure complete extraction. The solvent
phase was filtrated through Na2SO4 to
eliminate moisture. The absorbance was
measured at 480 nm and the astaxanthin
concentration was calculated using the
specific absorption coefficient
E1% = 1600. Total carbohydrate content
was also measured by the phenol-sulfuric
method [21].
3. RESULTS
Table I shows the effect of different
carbon sources on growth and
carotenogenesis of P. rhodozyma WG 07
(5 days). Higher biomass (dry cell mass)
was obtained in yeast P. rhodozyma WG
07 grown in sucrose (2% w/v) medium
(14.3 ± 0.98 gL1), followed by maltose
(12.5 ± 0.62 gL1), glucose (11.0 ±
0.4 gL1), and xylulose (10.6 ± 0.52 gL1).
2
Research & Reviews: A Journal of Life Sciences
Volume 1 Issue 2, December 2011, Pages 1–9.
___________________________________________________________________________

Production of carotenoid and astaxanthin carotenogenesis were seen in urea (0.25%

were significantly (P<0.05) higher in
sucrose medium (7.92 mg/L and 6.8 mgL1
respectively). Lower astaxanthin was
resulted in mannitol containing medium
(0.84 mgL1). In order to study the
astaxanthin production with different
nitrogen sources (Table II), significantly
(P<0.05) higher biomass and
w/v) containing medium. Higher
astaxanthin was extracted from urea-
containing medium (6.78 mgL1) whereas
lower biomass and astaxanthin were
obtained in ammonium nitrate-containing
medium (10.9 ± 0.29 and 3.21 ±
0.11 mgL1 respectively).

Table I Effect of Different Carbon Sources on Carotenogenesis in P. rhodozyma (5 days).

Total Carotenoid
Carbon Source Dry Cell Mass Culture Fluid Dry Cells Astaxanthine
gL1 1
(mgL ) 1
(µgL ) (mgL1)
1. Glucose 11.0 ± 0.4 2.73 ± 0.11 196 ± 7.21 1.80 ± 0.11
2. Maltose 12.5 ± 0.62* 6.81 ± 0.28 455 ± 9.64* 5.3 ± 0.14*
3. Sucrose 14.3 ± 0.98* 7.92 ± 0.21 604 ± 8.0* 6.8 ± 0.25*
4. Xylulose 10.6 ± 0.52 1.54 ± 0.1 124 ± 6.81 1.01 ± 0.19
5. Manitol 9.8 ± 0.3 1.38 ± 0.14 111 ± 5.21 0.84 ± 0.11
Mean values of triplicate trials with ± standard deviations. * Significantly different (P<0.05)

Table II Effect of Different Nitrogen Sources on Carotenogenesis in P. rhodozyma (5 days).

Total Carotenoid
Nitrogen Source Dry Cell Culture fluid Dry cells Astaxanthine
Mass (mgL )1 1
(µgL ) (mgL1)
1
gL
1. Yeast extract 11.9 ± 0.46 6.1 ± 0.11 408 ± 7.2 4.48 ± 0.27
2. (NH4)2SO4 12.8 ± 0.68 6.4 ± 0.22 421 ± 6.72 4.81 ± 0.15
3. Urea 13.9 ± 0.72* 7.61 ± 0.24* 594 ± 9.0* 6.78 ± 0.24*
4. Ammonium nitrate 10.9 ± 0.29 4.7 ± 0.21 310 ± 7.0 3.21 ± 0.11
Mean values of triplicate trials with ± standard deviations. * Significantly different (P<0.05)

4. DISCUSSION Astaxanthin is produced as a secondary

The dynamics of growth and synthesis of
the carotenoid astaxanthin during batch
fermentation was performed with various
carbon and nitrogen sources. Carbon and
nitrogen sources were the necessary
components in the fermentation medium,
and different components of the carbon
and nitrogen sources could greatly affect
the growth and yield of astaxanthin.
metabolite, which requires specific
nutritional conditions for its biosynthesis
[22]. In the industry, inexpensive
substrates [23] such as corn wet-milling
[24] and peat hydrolyzates [25] are used as
substrates for growth and carotenoid
production by P. rhodozyma. In this study,
isolate P. rhodozyma WG 07 produces
higher carotenoid fermentation (Table I).
Higher biomass (dry cell mass) was

3
Research & Reviews: A Journal of Life Sciences
Volume 1 Issue 2, December 2011, Pages 1–9.
___________________________________________________________________________
obtained for Phaffia rhodozyma WG 07 in
sucrose (2% W/V) medium (14.3 ± 0.98
gL1), followed by maltose (12.5% ±
0.62 gL1). Significantly higher (P<0.05)
carotenoid and astaxanthin were obtained
in the fermentation medium supplemented
with sucrose as carbon source (7.92 mgL1
and 6.8 mgL1 respectively), however, the
relatively low astaxanthin production in
cells cultured in media containing manitol,
xylulose, and glucose. But Fang and
Cheng [26] and Yamane et al. [27]
reported that higher astaxanthin production
was obtained in glucose-containing media
but carotenoid production was higher in
fructose media. The present study results
revealed that production media with
sucrose was the suitable carbon source for
maximum production of astaxanthin by P.
rhodozyma WG 07.
A fermentation medium containing corn
starch hydrolysate, corn steep liquor, and
urea have been devised for large-scale
production of astaxanthin by P. rhodozyma
[23]. In the present study, urea was the
best nitrogen source and significantly
(P<0.05) improves production of
astaxanthin and dry cell biomass (Table
II). Yeast extract was also a suitable
nitrogen source for higher production of
astaxanthin. Moriel et al. [20] reported that
urea supported optimum production of
biomass and astaxanthin by the yeast P.
rhodozyma ATCC 24202. Validation
experiments showed that the optimal
nitrogen source was composed of 0.28 g/L
(NH4)2SO4 [28]. Furthermore, the present
study provides positive evidence that the
new isolate WG 07 produces higher
astaxanthin.
5. CONCLUSIONS
We conclude that our new isolate P.
rhodozyma WG 07 produces higher
production of astaxanthin when media
supplemented with sucrose and urea was
the best carbon and nitrogen source.
Further strain improvement process may
be required for higher production of
astaxanthin.
ACKNOWLEDGEMENTS
We are thankful to the management of
THR College, Perambalur, Tamil Nadu,
for providing the laboratory to carry out
this work.
REFERENCES
1. Johnson E. A. and Schroeder W. A.
Advances in Biochemical Engineering
1995. 53. 119–178p.
2. Goodwin T. W. Biochemistry of
Carotenoids. vol 2. Animals. Chapman
and Hall, London, UK. 1984.
3. O’Connor I. and O’Brien N. Journal of
Dermatological Science 1998. 16.
226–230p.
4. Jyonouchi H. et al. Nutrition and
Cancer 1993. 19. 269–280p.
5. Johnson E. A. et al. Aquaculture 1980.
20. 123–134p.
6. Carvalho J. C. et al. Brazilian Archives
of Biology and Technology 2005. 48.
885–894p.
7. Carvalho J. C. et al. Agro Food Industry
Hi-Tech 2003. 4. 37–42p.
8. Joshi V. K. et al. Indian Journal of
Biotechnology 2003. 2. 362–369p.
9. Andrews A. G. et al. Phytochemistry
1976. 15. 1003–1007p.
10. Golubev. W. I. Yeast 1995. 11. 101–
110p.
11. Yamane Y. et al. Biotechnology Letters
1997. 19. 1109–1111p.
12. Flores-Cotera L. B. Applied
Microbiology and Biotechnology 2001.
55. 341–347p.
10. Haard N. F. Biotechnology Letters
1988. 10. 609–614p.
13. Hayman G. T. et al. Journal of
Industrial Microbiology and
Biotechnology 1995. 14. 389–395p.
4
 Research & Reviews: A Journal of Life Sciences
Volume 1 Issue 2, December 2011, Pages 1–9.
___________________________________________________________________________
14. Vagvolgyi C. S. et al. Canadian
Journal of Microbiology 1996. 42.
613–616p.
15. Florencio J. A. et al. Bioprocess and
Biosystems Engineering 1998. 19.
161–164p.
16. Barnett J. A. et al. Yeasts:
Characteristics and identification. 2nd
Edn. Cambridge University Press.
Cambridge. 1990.
17. Kurtzman C. P. and Fell J. W. The
Yeasts. A Taxonomic Study. North-
Holland. Amsterdam. 1999.
19. Gu W. L. et al. Journal of Industrial
Microbiology and Biotechnology 1997.
19. 114–117p.
20. Moriel D. G. et al. The Brazilian
Journal of Pharmaceutical Sciences
2004. 40(3). 421–424.
21. Dubois M. et al. Analytical Chemistry
1956. 28. 350–356p.
View publication stats
22. Johnson A. E. and Lewis. J. M.
Journal of General Microbiology
1979. 115. 173–183p.
23. Kesava S. S. et al. Bioprocess and
Biosystems Engineering 1998. 19.
165–170p.
24. Hayman G. T. et al. Journal of
Industrial Microbiology and
Biotechnology 1995. 14. 389–395p.
25. Vazquez M. and Martin. A. M. Journal
of the Science of Food and Agriculture
1998. 76. 481–487p.
26. Fang T. J. and Cheng. Y. S. Journal of
Fermentation and Bioengineering
1993. 75. 466–469p.
27. Yamane Y. et al. Biotechnology Letters
1997. 19. 1109–1111p.
28. Hui N. I. et al. Journal of Zhejiang
University - Science B 2007. 8(5).
365–370p.