REV. CHIM. (Bucharest) 63 No.3 2012 http://www.revistadechimie.

ro 316
Torularhodin Biosynthesis and Extraction by Yeast
Cells of Rhodotorula Rubra
CAMELIA UNGUREANU
1,2*
, MARIANA FERDES
2
, ANA AURELIA CHIRVASE
2
1
University of Agricultural Sciences and Veterinary Medicine Cluj-Napoca, 3 Manastur Str., 400372, Cluj-Napoca, Romania
2
University Politehnica of Bucharest, 1-7, Polizu Str., 011061, Bucharest, Romania
The paper presents the research work done to study the discontinuous bioprocess for the intracellular
carotenoidic pigment - torularhodin formation with the yeast Rhodotorula rubra ICCF 209. The growth and
carotenoid biosynthesis of the yeast Rhodotorula rubra was studied by cultivation in 3.7 L Bioengineering AG
lab bioreactor with mechanical stirring at an air flow rate of 200 L/h and agitation at 600 rpm for 4 days. The
medium composition, defined as MS3, was obtained with the formula: 30 g/L glucose, 1.5 g/L yeast extract,
5 g/L NH
4
NO
3
, 1 g/L KH
2
PO
4
, 0.4 g/L

MgSO
4
x 7H
2
O, 0.4 g/L NaCl and 1 g/L alanine. The bioreactor was
equipped with computerized control units for temperature, air flow, stirrer speed, pH, and pO
2
, and acquisition
data every 15 s was programmed.
Keywords: torularhodin, Rhodotorula rubra, pigmented yeast
* email: c_ungureanu@chim.upb.ro, Tel.: +40723239120
Facing the growing economic significance of
carotenoids, due to their use as food colorants, nutritional
supplements, in cosmetics or in human therapy as
antioxidants, much interest has been dedicated to new
supplies of this class of pigments [1-3]. In particular, the
development of carotenoid-producing bioprocesses is
regarded as a competitive solution, as it can provide
important quantities of pigments such as torularhodin and
-carotene produced by Rhodotorula rubra or astaxanthin
from Phaffia rhodozyma without facing the typical
problems generated by the weather dependency of the
agriculture production [4-5].
Due to the growing therapeutic importance of the
torularhodin as antioxidant product, much interest has been
devoted to prepare this pigment by red yeasts cultivation.
In particular, the preparation by applying yeasts
biotechnology methods (bioprocessing and extraction to
get intracellular bioproducts) can be considered as a
competitive solution, as it can be the only known source
until now to obtain the torularhodin pigment produced by
Rhodotorula or Sporobolomyces yeasts.
Rhodotorula is carotenoid biosynthetic yeast, part of the
Basidiomycota phyllum, easily identifiable by distinctive
yellow, pink, orange/red colonies [6]. The main carotenoids
produced were identified as torularhodin, torulene, -
carotene and -carotene in Rhodotorula species.
On MS3 agar-medium the cells are coral pink, usually
smooth, sometimes reticulate and rugose. Microscopic
morphology on Olympus U-CMAD 3, 500X shows spherical
or elongated budding yeast cells or blastoconidia, 2.5-6.5 x
6.5-14.0 m in size.
Natural colorants of microbial origin have attracted the
worldwide commercial interest due to the potential toxicity
caused by synthetic colors. With the help of biotechnology
intervention, production of some food grade natural
pigments such as torularhodin from Rhodotorula rubra, red
pigments from Monascus purpureus, Monascus ruber [7]
have gained considerable consumer acceptance.
Torularhodin (3', 4'-didehydro-, -caroten-16'-oic acid,
scheme 1) [8] is the unique carotenoid with a terminal
carboxylic group considered now-a-days as a powerful
antioxidant to be included in food and drugs formulations.
Fig. 1 Colony on MS3 agar medium (A)
and microscopic morphology of the
Rhodotorula rubra ICCF 209 yeast
cells (B)
Fig. 2 Chemical structure of torularhodin
The paper presents the research work done to study the
discontinuous bioprocess for the intracellular carotenoidic
pigment - torularhodin formation from the yeast
Rhodotorula rubra ICCF 209.
Experimental part
Biosynthesis of torularhodin
The experiments were carried out in 3.7 L (2 L working
volume) bioreactor Bioengineering AG, with computer-
controlled and recorded parameters. Rhodotorula rubra
ICCF 209 was employed in the study.
The bioreactor has mechanical stirring (Rushton
impellers) and the main parameters (temperature, pH,
mixing speed, air flow rate, pO
2
, and foam level) are
controlled. The operating parameters were 30
o
C, 600 rpm
and air flow rate of 200 L/h.
The medium composition, defined as MS3, was
obtained with the formula: 30 g/L glucose, 1.5 g/L yeast
REV. CHIM. (Bucharest) 63 No. 3 2012 http://www.revistadechimie.ro 317
extract, 5 g/L NH
4
NO
3
, 1 g/L KH
2
PO
4
, 0.4 g/L

MgSO
4
x7H
2
O,
0.4 g/L NaCl and 1 g/L alanine. Trace elements are assumed
to be taken from the tap water.
The bioprocess was operated in batch mode.
A suspension of the yeast cells in sterile water was used
for the inoculums preparation. The cells growth was
quantified by: optical density determination at = 600
nm, evolution of pH and dissolved oxygen. After cells
separation by centrifugation (20 min. at 8000 rpm) three
freeze-thaw cycles were performed.
Extraction and quantification of torularhodin
The pigments extraction procedure was done in
accordance with the dedicated literature [9, 10],
comprising acetone extraction of the total pigments
mixture including water soluble species, followed by n-
hexane extraction to separate the total carotenoids
content; another extraction with alkaline methanol
allowing the torularhodin (the only pigment with acid
structure) component isolation. The total carotenoids
concentration and the torularhodin concentration were
determined based on the spectrometric recording of the
extracts on the UV-VIS spectrophotometer (Jenway
Spectrophotometer). Adsorption spectra were drawn in
380-780 nm domain and the peaks were determined. To
calculate the torularhodin concentration, the specific
absorption coefficient E
1%
1932 was applied to the
difference between the absorbance of the hexane extract
before and after methanol phase extraction, at 515 nm.
Results and discussions
Several experiments were realized for growth and
carotenoids synthesis, including the torularhodin formation.
Growth and torularhodin synthesis were investigated based
on the results of the previously study [11] were the best
culure media was MS3.
The research methodology comprised: the cultivation
medium and bioprocessing conditions; determination of
yeast specific growth rate and torularhodin formation.
The cells growth was quantified by: optical density
(O.D.) determination at = 600 nm, evolution of pH and
dissolved oxygen (pO
2
, %).
The results of O.D. measurements (growth estimator),
pH medium and pO
2
% for a representative batch are
presented in figures 2-4.
The studied characteristics yeast growth, the total
carotenoids formation, and the torularhodin formation
recommend the initial pH range of 6-7 as being most
favorable; the torularhodin ratio from the total carotenoids
content being greater for the pH of 6-7. It was observed a
strong decrease a pH medium, until 2-1.5 units.
Figure 4 shows a phase lag followed by exponential
growth of approximately 25 h which is in accordance with
decreased of concentrations of dissolved oxygen in the
first 25 h of biosynthesis (fig. 5); this is due to accumulation
of yeast cells in the culture media.
The glucose concentration decrease as an indicator of
substrate consumption is represented in the figure 6.
Fig. 3. pH profile for torularhodin fermentation
Fig. 4. Evolution of optical density in time
Fig. 5. Variation of dissolved oxygen in time
Fig. 6. Evolution of glucose concentration in time
To express the growth kinetic and to calculate
max
(maximum specific growth rate [s
-1
; h
-1
])

an exponential
model is proposed.
The maximum specific growth rate
m
[h
-1
] was
calculated for a representative batch in accordance with
an exponential model (fig. 7) considering X-cells
concentration, g/L [12]:
(1)
After the integration and the linearization the equation
(1) becomes:
(2)
The linear regression representation is presented in the
figure 7.
The maximum specific growth rate for Rhodotorula
rubra ICCF 209 was obtained 0.18 h
-1
which can be
considered an appropriate value for yeast like Rhodotorula
rubra.
After cells separation by centrifugation (20 min at 8000
rpm) three freeze-thaw cycles were performed.
REV. CHIM. (Bucharest) 63 No.3 2012 http://www.revistadechimie.ro 318
Fig. 8. Visible absorption spectra of torularhodin extracted from
Rhodotorula rubra ICCF 209 in methanol
The total carotenoids concentration was determined as
-carotene content by using the extract absorbance values
similarly the torularhodin concentration was calculated
using methanol extracts absorbance values (fig. 8) with
the specific absorption coefficient, E
1%
1932.
In order to quantify the carotenoid pigments
concentration, the cell dry biomass was determined for
the final samples (19.51%, w/w) and the dry biomass/
culture liter was calculated (4.39 g/L medium).
For the tested Rhodotorula strain it has been determined
the highest pigments yield in the case of MS3 media; the
total carotenoid pigments was 871g/L and the
torularhodin concentration was 710 g/L.
Conclusions
The paper presents the research work done to study the
discontinuous bioprocess for the intracellular carotenoidic
pigment - torularhodin formation with the yeast Rhodotorula
rubra ICCF 209.
Fig. 7. The maximum specific growth rate of the studied yeast
Due to the growing therapeutic importance of the
torularhodin as antioxidant product, much interest has been
devoted to prepare this pigment by red yeasts cultivation.
The experiments were carried out in 3.7 L (2 L working
volume) bioreactor Bioengineering AG, with computer-
controlled and recorded parameters.
The maximum specific growth rate
m
[h
-1
] was
calculated for a representative batch in accordance with
an exponential model; the maximum specific growth rate
for Rhodotorula rubra ICCF 209 was obtained 0.18 h
-1
which
can be considered an appropriate value for yeast like
Rhodotorula rubra.
For the tested Rhodotorula strain it has been determined
the highest pigments yield in the case of MS3 media; the
total carotenoid pigments was 871g/L and the
torularhodin concentration was 710 g/L.
In the future research will be determined the antioxidant
activity of the extract obtained by the presented method.
Acknowledgments : The work was financially supported by the project
POSDRU/89/1.5/S/52432 from 1.04.2010 - Institutional organization of
a postdoctoral school of national interest Applied biotechnology
with impact in the Romanian economy; the project was cofunded by
the EU Social Fund in the framework of the Sectorial Operational
Programme 2007-2013 for Human Resources Development.
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Manuscript received: 11.11.2011