5/25/2018 Ungureanu Camelia 3 12

1/3

REV. CHIM. (Bucharest) 63 No.3 2012http://www.revistadechimie.ro316

Torularhodin Biosynthesis and Extraction by Yeast

Cells ofRhodotorula Rubra

CAMELIA UNGUREANU1,2*, MARIANA FERDES2, ANA AURELIA CHIRVASE2

1University of Agricultural Sciences and Veterinary Medicine Cluj-Napoca, 3 Manastur Str., 400372, Cluj-Napoca, Romania2University Politehnica of Bucharest, 1-7, Polizu Str., 011061, Bucharest, Romania

The paper presents the research work done to study the discontinuous bioprocess for the intracellularcarotenoidic pigment - torularhodin formation with the yeast Rhodotorula rubraICCF 209. The growth andcarotenoid biosynthesis of the yeast Rhodotorula rubra was studied by cultivation in 3.7 L Bioengineering AGlab bioreactor with mechanical stirring at an air flow rate of 200 L/h and agitation at 600 rpm for 4 days. Themedium composition, defined as MS3, was obtained with the formula: 30 g/L glucose, 1.5 g/L yeast extract,5 g/L NH

4NO

3, 1 g/L KH

2PO

4, 0.4 g/L

MgSO

4x 7H

2O, 0.4 g/L NaCl and 1 g/L alanine. The bioreactor was

equippedwith computerized control units for temperature, air flow, stirrer speed, pH, and pO2, 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 ofcarotenoids, due to their use as food colorants, nutritionalsupplements, in cosmetics or in human therapy asantioxidants, much interest has been dedicated to newsupplies of this class of pigments [1-3]. In particular, thedevelopment of carotenoid-producing bioprocesses isregarded as a competitive solution, as it can provideimportant quantities of pigments such as torularhodin and-carotene produced byRhodotorula rubraor astaxanthinfrom Phaffia rhodozyma without facing the typicalproblems generated by the weather dependency of theagriculture production [4-5].

Due to the growing therapeutic importance of thetorularhodin as antioxidant product, much interest has beendevoted to prepare this pigment by red yeasts cultivation.In particular, the preparation by applying yeastsbiotechnology methods (bioprocessing and extraction toget intracellular bioproducts) can be considered as acompetitive solution, as it can be the only known sourceuntil now to obtain the torularhodin pigment produced byRhodotorula or Sporobolomycesyeasts.

Rhodotorulais carotenoid biosynthetic yeast, part of theBasidiomycota phyllum, easily identifiable by distinctiveyellow, pink, orange/red colonies [6]. The main carotenoids

produced were identified as torularhodin, torulene, -carotene and -carotene inRhodotorulaspecies.

On MS3 agar-medium the cells are coral pink, usuallysmooth, sometimes reticulate and rugose. Microscopicmorphology on Olympus U-CMAD 3, 500X shows sphericalor elongated budding yeast cells or blastoconidia, 2.5-6.5 x6.5-14.0 m in size.

Natural colorants of microbial origin have attracted theworldwide commercial interest due to the potential toxicitycaused by synthetic colors. With the help of biotechnologyintervention, production of some food grade naturalpigments such as torularhodin fromRhodotorula rubra, redpigments fromMonascus 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 terminalcarboxylic group considered now-a-days as a powerfulantioxidant to be included in food and drugs formulations.

Fig. 1 Colony on MS3 agar medium (A)

and microscopic morphology of theRhodotorula rubra ICCF 209 yeastcells (B)

Fig. 2Chemical structure of torularhodin

The paper presents the research work done to study thediscontinuous bioprocess for the intracellular carotenoidicpigment - torularhodin formation from the yeastRhodotorula rubra ICCF 209.

Experimental partBiosynthesis of torularhodin

The experiments were carried out in 3.7 L (2 L workingvolume) bioreactor Bioengineering AG, with computer-controlled and recorded parameters. Rhodotorula rubraICCF 209was employed in the study.

The bioreactor has mechanical stirring (Rushtonimpellers) and the main parameters (temperature, pH,mixing speed, air flow rate, pO

2

, and foam level) arecontrolled. The operating parameters were 30 oC, 600 rpmand air flow rate of 200 L/h.

The medium composition, defined as MS3, wasobtained with the formula: 30 g/L glucose, 1.5 g/L yeast

5/25/2018 Ungureanu Camelia 3 12

2/3

REV. CHIM. (Bucharest) 63 No. 3 2012 http://www.revistadechimie.ro 317

extract, 5 g/L NH4NO

3, 1 g/L KH

2PO

4, 0.4 g/L

MgSO

4x7H

2O,

0.4 g/L NaCl and 1 g/L alanine. Trace elements are assumedto 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 wasquantified by: optical density determination at = 600nm, evolution of pH and dissolved oxygen. After cellsseparation by centrifugation (20 min. at 8000 rpm) three

freeze-thaw cycles were performed.

Extraction and quantification of torularhodinThe pigments extraction procedure was done in

accordance with the dedicated literature [9, 10],comprising acetone extraction of the total pigmentsmixture including water soluble species, followed by n-hexane extraction to separate the total carotenoidscontent; another extraction with alkaline methanolallowing the torularhodin (the only pigment with acidstructure) component isolation. The total carotenoidsconcentration and the torularhodin concentration weredetermined based on the spectrometric recording of theextracts on the UV-VIS spectrophotometer (JenwaySpectrophotometer). Adsorption spectra were drawn in380-780 nm domain and the peaks were determined. Tocalculate the torularhodin concentration, the specificabsorption coefficient E1%1932 was applied to thedifference between the absorbance of the hexane extractbefore and after methanol phase extraction, at 515 nm.

Results and discussionsSeveral experiments were realized for growth and

carotenoids synthesis, including the torularhodin formation.Growth and torularhodin synthesis were investigated basedon the results of the previously study [11] were the bestculure media was MS3.

The research methodology comprised: the cultivationmedium and bioprocessing conditions; determination ofyeast specific growth rate and torularhodin formation.

The cells growth was quantified by: optical density(O.D.) determination at = 600 nm, evolution of pH anddissolved 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 formationrecommend the initial pH range of 6-7 as being mostfavorable; the torularhodin ratio from the total carotenoidscontent being greater for thepH of 6-7. It was observed astrong decrease a pH medium, until 2-1.5 units.

Figure 4 shows a phase lag followed by exponentialgrowth of approximately 25 hwhich is in accordance withdecreased of concentrations of dissolved oxygen in the

first 25 h of biosynthesis (fig. 5); this is due to accumulationof yeast cells in the culture media.

The glucose concentration decrease as an indicator ofsubstrate 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 exponentialmodel is proposed.

The maximum specific growth rate m

[h -1] wascalculated for a representative batch in accordance with

an exponential model (fig. 7) considering X-cellsconcentration, g/L [12]:

(1)

After the integration and the linearization the equation(1) becomes:

(2)

The linear regression representation is presented in thefigure 7.

The maximum specific growth rate for Rhodotorula

rubra ICCF 209 was obtained 0.18 h-1

which can beconsidered an appropriate value for yeast likeRhodotorularubra.

After cells separation by centrifugation (20 min at 8000rpm) three freeze-thaw cycles were performed.

5/25/2018 Ungureanu Camelia 3 12

3/3

REV. CHIM. (Bucharest) 63 No.3 2012http://www.revistadechimie.ro318

Fig. 8.Visible absorption spectra of torularhodin extracted fromRhodotorula rubra ICCF 209 in methanol

The total carotenoids concentration was determined as-carotene content by using the extract absorbance valuessimilarly the torularhodin concentration was calculatedusing methanol extracts absorbance values (fig. 8) withthe specific absorption coefficient, E1%1932.

In order to quantify the carotenoid pigmentsconcentration, the cell dry biomass was determined forthe final samples (19.51%, w/w) and the dry biomass/culture liter was calculated (4.39g/L medium).

For the testedRhodotorulastrain it has been determinedthe highest pigments yield in the case of MS3 media; thetotal carotenoid pigments was 871g/L and thetorularhodin concentration was 710 g/L.

ConclusionsThe paper presents the research work done to study the

discontinuous bioprocess for the intracellular carotenoidicpigment - torularhodin formation with the yeastRhodotorula

rubra ICCF 209.

Fig. 7. The maximum specific growth rate of the studied yeast

Due to the growing therapeutic importance of thetorularhodin as antioxidant product, much interest has beendevoted to prepare this pigment by red yeasts cultivation.

The experiments were carried out in 3.7 L (2 L workingvolume) bioreactor Bioengineering AG, with computer-controlled and recorded parameters.

The maximum specific growth rate m

[h -1] wascalculated for a representative batch in accordance withan exponential model; the maximum specific growth rate

forRhodotorula rubraICCF 209 was obtained 0.18 h-1

whichcan be considered an appropriate value for yeast likeRhodotorula rubra.

For the testedRhodotorulastrain it has been determinedthe highest pigments yield in the case of MS3 media; thetotal carotenoid pigments was 871g/L and thetorularhodin concentration was 710 g/L.

In the future research will be determined the antioxidantactivity 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 - Inst itutional organization of

a postdoctoral school of national interest Applied biotechnology

with impact in the Romanian economy; the project was cofunded by

the EU Socia l Fund in the framework of the Sectorial Operational

Programme 2007-2013 for Human Resources Development.

References1. BHOSALE P., GADRE, R.V., Lett. Appl. Microbiol., 33,2001, p.122. DAVOLI, P., MILRAN, V., WEBER, R.W.S., Appl. Biochem. Microbiol.,40, no 4, 2004, p. 3923. MIHALCEA, A., UNGUREANU, C., FERDES, M., CHIRVASE, A.A.,TANASE, C. Rev. Chim. (Bucharest), 62, no 6, 2011, p. 6594. LIBKIND, D., VAN BROOCK, M., W.J. Microbiol. Biochem, 22, 2006,p. 6875. DEMAIN, A .L., PHAFF, H.J., KURTZMAN, C. P., In The Yeasts. ATaxonomic Study 4th edn, 1998, p. 1319

6. POSTGATE, J., The Outer Reaches of Life, Cambridge UniversityPress, 1994, p. 1327.UNGUREANU, C., FERDE, M., Chemical Engineering Transactions,20, 2010, p. 2238.SIMPSON, K. L., NAKAYAMA T. O. M., CHICHESTER, C. O., Biochem.

J., 92, 1964p. 5089. PETERSON, W. J., BELL, T.A., ETCHELLS, J.L., SMART, W.W.G., J.Bacteriol., 67, 1954, p. 70810. UNGUREANU, C., FERDES M., CHIRVASE, A.A., MOCANU, E.,Chemical Engineering Transactions. 24, 2011, p. 94311. FERDE, M., UNGUREANU, C., MIHALCEA, A., CHIRVASE, A. A.,MOCANU, E., Rev. Chim. (Bucharest), 62, no 3, 2011, p. 33912. BELLGARDT, K.H., 2000, Bioreaction Engineering, Ed. Springer,Berlin, 2000, p. 56

Manuscript received: 11.11.2011