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World Journa l o f Mic rob io logy and B io techno logy,8, 214-215

S h o r t C o m m u n i c a t io n

L ip id a c c u m u l a t i o n inR h o d o t o r u l a g l u t i n i s

o n s u g a r c a n e m o l a s s e s in s i n g l e - s t a g ec o n t i n u o u s c u l t u r e

R.M. Alvarez* , B . Ro dr iguez , J .M. Rom ano, A.O . Diaz , E . G 6m ez , D. Mir6 , L . Nav ar ro ,G. Saura and J .L . Garc ia

M i c r o b i a l l i p i d s p r o d u c e d b yRhodo to ru l a g lu t ln i sg r o w n i n c o n t i n u o u s c u l t u r e w i t h m o l a s s e s u n d e r n i t r o g e n -l im i t i n g c o n d i t i o n s w e r e e v a l u a t e d a n d t h e e f f e c ts o f g r o w t h r a t e o n f a t t y a c id c o m p o s i t i o n w e r e s t u d i e d . A s t h eg r o w t h r a t e d e c r e a s e d , c e l l b i o m a s s , l ip i d c o n t e n t a n d l i p id y i e l d g r a d u a l l y i n c r e a se d . T h e m a x i m u m l i p id c o n t e n tr e c o r d ed w a s 39% ( w / w ) o f d r y c e l l b i o m a s s a t a d i lu t i o n r a t e o f 0 . 0 4 h - 1 . T h e g r o w t h r a t e a l s o a f f e c te d f a t t ya c i d c o m p o s i t i o n : o l e i c a c i d d e c r e a s e d w i t h d e c r e a s i n g g r o w t h r a t e w h i l e s t e a r i c a c i d i n c r e a s e d .

Key words:Fatty acids, lipid,Rhodotorula,yeas t .

The wor ldwide impending demand fo r o i l s and fa t s has l edto a sea rch fo r uncon ven t iona l sources . Microb ia l p roduc t ionof lip ids appears to be an a t t r ac t ive so lu t ion to th i s p rob lem,part icular ly in those countr ies with restr ic ted supplies ofl ipids from natural sources (AlmazKnet al. 1 9 8 1; M o r e t o n1988). Rhod otorula glutinisis one o f the pr incipal olea gino usyeas t s used fo r l ip id p roduc t ion , be ing non- tox ic andre la t ive ly easy to g row and harves t (Ra t ledge 1979 , Mis raet al. 1984) . In th i s paper, we rep or t the e ffec t o f the g row thra te o f th is yeas t , wi th sugar cane m olasses a t d i ffe ren tconcen t ra t ion on the accumula t ion o f l ip ids , us ing thecon t inuous cu l tu re t echn ique .

M a t e r i a ls a n d M e t h o d s

Rhodotorula glu tinisL/24-2-1, from the collection of the CubanResearch Institute o f Sugar Cane By- Prod ucts, was used.Nitrogen-limiting med ium with the following com position (g/l):(NH4)2SO4, 0.29; (NH 4)2HP Oa, 0.7 and clarified molasse s(equivalent to 20 to 40 g total reducing sugars) was em ployed.A 251 Biolaffite fermenter was used and the op eration

R.M. Alvarez, B. Roddguez, J.M. Romano, A.O. Diaz, E. Gbmez, D. Mirb,G. Saura and J.L. Garcia are with the Instituto Cubano de Investigacionesde los Derivados de la Carla de Az0car, CUBA-10, Apartado 4026, C.Habana, Cuba. L. Navarro is with the Instituto Nacional de Higiene yEpidemiologia, Infanta y Manglar, C. Habana, Cuba. *Correspondingauthor.

(~ 1992 Rap id C ommunica t ions o f Oxfo rd L td

2 1 4 Wo rla Journal of Microbiology and Biotechnology, Vol 8, 1992

conditions and control set points were as follows: 171 workingvolume, temperature 32 -F 0.1~ pH 5 .5, 500 rev/min and airflow 0.7 v/v/rain. The following determinations were performed:yeast dry weight, total reducing sugars (TRS) by themodified Eynon-Lane method (D e W halley 194 9), ammonia andtotal nitrogen b y the m icro-Kjeldahl method (Tecator Unit). Forlipid extraction, 2 g d ry cells were hydrolysed with 80 ml 1 MHC1 for 30 rain at 1 21 ~ the deb ris was recovered,washed u ntil neutra l, drie d and then exhausted in a Soxhletapparatus with diethyl et he r. The extracted lip ids wereanalyzed for fatty a ci d composition as th eir methyl estersby gas chromatography.

R e s u l t s a n d D i s c u s s i o n

Table I show s tha t, a s the d i lu t ion ra te fo r g ro win g

Rhodotorula glutinisdecreased, cel l biomass, l ipid content andl ipid yield gradu al ly increased. Ho we ver, th e specific lipidproduc t ion ra te and l ip id p roduc t iv i ty ach ieved the i rmaximum va lues a t a d i lu t ion ra te o f 0 .05 h -1 . A s imi la rt rend, but a t a lower di lut ion rate , (0 .02 h-1) , was observedb y C h o i et al. (1982) and Yoon & Rhee (1983) , us ingRhodotorula glutinison g lucose .

T h e C : N r a t i o d i d n o t c h a n g e s i g n i f i c a n t l y w h e n t h esugar conce n t ra t ion was increased, because molasses con ta ins ign if icant amo unts o f o rgan ic n i trogen . H owe ver, a t thesame d i lut ion rate , the synthe sis o f lipids , cel l biomass, l ipid

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R . M . A l v a r e zet al.

Ta b l e 1 . Q u a n t i ta t i v e d a t a o n t h e c o n t i n u o u s c u l t u r e o fR o d o t o r u l a g l u t i n isa t d i ffe ren t d i lu t ion ra tes and to ta l reduc ing sugar (TRS)c o n c e n t r a t i o n s .

Run Di lu t ion Incom ing m ediu m (g / I ) TRs (g / I ) Biom ass Y,~s L ip id YLIs Dp D x Dp/xn o . r a t e r e m a i n i n g ( g / I ) c o n t e n t

( h - 1 ) T R S To t a l N 2 C : N ( % )

1 0.1 23 0.38 24 11.8 5.3 47.3 15.5 7.2 0.08 0.53 0.0182 0.06 18 0.34 21 4.4 7.1 47.3 27.0 12.8 0.12 0.43 0.0223 0.06 35 0.47 30 6.0 13.0 44.8 29.2 13.0 0.23 0.78 0.0254 0.05 20 0.36 22 2.0 8.1 45.0 32.4 14.5 0.13 0.40 0.0245 0.05 36 0.48 30 2.7 14.5 43.5 37.4 16.3 0.27 0.72 0.0306 0.04 20 0.36 22 1.7 10.5 57.4 34.0 19.5 0.14 0.42 0.0217 0.04 30 0.44 27 3.3 15.0 56.2 39.2 20.6 0.24 0.56 0.026

Yx/s:B i o m a s s y i e l d , ( g c e l l b i o m a s s / 1 0 0 g s u g a r c o n s u m e d ) .YL/s : L ip id y ie ld , (g l ip id /100 g sug ar cons um ed) .Dp: L ip id p roduc t iv i ty, (g l ip id / I /h ) .D x : B i o m a s s p r o d u c t i v i t y, ( g b i o m a s s / I / h ) .D p /x : S p e c i f i c l i p i d p r o d u c t i o n r a t e ( g l i p i d - f r e e b i o m a s s / h ) .

Tab le 2 . Fa t ty acy l compos i t ion of l ip ids f romR h o d o t o r u l ag lu t i n i$a t d i ffe ren t d i lu t ion ra tes in con t inuous cu l tu re and ba tchcu l tu re .

Run Di lu t ion Re l . % (w/w ) pr inc ipa l fa t ty ac idsno . ra te

( h 1 ) 1 4 : 0 1 8 : 0 1 8 : 0 1 8 : 1 1 8 : 2

Batc h* - 1.5 30 6 55 51 0.1 1.6 26 7 54 52 0.06 1.6 29 12 42 83 0.06 1.7 27 10 41 106 0.04 2.0 35 15 41 -

1 8 : 3

23

* Batch c u l tu re , 72 h in shak er.

prod uc t iv i ty and the spec i f ic l ip id p rod uc t ion ra te wereenhanced a t h igher concen t ra t ions o f molasses .

T h e m a x i m u m l i p id c o n t e n t o b t a i n e d w a s 3 9 % ( w / w ) o fd ry ce l l b iom ass a t a d i lu t ion ra te o f 0 .04 h i and 30 g /1of TRS. Un der these cond i t ions , b iomass y ie ld was 56 g ce l lb i o m a s s / 1 0 0 g s u g a r c o n s u m e d a n d t h e l i p i d y i e l d w a s2 0 .6 g / I O 0 g s u g a r c o n s u m e d .

The fa t ty ac id comp os i t ion o f the l ip ids va r i eds ign i f i can t ly wi th d i lu t ion ra te (Tab le 2 ). For ba tch andcon t inuous cu l tu re a t a d i lu t ion o f 0 .1 h -~ , f a t ty ac idcom pos i t ions w ere s imi la r bu t a s the d i lu t ion ra te dec reased ,the overa l l degree o f unsa tu ra t ion dec reased . Th i s e ffect was

main ly due to an inc rease in l eve l s o f s t ea r i c ac id and adecrease in oleic acid.

R e f e r e n c e s

Almazan, O., Ktibansky, M. & Otero, M.A. 198 1 Microbial fatsynthesis by Rhodolorula glutinisfrom blackstrap molasses incontinuous culture.Biotecknology Let ters 3 , 663-666.

Choi, S.Y., Dewey, D.Y ., Ryn & R hee, J.S. 1982 Production ofmicrobial lipids: Effects of grow th rate and oxy gen on lipidsynthesis and fatty acid composition ofRhodotorula gracilis.Biotechnology and Bioengineering24, 1165-1172.

De W alley, H.C.S . 1949 Testing of molasses and sugar syrup.

International Commission for Sugar Analysis 10th Session,Subject 6, pp. I3-26.Gonzalez, N. & K opecky, J. 19 72 Estudio de la calidad de las

levaduras cub ana s. Havana: Biotechnotogical Department,Cuban Research Institute of Sugar Cane By-Products.

Misra, S., Grosh, A. & D urra, J. 1984 Production and com positionof microbial fat fromRhodotorula glutinis. Journal of the Scienceof Food and Agricul ture35, 59-65.

Moreton, R.S. 1988 Physio logy of lipid-accumulating yeasts. InSingle Cell Oil,ed M oreton, R.S., pp. 1-3 2, Harlow, Essex:Longman.

Ratledge, C. 19 79 Resource conservation by novel biologicalprocesses. I. Grow fats from wastes.Chemical Socie ty Reviews8,283-296.

Yoon, S.H. & Rhe e, J.S. 1983 Lipids from yeast fermentation:Effects of cultural condition on lipid production and itscharacteristics ofRhod otorula glutinis. Journal of Am erical OilChemists ' Society60, 1281-I286.

( R e c e iv e d 2 A u g u s t 1 9 9 I ; a c c e p te d24 A u g u s t 1 9 9 1 )

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