World Journal of Microbiology and Biotechnology, 8, 214-215

Short C o m m u n i c a t i o n

Lipid accumulation in Rhodotorula glutinis on sugar cane molasses in single-stage continuous culture
R.M. Alvarez*, B. Rodriguez, J.M. Romano, A.O. Diaz, E. G6mez, D. Mir6, L. Navarro, G. Saura and J.L. Garcia
Microbial lipids produced by Rhodotorula glutlnis grown in continuous culture with molasses under nitrogenlimiting conditions were evaluated and the effects of growth rate on fatty acid composition were studied. As the growth rate decreased, cell biomass, lipid content and lipid yield gradually increased. The maximum lipid content recorded was 39% (w/w) of dry cell biomass at a dilution rate of 0.04 h -1. The growth rate also affected fatty acid composition: oleic acid decreased with decreasing growth rate while stearic acid increased.

Key words: Fatty acids, lipid, Rhodotorula, yeast.

The worldwide impending demand for oils and fats has led to a search for unconventional sources. Microbial production of lipids appears to be an attractive solution to this problem, particularly in those countries with restricted supplies of lipids from natural sources (AlmazKn et al. 1981; Moreton 1988). Rhodotorula glutinis is one of the principal oleaginous yeasts used for lipid production, being non-toxic and relatively easy to grow and harvest (Ratledge 1979, Misra et al. 1984). In this paper, we report the effect of the growth rate of this yeast, with sugar cane molasses at different concentration on the accumulation of lipids, using the continuous culture technique.

conditions and control set points were as follows: 171 working volume, temperature 32-F 0.1~ pH 5.5, 500 rev/min and air flow 0.7 v/v/rain. The following determinations were performed: yeast dry weight, total reducing sugars (TRS) by the modified Eynon-Lane method (De Whalley 1949), ammonia and total nitrogen by the micro-Kjeldahl method (Tecator Unit). For lipid extraction, 2 g dry cells were hydrolysed with 80 ml 1 M HC1 for 30 rain at 121~ the debris was recovered, washed until neutral, dried and then exhausted in a Soxhlet apparatus with diethyl ether. The extracted lipids were analyzed for fatty acid composition as their methyl esters by gas chromatography.

Results and Discussion Materials and Methods

Rhodotorula glutinis L/24-2-1, from the collection of the Cuban
Research Institute of Sugar Cane By-Products, was used. Nitrogen-limiting medium with the following composition (g/l): (NH4)2SO4, 0.29; (NH4)2HPOa, 0.7 and clarified molasses (equivalent to 20 to 40 g total reducing sugars) was employed. A 251 Biolaffite fermenter was used and the operation Table I shows that, as the dilution rate for growing Rhodotorula glutinis decreased, cell biomass, lipid content and lipid yield gradually increased. However, the specific lipid production rate and lipid productivity achieved their maximum values at a dilution rate of 0.05 h-1. A similar trend, but at a lower dilution rate, (0.02 h-1), was observed by Choi et al. (1982) and Yoon & Rhee (1983), using Rhodotorula glutinis on glucose. The C:N ratio did not change significantly when the sugar concentration was increased, because molasses contain significant amounts of organic nitrogen. However, at the same dilution rate, the synthesis of lipids, cell biomass, lipid

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 Investigaciones de los Derivados de la Carla de Az0car, CUBA-10, Apartado 4026, C. Habana, Cuba. L. Navarro is with the Instituto Nacional de Higiene y Epidemiologia, Infanta y Manglar, C. Habana, Cuba. *Corresponding author.

(~ 1992 Rapid Communications of Oxford Ltd

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Table 1. Quantitative data on the continuous culture of Rodotorula glutinis at different dilution rates and total reducing sugar (TRS) concentrations. Run no. Dilution rate (h -1)
0.1 0.06 0.06 0.05 0.05 0.04 0.04

Incoming medium (g/I) TRS

23 18 35 20 36 20 30

TRs (g/I) remaining

Biomass (g/I)

Y,~s

Total N 2
0.38 0.34 0.47 0.36 0.48 0.36 0.44

C:N
24 21 30 22 30 22 27 11.8 4.4 6.0 2.0 2.7 1.7 3.3 5.3 7.1 13.0 8.1 14.5 10.5 15.0 47.3 47.3 44.8 45.0 43.5 57.4 56.2

Lipid content (%)

15.5 27.0 29.2 32.4 37.4 34.0 39.2

YLIs

Dp

Dx

Dp/x

1 2 3 4 5 6 7

7.2 12.8 13.0 14.5 16.3 19.5 20.6

0.08 0.12 0.23 0.13 0.27 0.14 0.24

0.53 0.43 0.78 0.40 0.72 0.42 0.56

0.018 0.022 0.025 0.024 0.030 0.021 0.026

Yx/s: Biomass yield, (g cell biomass/100 g sugar consumed). YL/s: Lipid yield, (g lipid/100 g sugar consumed). Dp: Lipid productivity, (g lipid/I/h). Dx: Biomass productivity, (g biomass/I/h). Dp/x: Specific lipid production rate (g lipid-free biomass/h).

Table 2. Fatty acyl composition of lipids from Rhodotorula glutini$ at different dilution rates in continuous culture and batch culture. Run no. Dilution rate (h 1)
0.1 0.06 0.06 0.04

mainly due to an increase in levels of stearic acid and a decrease in oleic acid.

References
Almazan, O., Ktibansky, M. & Otero, M.A. 1981 Microbial fat synthesis by Rhodolorula glutinis from blackstrap molasses in continuous culture. Biotecknology Letters 3, 663-666. Choi, S.Y., Dewey, D.Y., Ryn & Rhee, J.S. 1982 Production of microbial lipids: Effects of growth rate and oxygen on lipid synthesis and fatty acid composition of Rhodotorula gracilis. Biotechnology and Bioengineering 24, 1165-1172. De Walley, H.C.S. 1949 Testing of molasses and sugar syrup. International Commission for Sugar Analysis 10th Session, Subject 6, pp. I3-26. Gonzalez, N. & Kopecky, J. 1972 Estudio de la calidad de las levaduras cubanas. Havana: Biotechnotogical Department, Cuban Research Institute of Sugar Cane By-Products. Misra, S., Grosh, A. & Durra, J. 1984 Production and composition of microbial fat from Rhodotorula glutinis. Journal of the Science of Food and Agriculture 35, 59-65. Moreton, R.S. 1988 Physiology of lipid-accumulating yeasts. In Single Cell Oil, ed Moreton, R.S., pp. 1-32, Harlow, Essex: Longman. Ratledge, C. 1979 Resource conservation by novel biological processes. I. Grow fats from wastes. Chemical Society Reviews 8, 283-296. Yoon, S.H. & Rhee, J.S. 1983 Lipids from yeast fermentation: Effects of cultural condition on lipid production and its characteristics of Rhodotorula glutinis. Journal of Americal Oil Chemists' Society 60, 1281-I286.

Rel. % (w/w) principal fatty acids 14:0

1.5 1.6 1.6 1.7 2.0

18:0
30 26 29 27 35

18:0
6 7 12 10 15

18:1
55 54 42 41 41

18:2
5 5 8 10 -

18:3

Batch* 1 2 3 6

2 3

* Batch culture, 72 h in shaker.

productivity and the specific lipid production rate were enhanced at higher concentrations of molasses. The maximum lipid content obtained was 39% (w/w) of dry cell biomass at a dilution rate of 0.04 h i and 30 g/1 of TRS. Under these conditions, biomass yield was 56 g cell biomass/100 g sugar consumed and the lipid yield was 20.6 g/IO0 g sugar consumed. The fatty acid composition of the lipids varied significantly with dilution rate (Table 2). For batch and continuous culture at a dilution of 0.1 h -~, fatty acid compositions were similar but as the dilution rate decreased, the overall degree of unsaturation decreased. This effect was

(Received 2 August 199I; accepted 24 August 1991)

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