EFFECT OF SODIUM ACETATE ON CITRIC ACID PRODUCTION FROM GLUCOSE BY YARROWIA LIPOLYTICA

Małgorzata Robak¹, Waldemar Rymowicz¹, Paweł Filipkowski^2
1 Department of Biotechnology and Food Microbiology, Wrocław University of Environmental and Life Sciences, Poland
² Department of Food Chemistry, Technology and Biotechnology, Gdansk University of Technology, Poland

ABSTRACT

The effect of sodium acetate addition on citric acid (CA) biosynthesis in batch fermentation process from glucose by acetate non utilizing (acu^-) mutant Yarrowia lipolytica A-101.1.31 was studied. Acetate as co-substrate in glucose medium, has improved the specific production rate in the first 24 hours of process, yield and productivity. Among the tested doses (1, 2, 8, 16, 20 gL^-1) the addition of 2 gL^-1 of sodium acetate had the most positive effect on the CA biosynthesis by Y.lipolytica A-101.1.31. In the future, a study on a continuous addition of sodium acetate could be taken into consideration because acetate was rapidly metabolized.

Key words: Yarrowia lipolytica, acetate utilisation, citrate production .

INTRODUCTION

Citric acid (CA) production by yeast could be in the future an alternative to Aspergillus niger one, specially if the yeast biomass became an additive to animal food and not a by-product. CA fermentation by various Y. lipolytica strains has been developed including free or immobilized-cells processes and continuous cell-recycle systems [1,2 12,17]. In order to improve CA secretion studies on glucose media and process parameters optimization have been effectuated for several mutants of Yarrowia lipolytica A-101, wide strain isolated from soil [16,17,18]. In some recently published studies the simultaneous utilization of glucose and acetate by Y. lipolytica was observed [14] and the use of two-substrates has emerged as a new possible way for CA biosynthesis optimization. Such co-utilization of glucose and acetate was reported by O’Beirne and Hamer in case of Escherichia coli [11] and by Wendisch et al. for Corynebacterium glutamicum [22].

The aim of this study was to evaluate the effect of sodium acetate addition on citric acid (CA) biosynthesis in batch fermentation process from glucose by acetate non utilizing (acu^-) mutant Y. lipolytica A-101.1.31.

MATERIAL AND METHODS

Microorganism and CA biosynthesis conditions. The strain Yarrowia lipolytica A-101.1.31 (acu^-, UV mutant) from the culture collection of the Department of Biotechnology and Food Microbiology, Wroclaw University of Environmental and Life Sciences of was used in the study. Biosynthesis medium in 1L of tap water contained: glucose (100 g), NH₄Cl (1.5 g), KH₂PO₄ (0.2 g), MgSO₄ x7H₂O (1 g), YE (0.9 g) and sodium acetate at the concentration of 0, 1, 2, 8, 16 and 20 g. The CA biosynthesis as batch fermentation (BC), was conducted in stirred jar bioreactor Bioflow III during 72 hours, at 30^oC with agitation rate of 550 rpm and aeration of 0.2 vvm. Culture acidity was automatically controlled at pH 5.5 (30% NaOH). For analysis, 10 mL samples were taken twice a day. The first samples were taken 15 minutes after the start of cultures.

Analysis. CA and acetate were determined by HPLC separation on Animex HPX-87H column (flow rate of 0.6 mLh^-1, elution with 0.01 N H₂SO₄, UV detection at 210 nm). Glucose was determined with glucose oxidase Kit (Poch, Gliwice, Poland) and biomass was determined by dry weight method.

RESULTS AND DISCUSSION

Biomass, citric acid accumulated in the medium and glucose residual level in 72 hours batch fermentation process with Y. lipolytica A-101.1.31 on glucose and sodium acetate (0-2%) were summarized in the Table 1. Three days accumulated biomass reached the level of ~12 gL^-1 for all of the tested doses of acetate (0-2%) and was comparable to that obtained during CA biosynthesis on ethanol [2], slightly lower than on glucose [17] and higher than on glycerol [12]. Yeast started to produce CA after 10-12 hours of process, independently of the acetate concentration. The maximal CA concentration obtained was 55 gL^-1 without acetate supplementation, 58 and 62 gL^-1 with 0.2 and 0.1% of acetate, respectively. It was comparable to the amount noted by others in BC processes [2,12,17].

The kinetics of biomass and citric acid accumulation, as well as substrates consumption during the process with the addition of 0.1 and 8 gL^-1 of sodium acetate as the second substrate, were presented on Fig. 1, 2 and 3, respectively. During the first 24 hours, the specific production rate -q_CA- reached the value of 0.088 gg^-1h^-1 without acetate and 0.096 gg¹h^-1 with the addition of 0.1 or 0.2% of sodium acetate (Table 2), therefore approaching the best value – 0.1 gg^-1h^-1 – reported for Y. lipolytica [7,17,23]. Arzumanow et al. in BC have reported decreasing specific productivity [2]. The highest value of q_CA, 0.16 gg^-1h^-1, was noted during the first 36-48 hours and the lowest 0.05 gg^-1h^-1 in 120-144 hours of BC.

It was shown earlier that the growth of acetate non utilizing mutants of Y. lipolytica was induced by the presence of 0.01% of glucose [13]. Now we have noticed that sodium acetate, even at the dose of 2 %, was rapidly (in 10-24 hours) metabolized by Y.lipolytica A-101-1.31 yeast from the glucose containing medium (Fig. 2, 3 and Table 1). So, the influence of that second C source on CA production must be taken into consideration only during the first 24 hours (Table 2). In that way, the volumetric process productivity increased about 30% and the yield nearly 47% in comparison to these parameters calculated for the same period of time for the process without acetate (Table 1 and 2). The maximal volumetric process productivity was 1.09-1.13 gL^-1 h^-1, and the yield was 0.59-0.66 gg^-1 for the culture with 0.2 and 0.1% of acetate respectively. At the end of the process (at 72 hours) the difference in these parameters was not so significant, although the yield increased 6.8-14.9%, respectively for the process with 0.1 and 0.2% of acetate. In the previous study, the yield of citric acid biosynthesis by Y.lipolytica A-101 strain on glucose was 0.51-0.78 gg^-1 in continuous and RBC process [16], but degeneration of yeast population leading to the decrease of CA production was observed. The addition of acetate may be the factor inducing a longer stability of Y. lipolytica population in the bioreactor. Such positive influence was observed by Chang et al. [3] for bacteria. They demonstrated the importance of acetate for the growth and survival of E.coli W3110. The beneficial action of acetate addition was reported by Dugelay et al [1999] in rabbit kidney tricarboxylic acids cycle activity, by Chen and Blaschek [4] in butanol production by Clostridium beijerinckii, by Nilsson et al [10] in the bacteriocin production by Carnobacterium piscicola, by Nezu and Shimokava [9] in orotic acid production by Candida albicans mutants and by Tunner et al [20] in betagalactosidase production by E.coli.

In the studies of yeasts acetate metabolism, Gentsch et al [6] and Tzschoppe et al [21] have described the mutation sites responsible for hypersensitivity to acetic acid in S. cerevisiae as well as in Y. lipolytica. Sousa et al [19] described transport of acetic acid in Zygosaccharomyces bailii. Rodrigues and Pais [15] have studied the influence of acetic and other weak carboxylic acids on the growth and cellular death of the yeast Y. lipolytica. Kujau et al. [8] have characterized Y. lipolytica acu^- mutants, showing that some of them were defective in acetyl-CoA synthetase and recently Robak [14] has shown differences in growth rate of acu^- mutants of Y. lipolytica on glucose, ethanol, glycerol and acetate. This paper is the first report about positive effect of acetate on citrate biosynthesis from glucose by Y. lipolytica.

CONCLUSION

Summarizing, the addition of acetate to the glucose medium has improved, especially in the first 24 hours, specific production rate, yield and productivity of citric acid biosynthesis by Yarrowia lipolytica yeast. Among the tested doses (1, 2, 8, 16, 20 gL^-1) the addition of 2 gL^-1 of sodium acetate has the most positive effect on the CA biosynthesis by Y. lipolytica A-101.1.31. This study has to be continued in order to precise the sodium acetate concentration leading to a more effective accumulation of CA by used yeast strain from glucose. Because the doses of sodium acetate between 2 and 8 gL^-1 were not tested, and a continuous addition can be taken into consideration.

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Accepted for print: 25.11.2007

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