Yarrowia lipolytica is one of the most extensively studied ‘‘non-conventional’’ yeast species, highlighted by their participation in bioremediation processes and production of lipases, γ-decalactone and citric acid. The use of immobilized cells presents several advantages over free cells in bioprocesses. In this work, Y. lipolytica entrapment in calcium alginate was applied for citric acid production from glycerol. The influence of sodium alginate and calcium chloride concentrations on the immobilization efficiency was investigated, being selected 4 % of sodium alginate and 0.5 M of calcium chloride, the best conditions. Afterwards, citric acid production was assessed at several spheres diameter and cells concentration, but no significant differences of final citric acid concentration were observed

Belo, Isabel

Braga, Adelaide

Ferreira, Patrícia

Gonçalves, Antónia

Universidade do Minho: RepositoriUM

 ChemPort2014 Poster session - Biological Engineering 10-14 Immobilization of whole cells of Yarrowia lipolytica for citric acid production  P-BE5 A. Gonçalves1,*, A. Braga1, P. Ferreira1, I. Belo1. (1) CEB- Center of Biological Engineering, University of Minho, Campus de Gualtar 4710-053, Braga, Portugal, *a.sales2712@gmail.com.   Yarrowia lipolytica is one of the most extensively studied ‘‘non-conventional’’ yeast species, highlighted by their participation in bioremediation processes and production of lipases, γ-decalactone and citric acid. The use of immobilized cells presents several advantages over free cells in bioprocesses. In this work, Y. lipolytica entrapment in calcium alginate was applied for citric acid production from glycerol. The influence of sodium alginate and calcium chloride concentrations on the immobilization efficiency was investigated, being selected 4 % of sodium alginate and 0.5 M of calcium chloride, the best conditions. Afterwards, citric acid production was assessed at several spheres diameter and cells concentration, but no significant differences of final citric acid concentration were observed.     Introduction  Yarrowia lipolytica is a eukaryotic microorganism, belonging to the Fungi kingdom. It is an obligate aerobic microorganism and one of the most extensively studied ‘‘non-conventional’’ yeast species. The ability of Y. lipolytica to grow under several environments (sewage, oil polluted media, marine and hypersaline environments and food) suggests the versatile metabolism of this yeast. This yeast is capable to use hydrophobic substrates such as n-alkanes, fatty acid methyl esters and triglycerides; and non-hydrophobic compounds like sugars, alcohols and organic acids. Beyond this feature, which made of Y. lipolytica a microorganism with biotechnological interest for bioremediation processes, this yeast is known as great producer of lipases [1], γ-decalactone [2], citric acid [3] and microbial lipids [4]. Citric acid, an organic acid intermediate of tricarboxylic acid cycle (TCA), is an acidulent flavouring agent and antioxidant, extensively used in food and beverage industry. The advantages on the use Y. lipolytica to biosynthesize citric acid, comparing with Aspergillus niger, include the several substrates that could be used as carbon source and the higher product yields. On traditional submerged fermentations, the use of free cells suspended in the medium influences the process yield, due to nutritional limitations, potential toxicity of reactants and products and difficulties in the recovery of final products. Thus, the use of immobilized cells constitutes a technology that has been explored, in order to solve those constraints. In nature, cells can be found immobilized as biofilms, microbial flocs and mycellial pellets. Artificially, the cells immobilization methods are based on surface attachment, entrapment within porous matrices, containment behind a barrier and self-aggregation. Immobilization of Y. lipolytica whole cells is possible, being described in the literature the use of agar-alginate composite beads for crude oil degradation [5]; methyl polymethacrylate and DupUM® for γ-decalactone production [6]; calcium alginate for oil wastewater degradation [7]; oil-absorbent polyurethane for the removal of oil films on surface waters [8]; among others. The central aim of this work is the production of citric acid using whole cells of Y. lipolytica entrapped into calcium alginate. Thus, strategies were delineated in order to select the best conditions for immobilization and production of citric acid.  Materials and methods  The strain used in this work was Y. lipolytica W29 (ATCC 20460), it was maintained on yeast-peptone-dextrose-agar medium (YPDA) at 4oC. Yeast cells were pre-grown in YPD medium (1 % yeast extract, 2 % peptone and 2 % glucose) at 27 °C and 170 rpm. A cell culture was used to prepare the cellular suspensions with 0.5 g·L-1, 1.5 g·L-1, 3 g·L-1 and 4.5 g·L-1, for experiments with free cells. For the immobilization experiments, it was used a cell culture of around 5 g·L-1 of cellular mass concentration (24 h of growth) to prepare the cellular suspension. The cells were separated from the medium by centrifugation and washed in NaCl (0.9 %). The cells for free  ChemPort2014 Poster session - Biological Engineering 10-15 experiments were resuspended in the medium, while the cells for immobilization experiments were resuspended in NaCl (0.9 %). Calcium alginate spheres were prepared as described in the literature [9] and as shown in the Graphical Illustration, in order to obtain a ratio between dry cell mass per unit mass of spheres and cells of 0.005±0.001.  In order to evaluate the effect of sodium alginate percentage and calcium chloride molarity in immobilization efficiency, spheres of around 3 mm of diameter were prepared with sodium alginate of 2 %, 3 % and 4 % and with calcium chloride molarity of 0.05 M, 0.1 M and 0.5 M. Afterwards, the spheres diameter was varied from around 2 to 4 mm. The ratios 0.005 gcells/gcells+support (~ 1.5 g·L-1 of cells in the medium), 0.010 gcells/gcells+support (~ 3 g·L-1 of cells in the medium) and 0.016 gcells/gcells+support (~ 4.5 g·L-1 of cells in the medium) were used in order to identify the best condition for citric acid biosynthesis. Citric acid production was carried out into 500 mL baffled Erlenmeyer flask with 200 mL of medium (glycerol (40 g·L-1), yeast extract (0.5 g·L-1), KH2PO4 (6 g·L-1), Na2HPO4 (0.5 g·L-1), MgSO4.7H20 (1.5 g·L-1), CaCl2 (0.15 g·L-1), ZnSO4.7H20 (0.02 g·L-1), FeCl3.6H20 (0.15 g·L-1) and MnSO4.H20 (0.06 g·L-1)) and maintained at 27 ºC, 170 rpm during 168 h. Periodically, analyses were made in order to monitor the pH, concentration of cells, glycerol consumption and citric acid production.  Results and discussion  The range of initial cells concentration used at experiments with free cells resulted on profile of citric acid concentrations presented in Figure 1. The increase of the initial cells concentration from 0.5 g·L-1 to 1.5 g·L-1 result in a best concentration of citric acid obtained (from 2.25 g·L-1 to 3.09 g·L-1). However, when this increase reaches to 3 g·L-1 or 4.5 g·L-1 of cells, around 104 h and 80 h after the fermentation, respectively, occurs a decrease in citric acid concentration. This reduction can be due to the consumption of citric acid, since the decrease starts after the depletion of glycerol. Thus, initial cells concentration that results in the better production of citric acid, within the range of tested concentrations, was 1.5 g·L-1, which was selected to use in further experiments with immobilized cells.   In cells immobilization, within the different percentage of sodium alginate and molarity of calcium chloride tested, the best retention of yeast cells in the spheres was obtained with 4 % of sodium alginate and 0.5 M chloride calcium. This result is supported by literature [10], which revealed that an increase in the concentration of sodium alginate and calcium chloride decrease the immobilized leakage.  Spheres in the above conditions were made with different diameters and its increase slightly reduced citric acid production (Table 1), but global productivity was unchanged (0.03 g·L-1·h-1). Thus, the spheres diameter did not influence the citric acid final concentration and 3 mm was selected.  Table 1. The higher concentrations of citric acid obtained for the range of spheres diameter tested. Spheres diameter (mm) Citric acid (g·L-1) 2.7 ± 0.2 5.54 3.4 ± 0.1 5.09 4.2 ± 0.1 4.97  Similar values of citric acid concentrations and equal global productivity (0.03 g·L-1·h-1) were obtained independently of cell concentration in the gel matrix (Table 2). Contrary to free cells, no consumption of citric acid was observed, and higher final citric acid concentration values were reached.   Figure 1. Citric acid concentrations obtained over 168 h using 0.5 g·L-1 (♦), 1.5 g·L-1 (▲), 3 g·L-1 (●) and 4.5 g·L-1 (■) of initial concentration of free cells in the medium.  ChemPort2014 Poster session - Biological Engineering 10-16 Table 2. The higher concentrations of citric acid obtained for the range of initial cells concentration immobilized into spheres. gcells/gcells+support Citric acid (g·L-1) 0.005 5,11 0.010 4,84 0.016 4,99  Conclusions  The work herein presented showed that for Y. lipolytica cells a strong gel matrix is needed to prevent cell leakage and to enable an efficient cell entrapment. For the strain W29 a 4% sodium alginate and 0.5 M calcium chloride was selected.  A final citric acid concentration of around 5 g·L-1 was possible to obtain with cells immobilized in spheres of around 3 mm of diameter and with a cells load of around 0.010 ± 0.005 dry cell mass per unit mass of spheres and cells. It was proven the advantage of using immobilized cells against free cells, since the citric acid consumption was prevent, and higher values of concentration were obtained.  Thus, this immobilization method presents great potential for citric acid continuous production by the Y. lipolytica from glycerol.  Acknowledgements Authors thank Fundação para a Ciência e a Tecnologia (FCT) for financial support through the project FCT Pest-OE/EQB/LA0023/2011 and SFRH/BD/63701/2009 and SFRH/BD/72621/2010 PhD grants. Also, authors thank the Project “BioInd - Biotechnology and Bioengineering for improved Industrial and Agro-Food processes, REF. NORTE-07-0124-FEDER-000028” Co-funded by the Programa Operacional Regional do Norte (ON.2 – O Novo Norte), QREN, FEDER.  References [1] G. Corzo, S. Revah, Bioresource Technology, 70 (1999) 173-180. [2] M. Aguedo, M. H. Ly, I. Belo, J. A. Teixeira, J-M. Belin, Y. Waché, Food Technology and Biotechnology, 42 (2004) 327-336. [3] S. Papanikolaou, I. Chevalot, M. Komaitis, G. Aggelis, I. Marc, Appl Microbiol Biotechnol, 58 (2002) 308-312. [4] P. Fickers, P. H. Benetti, Y. Wache, A. Marty, S. Mauersberger, M. S. Smit, J. M. Nicaud, FEMS Yeast Res, 5 (2005) 527-543. [5] S. S. Zinjarde, A. Pant, Journal of Environmental Science and Health, Part A: Toxic/Hazardous Substances and Environmental Engineering, 35 (2000) 755-763. [6] A. Braga, I. Belo. Appl Biochem Biotechnol, 169 (2013) 2202-2211. [7] L. Wu, G. Ge, J. Wan, Journal of Environmental Sciences, 21 (2009) 237-242 [8] Y-S. Oh, J. Maeng, S. J. Kim, Appl Biochem Biotechnol, 54 (2000) 418-423. [9] H. Kautola, W. Rymowicz, Y-Y. Linko, P Linko, Appl Microbiol Biotechnol, 35 (1991) 447-449. [10] A. Blandino, M. Macías, D. Cantero, Enzyme and Microbial Technology, 27 (2000) 319–324.  

Immobilization of whole cells of Yarrowia lipolytica for citric acid production

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