Downstream processing of mannosylerythritol lipids produced by ...

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378 U. Rau et al. Eur. J. Lipid Sci. Technol. 107 (2005) 373–380 4 Discussion Different microorganisms as, e.g., Kurtzmanomyces sp. I-11 [12], Candida antarctica [14] and Ustilago maydis [10] were used by other authors for shake flask production of MEL. Kitamoto’s group succeeded in the formation of 140 g MEL L 21 by additional feeding of n-octadecane using Pseudozyma (Candida) antarctica T 34 [19]. However, only rare information about bioreactor production of MEL is available. Kim et al. [15] used Candida sp. SY16 in a 5-L bioreactor for the formation of 100 g L 21 crude MEL phase containing only 4% (wt-%) pure MEL. Hitherto, 46 and 165 g L 21 are the highest reported yields of MEL obtained in a bioreactor by using Pseudozyma (Candida) antarctica ATCC 20509 [16] and Pseudozyma aphidis DSM 70725 [18], respectively. The choice of method for the isolation and purification of a particular biosurfactant depends on its ionic charge, its solubility in water, and on whether the product is cell bound or extracellular. The methods used include solvent extraction, adsorption followed by solvent extraction, precipitation, crystallisation, centrifugation and foam fractionation. Desai and Desai [20] as well as Syldatk and Wagner [21] gave an overview of these procedures. Solvent extraction is the most commonly used technique for the downstream processing of biosurfactants. We also described such a multi-step extraction (Fig. 3) for MEL that is further on absolutely necessary if, e.g., spectroscopic investigations are performed or an application as pharmaceuticals is intended [3, 4]. The disadvantage of this process is the production of huge amounts of waste solvents that have to be recycled. Beside the costs for bioreactor production, the recycling increases the manufacturing costs so that an acceptance of this bioprocess for industry is additionally inhibited. Fig. 5. HPLC and TLC of the solid MEL phase after heat treatment at 121 7C for 20 min. The HPLC signals correspond with the grouped bars of the solid phase of Fig. 4. Distribution of MEL (wt-%): A = 40.8, B = 43.4, C = 9.7, D = 6.1. The embedded TLC corresponds with the grouped bars of Fig. 4: Before heat treatment, 1 = viscous MEL phase; after heat treatment, 2 = aqueous phase, 3 = solid phase. Unfortunately, the specific adsorption of MEL or soybean oil and fatty acids failed by the use of different Amberlite resins. Only a general tendency to higher accumulation of MEL was observed in the order: XAD-16 . XAD-7HP . XAD-4. The MEL can also be isolated by preparative HPLC equipped with silica gel columns [15, 17, 22]. This is a superior method to produce a very pure MEL mixture or even to separate the individual MEL. However, the loss of product is substantial, and so this is not a beneficial solution in order to decrease the costs for the downstream process. A very easy, solvent-free separation of a MEL-enriched solid phase could be achieved by just heating the culture suspension to a temperature of 100 7C. Related to the total MEL content of the untreated culture suspension, the heating at 110 7C for 10 min led to the maximum transfer of 93% (wt-%) MEL into this solid phase. The supernatant could be poured off, which left a solid, highly viscous mass, on average composed of 87% (wt-%) MEL (Figs. 4, 5). The remaining 13% (wt-%) consisted of soybean oil and fatty acids in different amounts, depending on time and temperature throughout the treatment. Independent of the temperature, the longer the time of treatment, the more soybean oil was transferred to a new phase separated at the top. Furthermore, prolonged heating favoured the release of fatty acids from the remaining soybean oil. A comparison of known MEL downstream procedures is given in Tab. 1. Unfortunately, to the best knowledge of the authors, only two references are available with a quantitative description of the methods. The heat treatment attained the highest yield and is the fastest method by far, but only on average 87% (wt-%) MEL is contained © 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.ejlst.de
Eur. J. Lipid Sci. Technol. 107 (2005) 373–380 Downstream MEL 379 Tab. 1. Comparison of different methods for the downstream processing of MEL { . Ref. Method § Yield [wt-%] # Purity [wt-%] [22] Ethyl acetate extraction 1 preparative HPLC 79 100 [15] Ethyl acetate extraction 1 preparative HPLC 4 100 Stepwise extraction with different solvents (Fig. 3) 8 100 Heat treatment (Fig. 4) 93 87 { Preparative HPLC was performed with silica gel columns. § g MEL recovered Yield = g MEL before downstream 100 # Purity is related to the mass fraction of MEL. in the precipitated fraction. However, this solid-enriched MEL phase should be pure enough for the most industrial applications [5–8]. For example, pure MEL A, purified 100% (wt-%) MEL and a mixture of 88.3% MEL, 6.6% soybean oil and 5.1% (wt-%) fatty acids reduced the surface tension of water/air to similar data of 34.7, 26.7 and 31 mN m 21 , respectively [17]. 5 Conclusion Bioreactor production of mannosylerythritol lipids was performed by the use of Pseudozyma aphidis DSM 14930. Up to 93% (wt-%) MEL could be transferred into a solid, highly viscous phase by heating the culture suspension to 110 7C for 10 min. This phase contained on average 87% (wt-%) MEL and could be simply isolated by pouring off the supernatant, without producing solvent waste. Together with the high MEL yield of 165 g L 21 , obtained by foam-controlled addition of soybean oil [18], this facilitated downstream process should stimulate the industrial production of MEL. Acknowledgments We thank W. Grassl for technical assistance. References [1] G. Georgiou, S. C. Lin, M. Sharma: Surface-active compounds from microorganisms. Biotechnology 1992, 10,60–65. [2] D. Kitamoto, H. Isoda, T. Nakahara: Functions and potential applications of glycolipid biosurfactants – from energy-saving materials to gene delivery carriers. J Biosci Bioeng. 2002, 94, 187–201. [3] M. Shibahara, X. Zhao, Y. Wakamatsu, N. Nomura, T. Nakahara, C. Jin, H. Nagaso, T. Murata, K. K. Yokoyama: Mannosylerythritol lipid increases levels of galactoceramide in and neurite outgrowth from PC12 pheochromocytoma cells. Cytotechnol. 2000, 33, 247–251. [4] L. Vertesy, M. Kurz, J. Wink, G. Noelken: Patent US 6,472,158 (2002). [5] J. H. Im, H. Yanagishita, T. Ikegami, Y. Takeyama, Y. Idemoto, N. Koura, D. Kitamoto: Mannosylerythritol lipids, yeast glycolipid biosurfactants, are potential affinity ligand materials for human immunoglobulin G. J Biomed Mater Res. 2003, 65A, 379–385. [6] D. Kitamoto, H. Yanagishita, A. Endo, M. Nakaiwa, M. Nakane, T. Akiya: Remarkable antiagglomeration effect of a yeast biosurfactant, diacylmannosylerythritol, on ice-water slurry for cold thermal storage. Biotechnol Progress 2001, 17, 362–365. [7] Z. Hua, J. Chena, S. Luna, X. Wang: Influence of biosurfactants produced by Candida antarctica on surface properties of microorganism and biodegradation of n-alkanes. Water Research 2003, 37, 4143–4150. [8] Z. Hua, Y. Chen, G. Du, J. Chen: Effects of biosurfactants produced by Candida antarctica on the biodegradation of petroleum compounds. World J Microbiol Biotechnol. 2004, 20, 25–29. [9] D. Kitamoto, S. Akiba, C. Hioki, T. Tabuchi: Extracellular accumulation of mannosylerythritol lipids by a strain of Candida antarctica. Agric Biol Chem. 1990, 54, 31–36. [10] S. Spoeckner, V. Wray, M. Nimtz, S. Lang: Glycolipids of the smut fungus Ustilago maydis from cultivation on renewable resources. Appl Microbiol Biotechnol. 1999, 51, 33–39. [11] G. Deml, T. Anke, F. Oberwinkler, B. M. Gianetti, W. Steglich: Schizonellin A and B, new glycolipids from Schizonella melanogramma. Phytochem. 1980, 19, 83–87. [12] K. Kakugawa, M. Tamai, K. Imamura, K. Miyamoto, S. Miyoshi, Y. Morinaga, O. Suzuki, T. Miyakawa: Isolation of yeast Kurtzmanomyces sp. I-11, novel producer for mannosylerythritol lipid. Biosci Biotech Biochem. 2002, 62, 188– 191. [13] H. Kawashima, T. Nakahara, M. Oogaki, T. Tabuchi: Extracellular production of a mannosylerythritollipid by a mutant of Candida sp. from n-alkanes and triacylglycerols. J Ferment Technol. 1983, 61, 143–149. [14] D. Kitamoto, T. Yokoshima, H. Yanagishita, K. Haraya, H. K. Kitamoto: Formation of glycolipid biosurfactant, mannosylerythritol lipid, by Candida antarctica from aliphatic hydrocarbons via subterminal oxidation pathway. J Jpn Oil Chem Soc. 1999, 48, 1377–1384. [15] H.-S. Kim, B. D. Yoon, D. H. Choung, H.-M. Oh, T. Katsuragi, Y. Tani: Characterization of a biosurfactant, MEL, produced from Candida sp. SY16. Appl Microbiol Biotechnol. 1999, 52, 713–721. [16] M. Adamczak, W. Bednarski: Influence of medium composition and aeration on the synthesis of biosurfactant produced by Candida antarctica. Biotechnol Lett. 2000, 22, 313–316. [17] U. Rau, L. A. Nguyen, S. Schulz, V. Wray, M. Nimtz, H. Roeper, H. Koch, S. Lang: Formation and analysis of mannosylerythritol lipids secreted by Pseudozyma aphidis. Appl Microbiol Biotechnol. 2005, 66, 551–559. [18] U. Rau, L. A. Nguyen, H. Roeper, H. Koch, S. Lang: Fedbatch bioreactor production of mannosylerythritol lipids secreted by Pseudozyma aphidis. Appl Microbiol Biotechnol. 2005, DOI 10.1007/s00253-005-1906-5. © 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.ejlst.de
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