Downstream processing of mannosylerythritol lipids produced by ...

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374 U. Rau et al. Eur. J. Lipid Sci. Technol. 107 (2005) 373–380 2.2 Media and cultivation conditions The seed culture contained 100 mL medium in 500 mL baffled shake flasks and was inoculated from agar slants. The medium for precultivation contained (all media data are related to 1 L deionised water): 30 g glucose, 1 g NH 4NO 3, 0.3 g KH 2PO 4, 1 g yeast extract, pH = 6.0 (not controlled). After 2 d on a rotary shaker at 110 rpm and 30 7C, the preculture was transferred into the following bioreactor medium: 80 mL soybean oil (density 0.84 g mL 21 ), 2 g NaNO 3, 0.2 g KH 2PO 4, 0.2 g MgSO 4 7H 2O, 1 g yeast extract, pH = 6.2 (not controlled), temperature 27 7C. A 72-L bioreactor (Sartorius BBI Systems GmbH, Melsungen, Germany) equipped with three Rushton turbine impellers was used. The bioreactor was filled with only 30 L medium due to foam formation. As initial impeller speed and aeration rate, 300 rpm and 720 L h 21 , respectively, were applied. Soybean oil as antifoam agent and carbon source was additionally supplied throughout the cultivation. The bioreactor cultivation is described in detail elsewhere [18]. 2.3 Analyses In order to quantitatively detect MEL, triglycerides and fatty acids, 3 mL culture suspension was acidified with two drops of 5 N HCl to pH 2 and was subsequently extracted three times with 3 mL methyl tertiary butyl ether (MTBE). The mixture was vortexed for 1 min and centrifuged for 5 min at 6000 rpm. The organic phases were combined and analysed either by TLC or HPLC. The distinct separation of single spots for qualitative TLC analysis was carried out by the development of Silica gel 60 F 254 (Merck, Germany) plates with the ternary solvent system CHCl 3/MeOH/H 20 (65:15:2). HPLC was performed on a silica gel column (Nucleosil 100-5; CS-Chromatographie Service GmbH, Langerwehe, Germany) with an evaporative light scattering detector (ACS Mass Detector model 750/14; Houston, TX, USA) using a gradient solvent program consisting of various proportions of CHCl 3 and CH 3OH (from 99:1 to 0:100, vol/vol) at a flow rate of 1 mL min 21 [17]. The data of MEL, soybean oil, fatty acids and cell protein are mean values from at least two independent determinations. Differences of individual data varied between 2% and 7%. Due to non-uniform accumulation of storage material inside the cells, bio dry mass showed higher deviations from the mean value and, therefore, up to a fourfold determination was carried out. 2.4 Surface tension The influence of MEL on the surface tension of water at 25 7C was measured with a tensiomat (Lauda Wobser GmbH, Lauda-Königshofen, Germany) using the ring method. The detailed description of this method is published elsewhere [17]. 2.5 Downstream processing of MEL 2.5.1 Adsorption at XAD Different types of Amberlite XAD (Rohm and Haas, Philadelphia, PA, USA) were used as adsorbent. XAD-4 and XAD-16 are cross-linked polymers with different groups at their surface for the preferred adsorption of organic compounds with low and low-to-medium molecular weights, respectively. XAD-7HP can adsorb non-polar compounds from aqueous systems as well as polar compounds from non-polar solvents. Adsorption tests were carried out as follows: 10 mL culture suspension was added to 4 mL Amberlite beads. After 24 h of stirring, the beads were separated by filtration (glass fibre filter) and rinsed with 10 mL water. The rinsing solution was extracted by the addition of 10 mL MTBE under vigorous mixing. The beads were divided and subsequently extracted with 10 mL of either MTBE or methanol. The organic phases were analysed and compared by TLC. 2.5.2 Extraction with organic solvents The thoroughly mixed culture suspension was first extracted three times with MTBE. Aqueous and organic phases were separated by centrifugation. The collected organic phases were evaporated and vacuum dried (60 7C, 3000 Pa). After solving the extract in methanol, it was subsequently treated also three times, but with cyclohexane. The again evaporated and dried extract still contained minor amounts of soybean oil and fatty acids. These compounds were removed by using a mixture of n-hexane, methanol and water (1:6:3, pH 5.5) as well as subsequent threefold extraction of the aqueous phase by n-hexane. The lyophilised aqueous phase resulted in a pure MEL fraction as transparent, brown-coloured, highly viscous fluid. 2.5.3 Heating After heating of the culture suspension to 100–121 7C for 1–20 min, two MEL-containing phases, a solid sticky and an aqueous one, were formed. About 90% (wt/vol) of MEL was transferred into the solid phase by this procedure, which could be easily separated by pouring off the cell debris-containing aqueous phase. © 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.ejlst.de
Eur. J. Lipid Sci. Technol. 107 (2005) 373–380 Downstream MEL 375 3 Results 3.1 Bioreactor cultivation A representative example of bioreactor cultivation for the production of MEL using Pseudozyma aphidis DSM 14930 is shown in Fig. 2. After about 1 d, nitrate and yeast extract as nitrogen sources were consumed, and the course of cell protein indicated the approach to the stationary phase. However, bio dry mass continued to increase due to the accumulation of storage material inside the cells. After growth had ceased, the formation of foam cumulatively increased; therefore, impeller speed and aeration rate had to be reduced gradually from 300 to 250 rpm and from 720 to 100 L h 21 , respectively. In spite of these modifications, the pO 2 was not influenced essentially and remained at about 60%. The reduction of impeller speed and aeration rate was not sufficient to prevent overfoaming. For this reason, soybean oil was additionally fed in various rates, both as carbon source and as anti-foam agent. Depending on the addition and consumption rate, different amounts of soybean oil and fatty acids, released from soybean oil by the lipolytic activity of P. aphidis, were detected. However, repeated cultivations showed that after soybean oil addition was stopped, a prolongation of the cultivation time by 1 d was sufficient for the total assimilation of residual substrates. After 3 d, the first green to yellow MEL beads separated at the bottom of the sampling bottle. The number and width (2–10 mm) of these beads increased with time. Previous investigations [18] showed that the MEL beads were formed at a concentration greater than 40 g MEL L 21 and contained high quantities of MEL .60% (wt-%) and relatively small amounts of soybean oil ,20% (wt-%) as well as fatty acids ,10% (wt-%). After 8 d of cultivation, 90 g MEL L 21 was yielded. Fig. 2. Cultivation of P. aphidis in a 30-L bioreactor. After 1 d, the impeller speed and aeration rate were gradually decreased from 300 to 250 rpm and from 720 to 100 L h 21 , respectively, depending on the foam formation. Between days 2.5 and 8, soybean oil was fed at various rates (0.2– 0.67 g L 21 h 21 ). The first MEL beads were observed after 3 d. © 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.ejlst.de
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