Appl Microbiol Biotechnol DOI 10.1007/s00253-014-5585-y
APPLIED MICROBIAL AND CELL PHYSIOLOGY
The peculiarities of succinic acid production from rapeseed oil by Yarrowia lipolytica yeast Svetlana V. Kamzolova & Natalia G. Vinokurova & Emiliya G. Dedyukhina & Vladimir A. Samoilenko & Julia N. Lunina & Alexey A. Mironov & Ramil K. Allayarov & Igor G. Morgunov
Received: 21 December 2013 / Revised: 24 January 2014 / Accepted: 28 January 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract The process of succinic acid (SA) production represents the combination of microbial synthesis of αketoglutaric acid from rapeseed oil by yeast Yarrowia lipolytica VKM Y-2412 and subsequent decarboxylation of α-ketoglutaric acid by hydrogen peroxide to SA that leads to the production of 69.0 g l−1 of SA and 1.36 g l−1 of acetic acid. SA was isolated from the culture broth filtrate in a crystalline form. The SA recovery from the culture filtrate has certain difficulties due to the presence of residual triglycerides of rapeseed oil. The effect of different methods of the culture filtrate treatment and various sorption materials on the coagulation of triglycerides was studied, and as a result, the precipitation of residual triglycerides by acetone was chosen. The subsequent isolation procedures involved the decomposition of H2O2 in the filtrate followed by filtrate bleaching and acidification with a mineral acid, evaporation of filtrate, and SA extraction with ethanol from the residue. The purity of crystalline SA isolated from the culture broth filtrate achieved 97.6–100 %. The product yield varied from 62.6 to 71.6 % depending on the acidity of the supernatant.
Keywords Microbial production . Succinic acid (SA) . α-Ketoglutaric acid . Rapeseed oil . Yarrowia lipolytica . Purification S. V. Kamzolova (*) : N. G. Vinokurova : E. G. Dedyukhina : V. A. Samoilenko : J. N. Lunina : A. A. Mironov : R. K. Allayarov : I. G. Morgunov G.K. Skryabin Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, Prospect Nauki 5, Pushchino, Moscow Region, Russia 142290 e-mail: [email protected] A. A. Mironov : R. K. Allayarov : I. G. Morgunov Pushchino State Institute of Natural Sciences, Prospekt Nauki 3, Pushchino, Moscow Region, Russia 142290
Introduction Succinic acid (C4H6O4) (SA) has found wide applications in industry as a starting material for the synthesis of highperformance chemicals (1,4-butanediol, adipic acid, tetrahydrofuran, γ-butyrolactone, N-methylpyrrolidone) (Zeikus et al. 1999; Carole et al. 2004), as a fodder additive for stimulation of propionate formation in ruminant animals and enhancing the efficiency of farm animal production (Samuelov et al. 1999), and as a protector of humans against oxidative stress (Kondrashova 1991, 2002). The mono- or disodium salts of SA are used as a flavor to meats and soups; dioctyl sodium sulfosuccinate is used as a surfactant and wetting agent for textile and printing products and as a component of the Corexit dispersant applied in the recent Gulf oil spill cleanup operations (Thakker et al. 2012). The market potential for SA and its derivatives has been predicted to be up to 245×103 tons/year, with a market for the SA-derived polymers of 2×106 tons/year (Bozell and Petersen 2010). For industrial purposes, SA is produced by chemical synthesis from maleic anhydride; however, microbiological synthesis of SA is considered as more perspective due to its ecological and economical advantages (Zeikus et al. 1999; Carole et al. 2004; Werpy et al. 2006; McKinlay et al. 2007; Bechthold et al. 2008; Sauer et al. 2008; Bozell and Petersen 2010; Skorokhodova et al. 2013). It is noted that food and pharmaceutical industry will prefer to use bio-succinate to avoid potential health hazards (Gascon et al. 2006). The microbial production of SA is commonly based on the use of bacteria, such as Anaerobiospirillum succiniciproducens, Actinobacillus succinogenes, Mannheimia succiniciproducens, recombinant Escherichia coli strains, and Corynebacterium glutamicum. As an alternative of bacterial fermentations, the coauthors of the present study developed two-stage processes of SA production by yeast Yarrowia lipolytica that involve the biosynthesis of α-ketoglutaric acid from ethanol or n-alkanes,
Appl Microbiol Biotechnol
and the subsequent decarboxylation of α-ketoglutaric acid to SA in the presence of a strong oxidant—hydrogen peroxide (H2O2) (Kamzolova et al. 2009, 2012a, c). Currently, triglycerides of plant oils (rapeseed, sunflower, and palm oils) have attracted increased interest as substrates for the development of biotechnological processes; the products manufactured from plant oils are permissible for usage in the food industry and medicine. Moreover, plant oils provided high product yield (Glatz et al. 1984; Stottmeister et al. 2005; Otto et al. 2011; Aurich et al. 2012). Among plant oils, the rapeseed oil is considered as the most promising substrate for the development of biotechnological processes due to the high rape seed crop (from 1 ha, up to 1,000 kg of rapeseed oil can be obtained) and attracts increased interest as a raw material for biodiesel production. Moreover, rapeseed oil is rich in minerals and vitamins that may provide valuable nutrients for cell growth stimulation and production of valuable metabolites. The aim of the present work was to develop an efficient process of SA production from rapeseed oil, which includes microbial synthesis of α-ketoglutaric acid and its subsequent decarboxylation to SA and to elaborate effective method of SA extraction.
Materials and methods Microorganism For microbial synthesis of α-ketoglutaric acid from rapeseed oil, we used the strain Y. lipolytica VKM Y-2412 obtained from the All-Russian Collection of Microorganisms (VKM). This strain was characterized by a high production of α-ketoglutaric acid from ethanol (Kamzolova et al. 2009, 2012b), n-alkanes (Kamzolova et al. 2012a), and rapeseed oil (Kamzolova and Morgunov 2013). The strain was maintained at 4 °C on agar slants with n-alkanes as the carbon source. Chemicals All chemicals were of analytical grade (Sigma-Aldrich, St. Louis, MO, USA). Rapeseed oil was purchased from the “Russian seeds” Processing Plant (Vinev, Russia). The fatty acid profile of the rapeseed oil which had a total unsaturated fatty acid mass fraction of 93.6 % was as follows (percentage, by mass): C16:0, 4.0; C18:0, 1.2; C18:1, 58.8; C18:2, 28.1; and C18:3, 5.9. Cultivation Y. lipolytica VKM Y-2412 was cultivated in a 10-l ANKUM2M fermentor (Russia) with an initial volume of 5 l in the medium containing the following (grams per liter): rapeseed
oil, 20; (NH4)2SO4, 6.0; МgSO4·7H2O, 1.4; NaCl, 0.5; Ca(NO 3 ) 2 , 0.8; KH 2 PO 4 , 2.0; K 2 HPO 4 , 0.2; and Burkholder’s trace element solution (Burkholder et al. 1944). The medium contained increased concentrations of FeSO4·7H2O (5.96 mg l−1) and thiamine (1.3 μg l−1) in order to obtain a biomass of 20 g l−1. In the course of cultivation, pulsed additions of (NH4)2SO4 (by 1 g l−1) at 28, 48, 62, 75, 88, and 110 h were performed as the nitrogen level became less than 63 mg l−1. Pulsed additions of rapeseed oil (by 20 g l−1) to a final concentration of 160 g l−1 were performed as the pO2 value changed by 10 %. The fermentation conditions were maintained automatically at the constant level: temperature was 30±0.5 °C; pH was 4.5±0.1 during the first 72 h and 3.5±0.1 during the acid formation phase (from 48 to 156 h); pO2 was 25 % (from saturation) during the first 48 h and 50 % (from saturation) from 48 to 156 h; agitation was 800 rpm. Earlier, the optimization of cultivation conditions was described in details (Kamzolova and Morgunov 2013; Morgunov et al. 2013). Cultivation was carried out for 156 h. Separation of cells from the culture broth Yeast cells were separated from the culture broth by centrifugation at 5,000g for 10 min (4 °C). The aliquots of the culture broth filtrate containing α-ketoglutaric acid and pyruvic acid were treated with hydrogen peroxide as described in the Results section. Measurement techniques Yeast growth was followed by measuring the biomass dry weight: 1–3 ml of the culture broth was filtered through a membrane filter, and the yeast cells were washed with nhexane and dried under vacuum at 110 °C to a constant weight. To analyze organic acids, the culture broth was centrifuged (8,000g, 20 °C, 3 min); 1 ml of the supernatant was diluted with an equal volume of 8 % HClO4, and concentration of organic acids was analyzed on an HPLC chromatograph (LKB, Sweden) with an Inertsil ODS-3 reversed-phase column (250×4 mm, Elsiko, Russia) at 210 nm; 20 mM phosphoric acid was used as a mobile phase at a flow rate of 1.0 ml min−1. The column temperature was maintained at 35 °C. Quantitative determination was carried out using calibration curves constructed with the application of SA, αketoglutaric acid (KGA), pyruvic acid, citric acid, and other acids (Sigma-Aldrich, St. Louis, MO, USA) as standards. Authentic SA was purchased from Sigma (USA) (Cat. No. S-7501). Additionally, SA was analyzed enzymatically using a biochemical kit (Boehringer Mannheim/R-Biopharm). Protein was determined by the Lowry method.
Appl Microbiol Biotechnol
To determine fatty acid composition of lipids, biomass samples were washed thrice with n-hexane to remove oil contaminations. Methyl esters of fatty acids were obtained by the method of Sultanovich et al. (1982) and analyzed by gas-liquid chromatography on a Chrom-5 chromatograph (Czech Republic) with a flame ionization detector. The column (2 m×3 mm) was packed with 15 % Reoplex-400 coated on Chromaton N-AW (0.16−0.20 mm). The column temperature was 200 °C. Lipid content of biomass was determined as the sum of fatty acids by using docosane (C22H46) as an internal standard. Statistical analysis All the data presented are the mean values of three experiments and two measurements for each experiment; standard deviations were calculated (SD
APPLIED MICROBIAL AND CELL PHYSIOLOGY
The peculiarities of succinic acid production from rapeseed oil by Yarrowia lipolytica yeast Svetlana V. Kamzolova & Natalia G. Vinokurova & Emiliya G. Dedyukhina & Vladimir A. Samoilenko & Julia N. Lunina & Alexey A. Mironov & Ramil K. Allayarov & Igor G. Morgunov
Received: 21 December 2013 / Revised: 24 January 2014 / Accepted: 28 January 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract The process of succinic acid (SA) production represents the combination of microbial synthesis of αketoglutaric acid from rapeseed oil by yeast Yarrowia lipolytica VKM Y-2412 and subsequent decarboxylation of α-ketoglutaric acid by hydrogen peroxide to SA that leads to the production of 69.0 g l−1 of SA and 1.36 g l−1 of acetic acid. SA was isolated from the culture broth filtrate in a crystalline form. The SA recovery from the culture filtrate has certain difficulties due to the presence of residual triglycerides of rapeseed oil. The effect of different methods of the culture filtrate treatment and various sorption materials on the coagulation of triglycerides was studied, and as a result, the precipitation of residual triglycerides by acetone was chosen. The subsequent isolation procedures involved the decomposition of H2O2 in the filtrate followed by filtrate bleaching and acidification with a mineral acid, evaporation of filtrate, and SA extraction with ethanol from the residue. The purity of crystalline SA isolated from the culture broth filtrate achieved 97.6–100 %. The product yield varied from 62.6 to 71.6 % depending on the acidity of the supernatant.
Keywords Microbial production . Succinic acid (SA) . α-Ketoglutaric acid . Rapeseed oil . Yarrowia lipolytica . Purification S. V. Kamzolova (*) : N. G. Vinokurova : E. G. Dedyukhina : V. A. Samoilenko : J. N. Lunina : A. A. Mironov : R. K. Allayarov : I. G. Morgunov G.K. Skryabin Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, Prospect Nauki 5, Pushchino, Moscow Region, Russia 142290 e-mail: [email protected] A. A. Mironov : R. K. Allayarov : I. G. Morgunov Pushchino State Institute of Natural Sciences, Prospekt Nauki 3, Pushchino, Moscow Region, Russia 142290
Introduction Succinic acid (C4H6O4) (SA) has found wide applications in industry as a starting material for the synthesis of highperformance chemicals (1,4-butanediol, adipic acid, tetrahydrofuran, γ-butyrolactone, N-methylpyrrolidone) (Zeikus et al. 1999; Carole et al. 2004), as a fodder additive for stimulation of propionate formation in ruminant animals and enhancing the efficiency of farm animal production (Samuelov et al. 1999), and as a protector of humans against oxidative stress (Kondrashova 1991, 2002). The mono- or disodium salts of SA are used as a flavor to meats and soups; dioctyl sodium sulfosuccinate is used as a surfactant and wetting agent for textile and printing products and as a component of the Corexit dispersant applied in the recent Gulf oil spill cleanup operations (Thakker et al. 2012). The market potential for SA and its derivatives has been predicted to be up to 245×103 tons/year, with a market for the SA-derived polymers of 2×106 tons/year (Bozell and Petersen 2010). For industrial purposes, SA is produced by chemical synthesis from maleic anhydride; however, microbiological synthesis of SA is considered as more perspective due to its ecological and economical advantages (Zeikus et al. 1999; Carole et al. 2004; Werpy et al. 2006; McKinlay et al. 2007; Bechthold et al. 2008; Sauer et al. 2008; Bozell and Petersen 2010; Skorokhodova et al. 2013). It is noted that food and pharmaceutical industry will prefer to use bio-succinate to avoid potential health hazards (Gascon et al. 2006). The microbial production of SA is commonly based on the use of bacteria, such as Anaerobiospirillum succiniciproducens, Actinobacillus succinogenes, Mannheimia succiniciproducens, recombinant Escherichia coli strains, and Corynebacterium glutamicum. As an alternative of bacterial fermentations, the coauthors of the present study developed two-stage processes of SA production by yeast Yarrowia lipolytica that involve the biosynthesis of α-ketoglutaric acid from ethanol or n-alkanes,
Appl Microbiol Biotechnol
and the subsequent decarboxylation of α-ketoglutaric acid to SA in the presence of a strong oxidant—hydrogen peroxide (H2O2) (Kamzolova et al. 2009, 2012a, c). Currently, triglycerides of plant oils (rapeseed, sunflower, and palm oils) have attracted increased interest as substrates for the development of biotechnological processes; the products manufactured from plant oils are permissible for usage in the food industry and medicine. Moreover, plant oils provided high product yield (Glatz et al. 1984; Stottmeister et al. 2005; Otto et al. 2011; Aurich et al. 2012). Among plant oils, the rapeseed oil is considered as the most promising substrate for the development of biotechnological processes due to the high rape seed crop (from 1 ha, up to 1,000 kg of rapeseed oil can be obtained) and attracts increased interest as a raw material for biodiesel production. Moreover, rapeseed oil is rich in minerals and vitamins that may provide valuable nutrients for cell growth stimulation and production of valuable metabolites. The aim of the present work was to develop an efficient process of SA production from rapeseed oil, which includes microbial synthesis of α-ketoglutaric acid and its subsequent decarboxylation to SA and to elaborate effective method of SA extraction.
Materials and methods Microorganism For microbial synthesis of α-ketoglutaric acid from rapeseed oil, we used the strain Y. lipolytica VKM Y-2412 obtained from the All-Russian Collection of Microorganisms (VKM). This strain was characterized by a high production of α-ketoglutaric acid from ethanol (Kamzolova et al. 2009, 2012b), n-alkanes (Kamzolova et al. 2012a), and rapeseed oil (Kamzolova and Morgunov 2013). The strain was maintained at 4 °C on agar slants with n-alkanes as the carbon source. Chemicals All chemicals were of analytical grade (Sigma-Aldrich, St. Louis, MO, USA). Rapeseed oil was purchased from the “Russian seeds” Processing Plant (Vinev, Russia). The fatty acid profile of the rapeseed oil which had a total unsaturated fatty acid mass fraction of 93.6 % was as follows (percentage, by mass): C16:0, 4.0; C18:0, 1.2; C18:1, 58.8; C18:2, 28.1; and C18:3, 5.9. Cultivation Y. lipolytica VKM Y-2412 was cultivated in a 10-l ANKUM2M fermentor (Russia) with an initial volume of 5 l in the medium containing the following (grams per liter): rapeseed
oil, 20; (NH4)2SO4, 6.0; МgSO4·7H2O, 1.4; NaCl, 0.5; Ca(NO 3 ) 2 , 0.8; KH 2 PO 4 , 2.0; K 2 HPO 4 , 0.2; and Burkholder’s trace element solution (Burkholder et al. 1944). The medium contained increased concentrations of FeSO4·7H2O (5.96 mg l−1) and thiamine (1.3 μg l−1) in order to obtain a biomass of 20 g l−1. In the course of cultivation, pulsed additions of (NH4)2SO4 (by 1 g l−1) at 28, 48, 62, 75, 88, and 110 h were performed as the nitrogen level became less than 63 mg l−1. Pulsed additions of rapeseed oil (by 20 g l−1) to a final concentration of 160 g l−1 were performed as the pO2 value changed by 10 %. The fermentation conditions were maintained automatically at the constant level: temperature was 30±0.5 °C; pH was 4.5±0.1 during the first 72 h and 3.5±0.1 during the acid formation phase (from 48 to 156 h); pO2 was 25 % (from saturation) during the first 48 h and 50 % (from saturation) from 48 to 156 h; agitation was 800 rpm. Earlier, the optimization of cultivation conditions was described in details (Kamzolova and Morgunov 2013; Morgunov et al. 2013). Cultivation was carried out for 156 h. Separation of cells from the culture broth Yeast cells were separated from the culture broth by centrifugation at 5,000g for 10 min (4 °C). The aliquots of the culture broth filtrate containing α-ketoglutaric acid and pyruvic acid were treated with hydrogen peroxide as described in the Results section. Measurement techniques Yeast growth was followed by measuring the biomass dry weight: 1–3 ml of the culture broth was filtered through a membrane filter, and the yeast cells were washed with nhexane and dried under vacuum at 110 °C to a constant weight. To analyze organic acids, the culture broth was centrifuged (8,000g, 20 °C, 3 min); 1 ml of the supernatant was diluted with an equal volume of 8 % HClO4, and concentration of organic acids was analyzed on an HPLC chromatograph (LKB, Sweden) with an Inertsil ODS-3 reversed-phase column (250×4 mm, Elsiko, Russia) at 210 nm; 20 mM phosphoric acid was used as a mobile phase at a flow rate of 1.0 ml min−1. The column temperature was maintained at 35 °C. Quantitative determination was carried out using calibration curves constructed with the application of SA, αketoglutaric acid (KGA), pyruvic acid, citric acid, and other acids (Sigma-Aldrich, St. Louis, MO, USA) as standards. Authentic SA was purchased from Sigma (USA) (Cat. No. S-7501). Additionally, SA was analyzed enzymatically using a biochemical kit (Boehringer Mannheim/R-Biopharm). Protein was determined by the Lowry method.
Appl Microbiol Biotechnol
To determine fatty acid composition of lipids, biomass samples were washed thrice with n-hexane to remove oil contaminations. Methyl esters of fatty acids were obtained by the method of Sultanovich et al. (1982) and analyzed by gas-liquid chromatography on a Chrom-5 chromatograph (Czech Republic) with a flame ionization detector. The column (2 m×3 mm) was packed with 15 % Reoplex-400 coated on Chromaton N-AW (0.16−0.20 mm). The column temperature was 200 °C. Lipid content of biomass was determined as the sum of fatty acids by using docosane (C22H46) as an internal standard. Statistical analysis All the data presented are the mean values of three experiments and two measurements for each experiment; standard deviations were calculated (SD
