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Conversion of raw glycerol to microbial lipids by new Metschnikowia and Yarrowia lipolytica strains

Conversion of raw glycerol to microbial lipids by new Metschnikowia and Yarrowia lipolytica strains Ann Microbiol (2016) 66:1409–1418 DOI 10.1007/s13213-016-1228-0 ORIGINAL ARTICLE Conversion of raw glycerol to microbial lipids by new Metschnikowia and Yarrowia lipolytica strains 1 1 1 1 1 1 L. Canonico & S. Ashoor & M. Taccari & F. Comitini & M. Antonucci & C. Truzzi & 1 1 G. Scarponi & M. Ciani Received: 2 February 2016 /Accepted: 10 June 2016 /Published online: 30 June 2016 Springer-Verlag Berlin Heidelberg and the University of Milan 2016 Abstract Nine Metschnikowia spp. strains and six Yarrowia produced (Poli et al. 2014). However, the process of refining lipolytica strains were tested for lipid production on raw glyc- crude glycerol into a high purity product is expensive and erol from a biodiesel plant. Metschnikowia sp. 271 and energy consuming (Yen et al. 2012). Hence, the direct use of Y. lipolytica 347 were selected for biomass and lipid produc- crude glycerol would help to make biodiesel production more tion, and optimized using central composite factorial design profitable and sustainable (Uçkun Kiran et al. 2013). In this (CCD) and response surface methodology (RSM). The lipid regard, crude glycerol has been used as a sole carbon source production (2.60 g/L) of Y. lipolytica 347 was maximized at by various microalgae, yeasts, molds and bacteria, under both C/N 118, time 144 h and 90 g/L glycerol, while the maximum aerobic and anaerobic conditions (Rywinska et al. 2013), for lipid production (0.49 g/L) of Metschnikowia sp. 271 was the production of some value-added products such as 1,3- obtained under similar conditions but after a further incuba- propanediol (Papanikolaou et al. 2008; Chatzifragkou et al. tion for 7 days at 16 °C. The fatty acids profile of Y. lipolytica 2011), citric acid (Papanikolaou et al. 2008; Rymowicz et al. exhibited a considerable amount of C18:1n7 (~36 %), 2010), biopolymers (Ashby et al. 2011), succinic acid (Zhang C18:1n9 (~16 %), and C16:0 (~16 %), whereas et al. 2010), single cell protein (Taccari et al. 2012) and single Metschnikowia sp. produced mainly C18:1n9 (~33 %), cell oils (Papanikolaou and Aggelis 2009;Saenge et al. 2011). C16:0 (~21 %), and C16:1n7 (~21 %). Both yeasts showed In recent years, growing attention has been paid to the similar amounts of unsaturated fatty acids (~70 %). However, development of single cell oils, and it has been found that considerable amounts of polyunsaturated fatty acid (PUFAs) many microorganisms, such as algae, yeast, bacteria, and fun- were exhibited only by Metschnikowia sp. (~12 %). gi, have the ability to accumulate oils under appropriate culti- vation conditions. Microorganisms that can accumulate lipid to more than 20 % of their biomass are defined as oleaginous . . Keywords Crude glycerol Lipid production species. A number of oleaginous yeast species are known, Metschnikowia sp. Yarrowia lipolytica including Cryptococcus curvatus, Lipomyces starkeyi, Rhodosporidium toruloides, Rhodotorula glutinis and Yarrowia lipolytica (Sitepu et al. 2014). Introduction Lipid production in oleaginous yeasts occurs when a nutri- ent in the medium becomes limited and the carbon source is Crude glycerol is the main byproduct of the biodiesel produc- present in excess. Nitrogen limitation is the most efficient tion chain. The recent growth in biodiesel production has re- condition for inducing lipid accumulation (Ratledge 2004; sulted in a significant increase in the volume of glycerol Beopoulos et al. 2009). When nitrogen becomes unavailable the organism continues to assimilate the carbon source but the growth rate slows down, since nitrogen is essential for protein * M. Ciani and nucleic acid syntheses (Beopoulos et al. 2009). m.ciani@univpm.it These lipids usually consist of triacylglycerols (80–90 %) with a fatty acid composition similar to many plant seed oils Dipartimento Scienze della vita e dell’Ambiente (DISVA), Università Politecnica delle Marche, Via Brecce Bianche, 60131 Ancona, Italy (Ratledge and Evans 1984). Besides, microbial lipid 1410 Ann Microbiol (2016) 66:1409–1418 technology has many advantages, such as the short life cycle 72 h (Y. lipolytica). All experiments were carried out in of microbes as compared to plants, and lack of competition duplicate. with agriculture for land use. Thus, microbial lipids are con- sidered as a potential feedstock for biodiesel production (Liu Experimental design and process optimization and Zhao 2007;Meng et al. 2009). The aim of this work was to evaluate lipid accumulation of Response surface methodology (RSM) was conducted using a different Metschnikowia and Y. lipolytica strains using central composite factorial design (CCD) with three factors crude glycerol from biodiesel production as the sole carbon and two replicates of the central point. All factors and their source. In selected strains, optimal lipid accumulation respective levels are showed in Table 1. The experiments were conditiond and fatty acid composition were also determined. performedinbaffledflasks (250mL)containing50mL growth medium. Ammonium sulfate as a nitrogen source was added to obtain a C/N ratio of 63/145. To evaluate the effect of further incubation on lipid accumulation, Materials and methods Metschnikowia sp. 271 was incubated for an additional 7 days at 16 °C under static conditions. Strains and culture conditions Fifteen yeast strains were involved in this study: Analytical methods Metschnikowia sp. DiSVA 271 and DiSVA 272; Metschnikowia pulcherrima DiSVA 266, DiSVA 267, Yeast growth was estimated using a UV-1800 spectrophotom- DiSVA 268, DiSVA 269, DiSVA 273, DiSVA 274 and eter (Shimadzu, Japan) by measuring the optical density at DiSVA 275, andY. lipolytica DiSVA 347, DiSVA 352, 600 nm. Biomass concentration was evaluated as cell dry CBS 599, CBS 2073, CBS 2074 and CBS 2075. These weight. Glycerol concentrations (g/L) were determined using strains were obtained from the Yeast Collection of the Glycerol kit n° 148270 (Roche, Mannheim, Germany). Dipartimento di Scienze della Vita e dell’Ambiente Extraction of lipids from dry biomass was performed ac- (DiSVA), Università Politecnica delle Marche and the cording to the modified procedure of Bligh and Dyer (1959). Centraal Bureau voor Schimmelcultures, Netherlands (CBS). In short, lipids were extracted with a mixture of The yeast strains were grown on YPD medium, which is com- chloroform:methanol (2:1, v/v) for 24 h, centrifuged to obtain posed of (g/L): peptone, 20; yeast extract, 10; glucose, 20 and a clear supernatant, and the solvent removed by evaporation agar, 20, and maintained at 4 °C. under vacuum. Total cellular lipids (g/L) were determined gravimetrically. Lipids were converted to their fatty acid methyl esters Substrate source (FAMEs) according to a modified method of ISO 12966- 2:2011 (revision of ISO 5509:2000). Briefly, ~10 mg lipid Crude glycerol was obtained from a private biodiesel produc- extract was dissolved in n-heptane (0.5 mL), then 10 μL2 N tion company, and had the following characteristics: pH 7.10, sodium methylate was added. After vortexing for 3 min, the purity 88–92 %, water (3–5 %), potassium and sodium salts solution was centrifuged at 89 g for 1 min, then a few milli- (4–5 %), non-glycerol organic matter (0.5–1 %), methanol grams of sodium metabisulfite were added. Finally, 100 μLof (<0.2 %). the solution was transferred to a vial (1 mL), and diluted with 400 μL n-heptane. Screening of Metschnikowia and Yarrowia lipolytica strains Gas chromatography-mass spectrometry (GC-MS) on crude glycerol analyses were performed on an Agilent-6890 gas chro- matograph with split injection, coupled to an Agilent - Metschnikowia strains were screened on crude glycerol medi- 5973 N quadrupole mass selective detector. A CPS um with the following composition (g/L): glycerol, 40; am- ANALITICA CC-wax_MS (30 m, 0.25 mm ID, monium sulfate, 1 and yeast extract, 0.5 with C/N 60 and pH 6.0, whereas the medium used for Y. lipolytica strains Table 1 Experimental Variable Range and level consisted of 30 g/L glycerol, 0.5 g/L ammonium sulfate and range, and levels of the independent variables yeast extract with C/N 76 and pH 6.0. The experiments were −10 1 carried out in 250 mL baffled flasks containing 50 mL crude glycerol medium, sterilized at 121 °C/20 min and inoculated X , Glycerol (g/L) 30 60 90 from 24 h exponential yeasts pre-culture (2.5 mL). All flasks X ,C/N 63 118 145 were incubated at 28 °C under agitation speed 130 rpm and X , Time (h) 48 96 144 the results were obtained after 144 h (Metschnikowia) and Ann Microbiol (2016) 66:1409–1418 1411 0.25 μm film thickness) capillary column was used to and crude glycerol amounts on biomass and lipid production separate FAMEs. The inlet temperature was 250 °C, with of Metschnikowia and Y. lipolytica strains are shown in injection volume of 1 μL. The oven temperature was pro- Table 2. grammed from 100 °C (1 min), to 230 °C, initially at the Metschnikowia and Y. lipolytica strains showed notable rate of 25 °C/min from 100 to 150 °C, from 150 °C to differences in biomass production. Generally, 200 °C at the rate of 5 °C/min, and from 200 to 230 °C at Metschnikowia strains showed lower biomass production therateof1°C/min, andthetotal runtimewas 43min. compared with Y. lipolytica strains. M. pulcherrima 266 Helium (6.0, SOL, Italy) (8.0 p.s.i.) was used as the car- and M. pulcherrima 267 are the best producers of lipids rier gas. The ion source, transfer line and detector temper- [in absolute values (g/L), and relative values (% w/w)] atures were 230 °C, 250 °C and 150 °C, respectively. compared with the other Metschnikowia strains, but ex- Mass spectra (m/z 50 to m/z 400) were recorded at a rate hibited low crude glycerol consumption. Strains of three scans per second, with ionization energy of Y. lipolytica 347 and Y. lipolytica 2074, produced higher 70 eV. Data were collected under SIM mode. After a lipid amounts [in absolute values (g/L), and relative solvent delay of 2.0 min, the following fragment ions values (% w/w)] in comparison with the other were recorded: m/z 74 and 87 for saturated, m/z 74 and Y. lipolytica strains. For these reasons, and their ability 55 for monoenoic fatty acids, m/z 67 and 81 for dienoic to metabolize crude glycerol, the strains Metschnikowia fatty acids, and m/z 79 and 81 for polyunsaturated fatty sp. 271 and Y. lipolytica 347 were selected for biomass acids (Thurnhofer and Vetter 2006; Zhang et al. 2014). and lipid optimization trials (on the basis of glycerol Identification of fatty acids was performed using NIST consumed and biomass production). reference mass spectra database (NIST, Mass Spectral Database 02, National Institute of Standards and Technology, Gaithersburg, MD; http://www.nist.gov/srd) Biomass and lipid optimization of Metschnikowia sp. 271 MS search 2.0a (NIST 02.L, Ringoes, NJ). The retention and Y. lipolytica 347 strains by RSM time and mass spectra of standard fatty acid methyl esters (Supelco 37-component FAME Mix) were used to con- A Box-Behnken design with three variables (glycerol concen- firm the NIST identification of the fatty acids in the tration, C/N ratio and time process) at three levels was carried sample. out to determine the response pattern. The experimental de- sign matrixes and response values are shown in Tables 3 and 4. The second order polynomial equations for biomass (Y ), Statistical analysis lipid production (Y ) and lipid content (Y ), as a function of 2 3 glycerol concentration (x ), C/N ratio (x ) and time (x )are 1 2 3 The regression and statistical analyses were performed using presented as follows for: the statistical software package JMP 12 (http://www.jmp. com). The mathematical relationship of the independent (1) Yarrowia lipolytica variable was determined by fitting a second order polynomial equation to data obtained from the 15 runs: 2 BiomassðÞ Y ¼ 6:9191 þ 2:2738x –0:0313x 1 1 2 Y ¼ β0 þ ∑βixi þ ∑βiixi þ ∑βijxixj 2 2 þ 1:3401x –0:0521x þ 0:0705x 3 1 2 Where Y is the predicted response, the independent vari- ables (xi and xj), the intercept term (β0), the linear effects (βi), þ 0:5104x –0:3227x x þ 0:2500x x 3 1 2 1 3 the squared effects (βii) and the first order interaction effect þ 0:5704x x Lipid productionðÞ Y 2 3 2 (βij). ¼ 1:4479 þ 0:1862x þ 0:1795x þ 0:6775x 1 2 3 2 2 2 þ 0:0832x –0:0071x –0:3688x 1 2 3 Results and discussion þ 0:0451x x þ 0:2588x x 1 2 1 3 Screening of Metschnikowia and Y. lipolytica strains for lipid production þ 0:2114x x Lipid contentðÞ Y 2 3 3 ¼ 21:3103–4:1165x þ 1:1538x þ 6:3442x 1 2 3 Carbon and nitrogen source, C/N molar ratio, and culture con- 2 2 2 ditions (temperature and pH) have a significant influence on þ 2:0225x þ 0:3922x –8:2350x 1 2 3 cell growth and lipid accumulation of oleaginous yeasts. The þ 2:9151x x þ 0:5125x x þ 1:5569x x 1 2 1 3 2 3 effects of nitrogen sources (yeast extract, ammonium sulfate) 1412 Ann Microbiol (2016) 66:1409–1418 (2) Metschnikowia sp. were used as an instrument to verify the significance of each coefficient, which also shows the interaction strength of each BiomassðÞ Y ¼ 3:3462 þ 0:3515x –0:0938x 1 1 2 parameter. 2 2 2 For Y. lipolytica, the linear terms glycerol concentration þ 0:5431x þ 0:1146x þ 0:1642x þ 0:5521x 3 1 2 3 (x )and time (x ) were significant in terms of biomass, 1 3 þ 0:8700x x lipid production and lipid content. In contrast, the linear 1 2 term C/N ratio (x ) was significant only for lipid produc- þ 0:0x x – 0:0691x x Lipid productionðÞ Y 1 3 2 3 2 tion. The interaction term x x showed a significant ef- 1 2 fect on lipid content, while other interaction terms were ¼ 0:0918–0:0113x –0:0035x –0:0339x 1 2 3 significant for lipid production. The results presented in 2 2 þ 0:0468x þ 0:0004x 1 2 Table 3 show the effect of glycerol concentration, C/N ratio and time on biomass, lipid production and lipid þ 0:0958x –0:0065x x –0:0943x x 3 1 2 1 3 content of Y. lipolytica. The lipids and biomass were þ 0:0277x x Lipid contentðÞ Y maximized (2.60 g/L, 11.5 g/L) at C/N 118, time 144 h, 2 3 3 with 90 g/L glycerol (Fig. 1a,b). The maximum lipid con- ¼ 2:7149–0:9355x –0:0375x –1:4245x 1 2 3 tent (32.6 % w/w) was obtained with a glycerol concen- 2 2 tration of 30 g/L, C/N 63 and time 96 h (Fig. 1c). þ 1:3229x þ 0:1246x 1 2 Moreover, good results of biomass production, lipid ac- þ 2:2729x –1:1832x x –2:1875x x 3 1 2 1 3 cumulation and lipid content (9.0 g/L, 2.07 g/L, 22.8 % w/w) were achieved with a glycerol concentration of þ 1:1688x x 2 3 60 g/L, C/N 145 and time 144 h. These amounts are higher than those achieved by Poli et al. (2014), who obtained 6.7 g/L biomass and 1.27 g/L lipid from The models fit satisfactorily with the experimental data, as Y. lipolytica QU21 using crude glycerol as a carbon indicated from R and P values (Table 5). The values of prob- source, and ammonium sulfate as a nitrogen source. ability (P)> F for biomass, lipid production and lipid content, Also, the yeast strain Y. lipolytica ACA-DC 50109 indicated that the results of the models were significant. The achieved lipid production of 1.37 g/L and 0.79 g/L signs of the factor coefficients in the model equations indicat- (20.4 % and 13.4 % of the dry cell mass) after a fermenta- ed their relative effects on biomass, lipid production and lipid tion time of 50 h and 240 h, respectively, with an initial content, and the significance of each coefficient, as deter- glycerol concentration of 104.9 g/L under bioreactor con- mined by P-values, are illustrated in Table 5.The P-values ditions (Makri et al. 2010). Moreover, Cheirsilp and Table 2 Biomass and lipid Strain Biomass (g/L) Lipid (g/L) Lipid content Glycerol production from different dry weight (%) (w/w) consumed (g/L) Metschnikowia and Yarrowia lipolytica strains grown on crude M. pulcherrima 266 4.1 ± 0.2 0.45 ± 0.10 10.8 ± 2.7 6.0 ± 0.2 glycerol as carbon source M. pulcherrima 267 4.4 ± 0.4 0.38 ± 0.20 8.7 ± 3.7 4.0 ± 0.8 M. pulcherrima 268 4.9 ± 0.2 0.19 ± 0.00 3.8 ± 0.3 15.6 ± 1.5 M. pulcherrima 269 5.0 ± 0.8 0.24 ± 0.01 5.2 ± 1.5 15.6 ± 0.3 Metschnikowia sp. 271 6.4 ± 0.6 0.26 ± 0.01 4.3 ± 0.6 23.0 ± 1.6 Metschnikowia sp. 272 5.7 ± 0.4 0.27 ± 0.10 4.6 ± 1.0 16.4 ± 0.5 M. pulcherrima 273 4.8 ± 0.2 0.25 ± 0.00 4.8 ± 1.1 9.4 ± 1.4 M. pulcherrima 274 4.1 ± 0.4 0.19 ± 0.01 4.6 ± 0.9 19.0 ± 1.1 M. pulcherrima 275 6.1 ± 0.4 0.19 ± 0.10 3.1 ± 1.4 16.4 ± 0.8 Y. lipolytica 347 9.4 ± 0.1 0.43 ± 0.00 4.6 ± 0.1 24.0 ± 2.8 Y. lipolytica 352 8.5 ± 0.7 0.09 ± 0.01 1.0 ± 0.3 12.4 ± 0.2 Y. lipolytica 599 8.7 ± 0.4 0.22 ± 0.02 2.4 ± 0.2 26.5 ± 0.7 Y. lipolytica 2073 6.6 ± 0.2 0.27 ± 0.06 4.2 ± 0.9 28.6 ± 0.1 Y. lipolytica 2074 8.0 ± 0.1 0.65 ± 0.11 8.0 ± 1.2 13.7 ± 0.1 Y. lipolytica 2075 9.0 ± 0.1 0.35 ± 0.08 4.0 ± 0.8 10.6 ± 0.2 Lipid (g/L)/ biomass (g/L) dry weight × 100 Ann Microbiol (2016) 66:1409–1418 1413 Table 3 Experimental designs Run order Glycerol (g) C/N ratio Time (h) Biomass (g/L) Lipid (g/L) Lipid content and response based on (%) (w/w) experimental runs of Y. lipolytica 1 30 63 96 4.0 ± 0.71 1.29 ± 0.07 32.6 ± 4.03 2 30 145 96 6.0 ± 0.00 1.57 ± 0.28 26.2 ± 4.74 3 90 63 96 8.8 ± 0.35 1.45 ± 0.24 16.5 ± 2.12 4 90 145 96 9.0 ± 0.71 1.79 ± 0.19 19.7 ± 0.42 5 60 63 48 7.3 ± 3.89 0.45 ± 0.20 6.4 ± 0.59 6 60 63 144 9.0 ± 0.01 1.28 ± 0.29 14.3 ± 3.25 7 60 145 48 4.8 ± 1.06 0.49 ± 0.01 10.4 ± 2.26 8 60 145 144 9.0 ± 0.00 2.07 ± 0.02 22.8 ± 0.35 9 30 118 48 3.8 ± 0.35 0.36 ± 0.01 9.5 ± 0.71 10 90 118 48 8.3 ± 1.06 0.43 ± 0.03 5.3 ± 0.35 11 30 118 144 6.0 ± 0.01 1.49 ± 0.20 24.8 ± 3.11 12 90 118 144 11.5 ± 0.00 2.60 ± 0.18 22.6 ± 1.56 13 60 118 96 6.3 ± 0.35 1.35 ± 0.07 21.6 ± 0.14 14 60 118 96 7.3 ± 0.35 1.86 ± 0.03 25.7 ± 1.63 15 60 118 96 7.3 ± 0.35 1.32 ± 0.06 18.0 ± 0.00 Lipid (g/L)/ biomass (g/L) dry weight × 100 Louhasakul (2013), using Y. lipolytica TISTR 5151, have a wide margin for improvement in lipid production. showed a lipid production of approximately 2 g/L and lipid In this regard, some genetically engineered Y. lipolitica content of up to 64 % on crude glycerol. In a more recent strains, such as Y. lipolitica PO1f, exhibited lipid accu- study, in a flask experiment using Y. lipolytica A101 strain, mulation approaching 90 % of cell mass (Blazeck et al. lipid production of 2.31 g/L and lipid content of 27.3 % 2014), and the yeast strain Y. lipolitica NS432 achieved were achieved (Dobrowolski et al. 2016). lipid production of up to 85 g/L with lipid content of On the other hand, most of the highest lipid contents 73 % using glucose as a carbon source (Friedlander obtained were from genetically engineered Y. lipolitica et al. 2016). Also, the yeast strain Y. lipolytica strains, while the wild type strains used in this work JMY4086 produced about 24 g/L lipids (40 % of dry Table 4 Experimental designs Run Glycerol (g) C/N Time (h) Biomass Lipid (g/L) Lipid content and response based on order ratio (g/L) (%) (w/w) experimental runs of Metschnikowia sp. 271 1 30 63 96 5.0 ± 0.71 0.14 ± 0.00 2.9 ± 0.49 2 30 145 96 2.3 ± 0.35 0.14 ± 0.01 6.2 ± 0.59 3 90 63 96 3.5 ± 0.01 0.15 ± 0.00 4.2 ± 0.07 4 90 145 96 3.8 ± 0.35 0.13 ± 0.00 3.5 ± 0.42 5 60 63 48 3.0 ± 0.01 0.28 ± 0.16 9.3 ± 5.23 6 60 63 144 4.3 ± 1.06 0.10 ± 0.01 2.5 ± 0.74 7 60 145 48 4.0 ± 0.00 0.25 ± 0.03 6.3 ± 0.71 8 60 145 144 5.0 ± 0.00 0.12 ± 0.01 2.4 ± 0.28 9 30 118 48 2.5 ± 0.01 0.14 ± 0.03 5.8 ± 1.13 10 90 118 48 4.5 ± 0.01 0.29 ± 0.11 6.3 ± 2.36 11 30 118 144 3.5 ± 0.00 0.37 ± 0.01 10.7 ± 0.49 12 90 118 144 5.5 ± 0.01 0.13 ± 0.01 2.4 ± 0.26 13 60 118 96 3.3 ± 0.35 0.10 ± 0.01 3.2 ± 0.21 14 60 118 96 3.3 ± 0.35 0.09 ± 0.01 2.7 ± 0.14 15 60 118 96 3.5 ± 0.00 0.08 ± 0.01 2.3 ± 0.14 Lipid (g/L)/ biomass (g/L) dry weight × 100 1414 Ann Microbiol (2016) 66:1409–1418 Table 5 Estimated regression coefficients and analysis of variance for response variables Coefficient Biomass (Y ) Lipid (Y)Lipid Coefficient Biomass (Y)Lipid(Y)Lipid 1 2 1 2 content (Y ) content (Y ) 3 3 Y. lipolytica Metschnikowia sp. Intercept 6.9191* 1.4479* 21.3103* Intercept 3.3462* 0.0918* 2.7149* x 2.2738* 0.1862* – 4.1165* x 0.3515 – 0.0113 – 0.9355 1 1 x – 0.0313 0.1795* 1.1538 x – 0.0938 – 0.0035 – 0.0375 2 2 x 1.3401* 0.6775* 6.3442* x 0.5431* – 0.0339* – 1.4245* 3 3 x x – 0.3227 0.0451 2.9151* x x 0.8700* – 0.0065 – 1.1832 1 2 1 2 x x 0.2500 0.2588* 0.5125 x x 0.0 – 0.0943* – 2.1875* 1 3 1 3 x x 0.5704 0.2114* 1.5569 x x – 0.0691 0.0277 1.1688 2 3 2 3 2 2 x – 0.0521 0.0832 2.0225 x 0.1146 0.0468* 1.3229 1 1 2 2 x 0.0705 – 0.0071 0.3922 x 0.1642 0.0004 0.1246 2 2 2 2 x 0.5104 – 0.3688* – 8.235* x 0.5521* 0.0958* 2.2729* 3 3 Variability Variability 2 2 R of the model 0.806 0.918 0.858 R of the model 0.665 0.705 0.664 2 2 Adjusted R 0.719 0.881 0.794 Adjusted R 0.514 0.573 0.513 F-ratio 9.26 24.87 13.43 F-ratio 4.41 5.32 4.39 Prob > F <0.0001* <0.0001* <0.0001* Prob > F 0.0027* 0.0009* 0.0028* * P-values significant cell mass) using crude glycerol as a carbon source Indeed, Santamauro et al. (2014) obtained the maximum (Rakicka et al. 2015). In contrast, the wild type strain lipid production from M. pulcherrima in raw glycerol Y. lipolytica NS18 achieved lipid production of 12.8 g/ after an incubation at 25 °C for 3 days followed by L with lipid content of 25 % using glucose as a carbon further 12 days at 15 °C. source (Friedlander et al. 2016). Regarding Metschnikowia sp. 271, the linear effect of time Fatty acid composition (x ) was significant for all parameters, whereas other linear terms were not significant for any parameter. The interaction The major fatty acids species identified in Y. lipolytica term x x gave a positive and significant effect on biomass 347 and Metschnikowia sp. 271 are presented in Table 6. 1 2 production, while the interaction term x x was significant for Y. lipolytica is enriched in C16:0, C16:1n7, C18:0, 1 3 lipid production and lipid content. In this regard, C18:1n9, C18:1n7 and C24:0, whereas the sum of others Metschnikowia sp. 271 showed the optimal crude glycerol fatty acids is below 15 %. Metschnikowia sp. is enriched concentration, C/N ratio and fermentation time of 30 g/L, C/ in C16:0, C16:1n7, C18:0, C18:1n9 and C18:2n6, and N 118 and 144 h, respectively, both for lipid production and the sum of remaining acids is below 6 %. Y. lipolytica lipid content (Table 4). Under these conditions, 0.37 g/L lipids exhibited a considerable amount of C18:1n7 (~36 %), was obtained (Fig. 2a), with lipid accumulation in terms of C18:1n9 (~16 %), and C16:0 (~16 %), whereas relative values (lipid content %) of 10.7 % w/w (Fig. 2b). An Metschnikowia sp. produced mainly C18:1n9 (~33 %), increase of glycerol concentration (90 g/L), under the same C16:0 (~21 %), and C16:1n7 (~21 %). Metschnikowia fermentation conditions, was favorable for higher biomass sp. showed a content of C16:1n7, C18:0 and C18:1n9 production (5.5 g/L), as also reported by Santamauro et al. two-fold higher than that of Y. lipolytica. In this respect, (2014)(Fig. 2c). Meng et al. (2009) reported that oils accumulated by Even though the maximum lipid production of yeasts are predominantly oleic (C18:1), linoleic (C18:2), Metschnikowia sp. was 0.37 g/L, a further incubation stearic (C18:0), palmitic (C16:0) or palmitoleic acids for 7 days at 16 °C under static conditions determined (C16:1). There are some differences between the two an increase in lipid production of 32 % (0.49 g/L). strains. In particular, oleic acid (C18:1) is produced more Ann Microbiol (2016) 66:1409–1418 1415 Fig. 1 Response surface and contour plot of the combined effects of glycerol concentration, C/N ratio, time on a lipid production, b biomass and c lipid content of Yarrowia lipolytica 347 abundantly by Y. lipolytica (52 % of total fatty acids), The results of analysis of the fatty acid composition of Y. while in Metschnikowia sp. C18:1 represents a lower lipolytica DISVA 347 are in agreement with previous results percentage (33 %). Moreover, the fatty acid C18:2n6 (Makri et al. 2010;Poli et al. 2014) except for linoleic acid was found only in Metschnikowia sp., whereas C18:1n7 (C18:2), which was not produced by this strain under the and C24:0 were produced only by Y. lipolytica.The sum conditions tested. of saturated fatty acids is almost comparable between the Regarding PUFAs, the fungus strain Mortierella alpina 1S-4 two strains, while Y. lipolytica showed higher content of exhibited PUFA production ranging from 30 % to 70 % of the monounsaturated fatty acids (MUFAs), and, consequent- total fatty acids (Sakuradani and Shimizu 2009). Also, the ge- ly, lower content of polyunsaturated fatty acids (PUFAs) netically engineered Y. lipolitica Y4053producedhighamount with respect to Metschnikowia sp. of PUFAs, up to 56.6 % of the total fatty acids (Xue et al. 2013). 1416 Ann Microbiol (2016) 66:1409–1418 Fig. 2 Response surface and contour plot of the combined effects of glycerol concentration, C/N ratio, time on a lipid production, b lipid content and c biomass by Metschnikowia sp. 271 The high amount of saturated and monounsaturated C16 of use in lipid applications in food, and in pharmaceutical and C18 fatty acids in the lipids produced by both yeast and cosmetic formulations (Carvalho et al. 2015). On the strains, similar to the vegetable oil feedstock commonly other hand, the use of low-cost lipid production by used for biodiesel (rapeseed, soybean, sunflower and palm) Metschnikowia yeast in non-sterile conditions (Santamauro (Leung et al. 2010), indicates the potential use of these et al. 2014), makes the cultivation of this oleaginous yeast lipids for biodiesel production. On the other hand, the in crude glycerol very attractive. However, further investi- ability of Metschnikowia sp. to produce PUFAs at a con- gations are needed to optimize the production of PUFAs centration of > 10 % is an interesting feature that could be by the Metschnikowia strain used. Ann Microbiol (2016) 66:1409–1418 1417 Table 6 Fatty acid profile (%) of lipid content produced by Y. lipolytica Beopoulos A, Cescut J, Haddouche R, Uribelarrea JL, Molina-Jouve C, 347 and Metschnikowia sp. 271 Nicaud JM (2009) Yarrowia lipolytica as a model for bio-oil pro- duction. Prog Lipid Res 48:375–387 Fatty acid Y. lipolytica Metschnikowia sp. Blazeck J, Hill A, Liu L, Knight R, Miller J, Pan A, Otoupal P, Alper HS (2014) Harnessing Yarrowia lipolytica lipogenesis to create a plat- C16:0 15.9 ± 1.30 21.2 ± 0.43 form for lipid and biofuel production. Nat Commun 5:3131 Bligh EG, Dyer WJ (1959) A rapid method of total lipid extraction and C16:1n7 10.5 ± 0.85 20.6 ± 0.17 purification. Can J Biochem Physiol 37:911–917 C18:0 4.3 ± 0.00 9.3 ± 0.17 Carvalho AKF, Rivaldi JD, Barbosa JC, De Castro HF (2015) C18:1n9 15.6 ± 0.40 32.9 ± 0.07 Biosynthesis, characterization and enzymatic transesterification of C18:1n7 36.5 ± 1.02 — single cell oil of Mucor circinelloides—asustainablepathway for biofuel production. Bioresour Technol 181:47–53 C18:2n6 — 10.5 ± 0.09 Chatzifragkou A, Papanikolaou S, Dietz D, Doulgeraki A, Nychas GJE, C18:3n3 1.2 ± 0.01 1.8 ± 0.03 Zeng AP (2011) Production of 1,3-propanediol by Clostridium C24:0 4.0 ± 0.03 — butyricum growing on biodiesel-derived crude glycerol through a Saturated 31.9 33.0 non-sterilized fermentation process. Appl Microbiol Biotechnol 91:101–112 MUFAs 66.7 54.5 Cheirsilp B, Louhasakul Y (2013) Industrial wastes as a promising re- PUFAs 1.4 12.5 newable source for production of microbial lipid and direct transesterification of the lipid into biodiesel. Bioresour Technol Monounsaturated fatty acids 142:329–337 Polyunsaturated fatty acids Dobrowolski A, Mituła P, Rymowicz W, Miro czuk AM (2016) Efficient conversion of crude glycerol from various industrial wastes into single cell oil by yeast Yarrowia lipolytica. Bioresour Technol 207: Conclusions 237–243 Friedlander J, Tsakraklides V, Kamineni A, Greenhagen EH, Consiglio RSM was used to optimize biomass and lipid produc- AL, MacEwen K, Crabtree DV, Afshar J, Nugent RL, Hamilton MA, Joe Shaw A, South CR, Stephanopoulos G, Brevnova EE tion of selected Y. lipolytica 347 and Metschnikowia sp. (2016) Engineering of a high lipid producing Yarrowia lipolytica 271 strains. The maximum lipid production of both strain. Biotechnol Biofuels 9:77 strains was achieved at C/N 118. To promote lipid ac- Leung D, Wu X, Leung MKH (2010) A review on biodiesel production cumulation in Metschnikowia sp.271,a furtherincuba- using catalyzed transesterification. Appl Energy 87:1083–1095 Liu B, Zhao ZK (2007) Biodiesel production by direct methanolysis of ole- tion at low temperature (16 °C) was needed. Both aginous microbial biomass. J Chem Technol Biotechnol 82:775–780 strains exhibited high amounts of C16 and C18 fatty Makri A, Fakas S, Aggelis G (2010) Metabolic activities of biotechno- acids, which indicates the potential use of this lipid logical interest in Yarrowia lipolytica grown on glycerol in repeated for biodiesel production. Metschnikowia sp. 271 strain batch cultures. Bioresour Technol 101:2351–2358 Meng X, Yang J, Xu X, Zhang L, Nie Q, Xian M (2009) Biodiesel produced acceptable amount of PUFAs. This feature production from oleaginous microorganisms. Renew Energy 34:1–5 could be considered a starting point for further investi- Papanikolaou S, Aggelis G (2009) Biotechnological valorization of bio- gations since its lipid could be profitably used in the diesel derived glycerol waste through production of single cell oil food industry, and in pharmaceutical and cosmetic and citric acid by Yarrowia lipolytica. Lipid Technol 21(4):83–87 formulations. Papanikolaou S, Fakas S, Fick M, Chevalot I, Galiotou-Panayotou M, Komaitis M, Marc I, Aggelis G (2008) Biotechnological valoriza- tion of raw glycerol discharged after bio-diesel (fatty acid methyl Acknowledgments This work was supported financially by Ministero esters) manufacturing process: production of 1,3 propanediol, citric delle Politiche Agricole e Forestali (MIPAF) (D.M. 26285/7303/2009) acid and single cell oil. Biomass Bioenergy 32:60–71 project BI lieviti nel recupero e valorizzazione del glicerolo grezzo Poli JS, Da Silva MAN, Siqueira EP, Pasa VMD, Rosa CA, Valente P derivante dalla produzione di biodiesel (LIEBIG). (2014) Microbial lipid produced by Yarrowia lipolytica QU21 using industrial waste: a potential feedstock for biodiesel production. Bioresour Technol 161:320–326 Rakicka M, Lazar Z, Dulermo T, Fickers P, Nicaud JM (2015) Lipid References production by the oleaginous yeast Yarrowia lipolytica using indus- trial by-products under different culture conditions. Biotechnol Ashby RD, Solaiman DKY, Strahan GD (2011) Efficient utilization of Biofuels 8:104 crude glycerol as fermentation substrate in the synthesis of poly(3- Ratledge C (2004) Fatty acid biosynthesis in microorganisms being used hydroxybutyrate) biopolymers. J Am Oil Chem Soc 88:949–959 for Single Cell Oil production. Biochimie 86:807–815 1418 Ann Microbiol (2016) 66:1409–1418 Ratledge C, Evans CT (1984) Influence of nitrogen metabolism on lipid Taccari M, Canonico L, Comitini F, Mannazzu I, Ciani M (2012) Screening of yeasts for growth on crude glycerol and optimization accumulation in oleaginous yeasts. J Gen Microbiol 130:1693–1704 Rymowicz W, Fatykhova AR, Kamzolova SV, Rywinska A, of biomass production. Bioresour Technol 110:488–495 Morgunov IG (2010) Citric acid production from glycerol- Thurnhofer S, Vetter W (2006) Application of ethyl esters and d3-methyl containing waste of biodiesel industry by Yarrowia lipolytica in esters as internal standards for the gas chromatographic quantifica- batch, repeated batch, and cell recycle regimes. Appl Microbiol tion of transesterified fatty acid methyl esters in food. J Agric Food Biotechnol 87(3):971–979 Chem 54:3209–3214 Rywinska A, Juszczyk P, Wojtatowicz M, Robak M, Lazar Z, Uçkun Kiran E, Trzcinski A, Webb C (2013) Microbial oil produced from Tomaszewska L, Rymowicz W (2013) Glycerol as a promising sub- biodiesel by-products could enhance overall production. Bioresour strate for Yarrowia lipolytica biotechnological applications. Biomass Technol 129:650–654 Bioenergy 48:148–166 Xue Z, Sharpe PL, Hong SP, Yadav NS, Xie D, Short DR, Damude HG, Saenge C, Cheirsilp B, Suksaroge TT, Bourtoom T (2011) Potential use Rupert RA, Seip JE, Wang J, Pollak DW, Bostick MW, Bosak MD, of oleaginous red yeast Rhodotorula glutinis for the bioconversion Macool DJ, Hollerbach DH, Zhang H, Arcilla DM, Bledsoe SA, of crude glycerol from biodiesel plant to lipids and carotenoids. Croker K, McCord EF, Tyreus BD, Jackson EN, Zhu Q (2013) Process Biochem 46(1):210–218 Production of omega-3 eicosapentaenoic acid by metabolic engi- Sakuradani E, Shimizu S (2009) Single cell oil production by Mortierella neering of Yarrowia lipolytica. Nat Biotechnol 31:734–740 alpina. J Biotechnol 144:31–36 Yen H-W, Yang Y-C, Yu Y-H (2012) Using crude glycerol and thin Santamauro F, Whiffin FM, Scott RJ, Chuck CJ (2014) Low-cost stillage for the production of microbial lipids through the cultivation lipid production by an oleaginous yeast cultured in non-sterile of Rhodotorula glutinis. J Biosci Bioeng 114:453–456 conditions using model waste resources. Biotechnol Biofuels 7: Zhang X, Shanmugam KT, Ingram LO (2010) Fermentation of glycerol 34–44 to succinate by metabolically engineered strains of Escherichia coli. Sitepu IR, Garay LA, Sestric R, Levin D, Block DE, Bruce German J, Appl Environ Microbiol 76:2397–2401 Boundy-Mills KL (2014) Oleaginous yeasts for biodiesel: current Zhang L, Li P, Sun X, Hu W, Wang X, Zhang Q, Ding X (2014) and future trends in biology and production. Biotechnol Adv 32(7): Untargeted fatty acid profiles based on the selected ion monitoring 1336–1360 mode. Anal Chim Acta 839:44–50 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Annals of Microbiology Springer Journals

Conversion of raw glycerol to microbial lipids by new Metschnikowia and Yarrowia lipolytica strains

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Springer Journals
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Copyright © 2016 by Springer-Verlag Berlin Heidelberg and the University of Milan
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Life Sciences; Microbiology; Microbial Genetics and Genomics; Microbial Ecology; Mycology; Medical Microbiology; Applied Microbiology
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1869-2044
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10.1007/s13213-016-1228-0
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Ann Microbiol (2016) 66:1409–1418 DOI 10.1007/s13213-016-1228-0 ORIGINAL ARTICLE Conversion of raw glycerol to microbial lipids by new Metschnikowia and Yarrowia lipolytica strains 1 1 1 1 1 1 L. Canonico & S. Ashoor & M. Taccari & F. Comitini & M. Antonucci & C. Truzzi & 1 1 G. Scarponi & M. Ciani Received: 2 February 2016 /Accepted: 10 June 2016 /Published online: 30 June 2016 Springer-Verlag Berlin Heidelberg and the University of Milan 2016 Abstract Nine Metschnikowia spp. strains and six Yarrowia produced (Poli et al. 2014). However, the process of refining lipolytica strains were tested for lipid production on raw glyc- crude glycerol into a high purity product is expensive and erol from a biodiesel plant. Metschnikowia sp. 271 and energy consuming (Yen et al. 2012). Hence, the direct use of Y. lipolytica 347 were selected for biomass and lipid produc- crude glycerol would help to make biodiesel production more tion, and optimized using central composite factorial design profitable and sustainable (Uçkun Kiran et al. 2013). In this (CCD) and response surface methodology (RSM). The lipid regard, crude glycerol has been used as a sole carbon source production (2.60 g/L) of Y. lipolytica 347 was maximized at by various microalgae, yeasts, molds and bacteria, under both C/N 118, time 144 h and 90 g/L glycerol, while the maximum aerobic and anaerobic conditions (Rywinska et al. 2013), for lipid production (0.49 g/L) of Metschnikowia sp. 271 was the production of some value-added products such as 1,3- obtained under similar conditions but after a further incuba- propanediol (Papanikolaou et al. 2008; Chatzifragkou et al. tion for 7 days at 16 °C. The fatty acids profile of Y. lipolytica 2011), citric acid (Papanikolaou et al. 2008; Rymowicz et al. exhibited a considerable amount of C18:1n7 (~36 %), 2010), biopolymers (Ashby et al. 2011), succinic acid (Zhang C18:1n9 (~16 %), and C16:0 (~16 %), whereas et al. 2010), single cell protein (Taccari et al. 2012) and single Metschnikowia sp. produced mainly C18:1n9 (~33 %), cell oils (Papanikolaou and Aggelis 2009;Saenge et al. 2011). C16:0 (~21 %), and C16:1n7 (~21 %). Both yeasts showed In recent years, growing attention has been paid to the similar amounts of unsaturated fatty acids (~70 %). However, development of single cell oils, and it has been found that considerable amounts of polyunsaturated fatty acid (PUFAs) many microorganisms, such as algae, yeast, bacteria, and fun- were exhibited only by Metschnikowia sp. (~12 %). gi, have the ability to accumulate oils under appropriate culti- vation conditions. Microorganisms that can accumulate lipid to more than 20 % of their biomass are defined as oleaginous . . Keywords Crude glycerol Lipid production species. A number of oleaginous yeast species are known, Metschnikowia sp. Yarrowia lipolytica including Cryptococcus curvatus, Lipomyces starkeyi, Rhodosporidium toruloides, Rhodotorula glutinis and Yarrowia lipolytica (Sitepu et al. 2014). Introduction Lipid production in oleaginous yeasts occurs when a nutri- ent in the medium becomes limited and the carbon source is Crude glycerol is the main byproduct of the biodiesel produc- present in excess. Nitrogen limitation is the most efficient tion chain. The recent growth in biodiesel production has re- condition for inducing lipid accumulation (Ratledge 2004; sulted in a significant increase in the volume of glycerol Beopoulos et al. 2009). When nitrogen becomes unavailable the organism continues to assimilate the carbon source but the growth rate slows down, since nitrogen is essential for protein * M. Ciani and nucleic acid syntheses (Beopoulos et al. 2009). m.ciani@univpm.it These lipids usually consist of triacylglycerols (80–90 %) with a fatty acid composition similar to many plant seed oils Dipartimento Scienze della vita e dell’Ambiente (DISVA), Università Politecnica delle Marche, Via Brecce Bianche, 60131 Ancona, Italy (Ratledge and Evans 1984). Besides, microbial lipid 1410 Ann Microbiol (2016) 66:1409–1418 technology has many advantages, such as the short life cycle 72 h (Y. lipolytica). All experiments were carried out in of microbes as compared to plants, and lack of competition duplicate. with agriculture for land use. Thus, microbial lipids are con- sidered as a potential feedstock for biodiesel production (Liu Experimental design and process optimization and Zhao 2007;Meng et al. 2009). The aim of this work was to evaluate lipid accumulation of Response surface methodology (RSM) was conducted using a different Metschnikowia and Y. lipolytica strains using central composite factorial design (CCD) with three factors crude glycerol from biodiesel production as the sole carbon and two replicates of the central point. All factors and their source. In selected strains, optimal lipid accumulation respective levels are showed in Table 1. The experiments were conditiond and fatty acid composition were also determined. performedinbaffledflasks (250mL)containing50mL growth medium. Ammonium sulfate as a nitrogen source was added to obtain a C/N ratio of 63/145. To evaluate the effect of further incubation on lipid accumulation, Materials and methods Metschnikowia sp. 271 was incubated for an additional 7 days at 16 °C under static conditions. Strains and culture conditions Fifteen yeast strains were involved in this study: Analytical methods Metschnikowia sp. DiSVA 271 and DiSVA 272; Metschnikowia pulcherrima DiSVA 266, DiSVA 267, Yeast growth was estimated using a UV-1800 spectrophotom- DiSVA 268, DiSVA 269, DiSVA 273, DiSVA 274 and eter (Shimadzu, Japan) by measuring the optical density at DiSVA 275, andY. lipolytica DiSVA 347, DiSVA 352, 600 nm. Biomass concentration was evaluated as cell dry CBS 599, CBS 2073, CBS 2074 and CBS 2075. These weight. Glycerol concentrations (g/L) were determined using strains were obtained from the Yeast Collection of the Glycerol kit n° 148270 (Roche, Mannheim, Germany). Dipartimento di Scienze della Vita e dell’Ambiente Extraction of lipids from dry biomass was performed ac- (DiSVA), Università Politecnica delle Marche and the cording to the modified procedure of Bligh and Dyer (1959). Centraal Bureau voor Schimmelcultures, Netherlands (CBS). In short, lipids were extracted with a mixture of The yeast strains were grown on YPD medium, which is com- chloroform:methanol (2:1, v/v) for 24 h, centrifuged to obtain posed of (g/L): peptone, 20; yeast extract, 10; glucose, 20 and a clear supernatant, and the solvent removed by evaporation agar, 20, and maintained at 4 °C. under vacuum. Total cellular lipids (g/L) were determined gravimetrically. Lipids were converted to their fatty acid methyl esters Substrate source (FAMEs) according to a modified method of ISO 12966- 2:2011 (revision of ISO 5509:2000). Briefly, ~10 mg lipid Crude glycerol was obtained from a private biodiesel produc- extract was dissolved in n-heptane (0.5 mL), then 10 μL2 N tion company, and had the following characteristics: pH 7.10, sodium methylate was added. After vortexing for 3 min, the purity 88–92 %, water (3–5 %), potassium and sodium salts solution was centrifuged at 89 g for 1 min, then a few milli- (4–5 %), non-glycerol organic matter (0.5–1 %), methanol grams of sodium metabisulfite were added. Finally, 100 μLof (<0.2 %). the solution was transferred to a vial (1 mL), and diluted with 400 μL n-heptane. Screening of Metschnikowia and Yarrowia lipolytica strains Gas chromatography-mass spectrometry (GC-MS) on crude glycerol analyses were performed on an Agilent-6890 gas chro- matograph with split injection, coupled to an Agilent - Metschnikowia strains were screened on crude glycerol medi- 5973 N quadrupole mass selective detector. A CPS um with the following composition (g/L): glycerol, 40; am- ANALITICA CC-wax_MS (30 m, 0.25 mm ID, monium sulfate, 1 and yeast extract, 0.5 with C/N 60 and pH 6.0, whereas the medium used for Y. lipolytica strains Table 1 Experimental Variable Range and level consisted of 30 g/L glycerol, 0.5 g/L ammonium sulfate and range, and levels of the independent variables yeast extract with C/N 76 and pH 6.0. The experiments were −10 1 carried out in 250 mL baffled flasks containing 50 mL crude glycerol medium, sterilized at 121 °C/20 min and inoculated X , Glycerol (g/L) 30 60 90 from 24 h exponential yeasts pre-culture (2.5 mL). All flasks X ,C/N 63 118 145 were incubated at 28 °C under agitation speed 130 rpm and X , Time (h) 48 96 144 the results were obtained after 144 h (Metschnikowia) and Ann Microbiol (2016) 66:1409–1418 1411 0.25 μm film thickness) capillary column was used to and crude glycerol amounts on biomass and lipid production separate FAMEs. The inlet temperature was 250 °C, with of Metschnikowia and Y. lipolytica strains are shown in injection volume of 1 μL. The oven temperature was pro- Table 2. grammed from 100 °C (1 min), to 230 °C, initially at the Metschnikowia and Y. lipolytica strains showed notable rate of 25 °C/min from 100 to 150 °C, from 150 °C to differences in biomass production. Generally, 200 °C at the rate of 5 °C/min, and from 200 to 230 °C at Metschnikowia strains showed lower biomass production therateof1°C/min, andthetotal runtimewas 43min. compared with Y. lipolytica strains. M. pulcherrima 266 Helium (6.0, SOL, Italy) (8.0 p.s.i.) was used as the car- and M. pulcherrima 267 are the best producers of lipids rier gas. The ion source, transfer line and detector temper- [in absolute values (g/L), and relative values (% w/w)] atures were 230 °C, 250 °C and 150 °C, respectively. compared with the other Metschnikowia strains, but ex- Mass spectra (m/z 50 to m/z 400) were recorded at a rate hibited low crude glycerol consumption. Strains of three scans per second, with ionization energy of Y. lipolytica 347 and Y. lipolytica 2074, produced higher 70 eV. Data were collected under SIM mode. After a lipid amounts [in absolute values (g/L), and relative solvent delay of 2.0 min, the following fragment ions values (% w/w)] in comparison with the other were recorded: m/z 74 and 87 for saturated, m/z 74 and Y. lipolytica strains. For these reasons, and their ability 55 for monoenoic fatty acids, m/z 67 and 81 for dienoic to metabolize crude glycerol, the strains Metschnikowia fatty acids, and m/z 79 and 81 for polyunsaturated fatty sp. 271 and Y. lipolytica 347 were selected for biomass acids (Thurnhofer and Vetter 2006; Zhang et al. 2014). and lipid optimization trials (on the basis of glycerol Identification of fatty acids was performed using NIST consumed and biomass production). reference mass spectra database (NIST, Mass Spectral Database 02, National Institute of Standards and Technology, Gaithersburg, MD; http://www.nist.gov/srd) Biomass and lipid optimization of Metschnikowia sp. 271 MS search 2.0a (NIST 02.L, Ringoes, NJ). The retention and Y. lipolytica 347 strains by RSM time and mass spectra of standard fatty acid methyl esters (Supelco 37-component FAME Mix) were used to con- A Box-Behnken design with three variables (glycerol concen- firm the NIST identification of the fatty acids in the tration, C/N ratio and time process) at three levels was carried sample. out to determine the response pattern. The experimental de- sign matrixes and response values are shown in Tables 3 and 4. The second order polynomial equations for biomass (Y ), Statistical analysis lipid production (Y ) and lipid content (Y ), as a function of 2 3 glycerol concentration (x ), C/N ratio (x ) and time (x )are 1 2 3 The regression and statistical analyses were performed using presented as follows for: the statistical software package JMP 12 (http://www.jmp. com). The mathematical relationship of the independent (1) Yarrowia lipolytica variable was determined by fitting a second order polynomial equation to data obtained from the 15 runs: 2 BiomassðÞ Y ¼ 6:9191 þ 2:2738x –0:0313x 1 1 2 Y ¼ β0 þ ∑βixi þ ∑βiixi þ ∑βijxixj 2 2 þ 1:3401x –0:0521x þ 0:0705x 3 1 2 Where Y is the predicted response, the independent vari- ables (xi and xj), the intercept term (β0), the linear effects (βi), þ 0:5104x –0:3227x x þ 0:2500x x 3 1 2 1 3 the squared effects (βii) and the first order interaction effect þ 0:5704x x Lipid productionðÞ Y 2 3 2 (βij). ¼ 1:4479 þ 0:1862x þ 0:1795x þ 0:6775x 1 2 3 2 2 2 þ 0:0832x –0:0071x –0:3688x 1 2 3 Results and discussion þ 0:0451x x þ 0:2588x x 1 2 1 3 Screening of Metschnikowia and Y. lipolytica strains for lipid production þ 0:2114x x Lipid contentðÞ Y 2 3 3 ¼ 21:3103–4:1165x þ 1:1538x þ 6:3442x 1 2 3 Carbon and nitrogen source, C/N molar ratio, and culture con- 2 2 2 ditions (temperature and pH) have a significant influence on þ 2:0225x þ 0:3922x –8:2350x 1 2 3 cell growth and lipid accumulation of oleaginous yeasts. The þ 2:9151x x þ 0:5125x x þ 1:5569x x 1 2 1 3 2 3 effects of nitrogen sources (yeast extract, ammonium sulfate) 1412 Ann Microbiol (2016) 66:1409–1418 (2) Metschnikowia sp. were used as an instrument to verify the significance of each coefficient, which also shows the interaction strength of each BiomassðÞ Y ¼ 3:3462 þ 0:3515x –0:0938x 1 1 2 parameter. 2 2 2 For Y. lipolytica, the linear terms glycerol concentration þ 0:5431x þ 0:1146x þ 0:1642x þ 0:5521x 3 1 2 3 (x )and time (x ) were significant in terms of biomass, 1 3 þ 0:8700x x lipid production and lipid content. In contrast, the linear 1 2 term C/N ratio (x ) was significant only for lipid produc- þ 0:0x x – 0:0691x x Lipid productionðÞ Y 1 3 2 3 2 tion. The interaction term x x showed a significant ef- 1 2 fect on lipid content, while other interaction terms were ¼ 0:0918–0:0113x –0:0035x –0:0339x 1 2 3 significant for lipid production. The results presented in 2 2 þ 0:0468x þ 0:0004x 1 2 Table 3 show the effect of glycerol concentration, C/N ratio and time on biomass, lipid production and lipid þ 0:0958x –0:0065x x –0:0943x x 3 1 2 1 3 content of Y. lipolytica. The lipids and biomass were þ 0:0277x x Lipid contentðÞ Y maximized (2.60 g/L, 11.5 g/L) at C/N 118, time 144 h, 2 3 3 with 90 g/L glycerol (Fig. 1a,b). The maximum lipid con- ¼ 2:7149–0:9355x –0:0375x –1:4245x 1 2 3 tent (32.6 % w/w) was obtained with a glycerol concen- 2 2 tration of 30 g/L, C/N 63 and time 96 h (Fig. 1c). þ 1:3229x þ 0:1246x 1 2 Moreover, good results of biomass production, lipid ac- þ 2:2729x –1:1832x x –2:1875x x 3 1 2 1 3 cumulation and lipid content (9.0 g/L, 2.07 g/L, 22.8 % w/w) were achieved with a glycerol concentration of þ 1:1688x x 2 3 60 g/L, C/N 145 and time 144 h. These amounts are higher than those achieved by Poli et al. (2014), who obtained 6.7 g/L biomass and 1.27 g/L lipid from The models fit satisfactorily with the experimental data, as Y. lipolytica QU21 using crude glycerol as a carbon indicated from R and P values (Table 5). The values of prob- source, and ammonium sulfate as a nitrogen source. ability (P)> F for biomass, lipid production and lipid content, Also, the yeast strain Y. lipolytica ACA-DC 50109 indicated that the results of the models were significant. The achieved lipid production of 1.37 g/L and 0.79 g/L signs of the factor coefficients in the model equations indicat- (20.4 % and 13.4 % of the dry cell mass) after a fermenta- ed their relative effects on biomass, lipid production and lipid tion time of 50 h and 240 h, respectively, with an initial content, and the significance of each coefficient, as deter- glycerol concentration of 104.9 g/L under bioreactor con- mined by P-values, are illustrated in Table 5.The P-values ditions (Makri et al. 2010). Moreover, Cheirsilp and Table 2 Biomass and lipid Strain Biomass (g/L) Lipid (g/L) Lipid content Glycerol production from different dry weight (%) (w/w) consumed (g/L) Metschnikowia and Yarrowia lipolytica strains grown on crude M. pulcherrima 266 4.1 ± 0.2 0.45 ± 0.10 10.8 ± 2.7 6.0 ± 0.2 glycerol as carbon source M. pulcherrima 267 4.4 ± 0.4 0.38 ± 0.20 8.7 ± 3.7 4.0 ± 0.8 M. pulcherrima 268 4.9 ± 0.2 0.19 ± 0.00 3.8 ± 0.3 15.6 ± 1.5 M. pulcherrima 269 5.0 ± 0.8 0.24 ± 0.01 5.2 ± 1.5 15.6 ± 0.3 Metschnikowia sp. 271 6.4 ± 0.6 0.26 ± 0.01 4.3 ± 0.6 23.0 ± 1.6 Metschnikowia sp. 272 5.7 ± 0.4 0.27 ± 0.10 4.6 ± 1.0 16.4 ± 0.5 M. pulcherrima 273 4.8 ± 0.2 0.25 ± 0.00 4.8 ± 1.1 9.4 ± 1.4 M. pulcherrima 274 4.1 ± 0.4 0.19 ± 0.01 4.6 ± 0.9 19.0 ± 1.1 M. pulcherrima 275 6.1 ± 0.4 0.19 ± 0.10 3.1 ± 1.4 16.4 ± 0.8 Y. lipolytica 347 9.4 ± 0.1 0.43 ± 0.00 4.6 ± 0.1 24.0 ± 2.8 Y. lipolytica 352 8.5 ± 0.7 0.09 ± 0.01 1.0 ± 0.3 12.4 ± 0.2 Y. lipolytica 599 8.7 ± 0.4 0.22 ± 0.02 2.4 ± 0.2 26.5 ± 0.7 Y. lipolytica 2073 6.6 ± 0.2 0.27 ± 0.06 4.2 ± 0.9 28.6 ± 0.1 Y. lipolytica 2074 8.0 ± 0.1 0.65 ± 0.11 8.0 ± 1.2 13.7 ± 0.1 Y. lipolytica 2075 9.0 ± 0.1 0.35 ± 0.08 4.0 ± 0.8 10.6 ± 0.2 Lipid (g/L)/ biomass (g/L) dry weight × 100 Ann Microbiol (2016) 66:1409–1418 1413 Table 3 Experimental designs Run order Glycerol (g) C/N ratio Time (h) Biomass (g/L) Lipid (g/L) Lipid content and response based on (%) (w/w) experimental runs of Y. lipolytica 1 30 63 96 4.0 ± 0.71 1.29 ± 0.07 32.6 ± 4.03 2 30 145 96 6.0 ± 0.00 1.57 ± 0.28 26.2 ± 4.74 3 90 63 96 8.8 ± 0.35 1.45 ± 0.24 16.5 ± 2.12 4 90 145 96 9.0 ± 0.71 1.79 ± 0.19 19.7 ± 0.42 5 60 63 48 7.3 ± 3.89 0.45 ± 0.20 6.4 ± 0.59 6 60 63 144 9.0 ± 0.01 1.28 ± 0.29 14.3 ± 3.25 7 60 145 48 4.8 ± 1.06 0.49 ± 0.01 10.4 ± 2.26 8 60 145 144 9.0 ± 0.00 2.07 ± 0.02 22.8 ± 0.35 9 30 118 48 3.8 ± 0.35 0.36 ± 0.01 9.5 ± 0.71 10 90 118 48 8.3 ± 1.06 0.43 ± 0.03 5.3 ± 0.35 11 30 118 144 6.0 ± 0.01 1.49 ± 0.20 24.8 ± 3.11 12 90 118 144 11.5 ± 0.00 2.60 ± 0.18 22.6 ± 1.56 13 60 118 96 6.3 ± 0.35 1.35 ± 0.07 21.6 ± 0.14 14 60 118 96 7.3 ± 0.35 1.86 ± 0.03 25.7 ± 1.63 15 60 118 96 7.3 ± 0.35 1.32 ± 0.06 18.0 ± 0.00 Lipid (g/L)/ biomass (g/L) dry weight × 100 Louhasakul (2013), using Y. lipolytica TISTR 5151, have a wide margin for improvement in lipid production. showed a lipid production of approximately 2 g/L and lipid In this regard, some genetically engineered Y. lipolitica content of up to 64 % on crude glycerol. In a more recent strains, such as Y. lipolitica PO1f, exhibited lipid accu- study, in a flask experiment using Y. lipolytica A101 strain, mulation approaching 90 % of cell mass (Blazeck et al. lipid production of 2.31 g/L and lipid content of 27.3 % 2014), and the yeast strain Y. lipolitica NS432 achieved were achieved (Dobrowolski et al. 2016). lipid production of up to 85 g/L with lipid content of On the other hand, most of the highest lipid contents 73 % using glucose as a carbon source (Friedlander obtained were from genetically engineered Y. lipolitica et al. 2016). Also, the yeast strain Y. lipolytica strains, while the wild type strains used in this work JMY4086 produced about 24 g/L lipids (40 % of dry Table 4 Experimental designs Run Glycerol (g) C/N Time (h) Biomass Lipid (g/L) Lipid content and response based on order ratio (g/L) (%) (w/w) experimental runs of Metschnikowia sp. 271 1 30 63 96 5.0 ± 0.71 0.14 ± 0.00 2.9 ± 0.49 2 30 145 96 2.3 ± 0.35 0.14 ± 0.01 6.2 ± 0.59 3 90 63 96 3.5 ± 0.01 0.15 ± 0.00 4.2 ± 0.07 4 90 145 96 3.8 ± 0.35 0.13 ± 0.00 3.5 ± 0.42 5 60 63 48 3.0 ± 0.01 0.28 ± 0.16 9.3 ± 5.23 6 60 63 144 4.3 ± 1.06 0.10 ± 0.01 2.5 ± 0.74 7 60 145 48 4.0 ± 0.00 0.25 ± 0.03 6.3 ± 0.71 8 60 145 144 5.0 ± 0.00 0.12 ± 0.01 2.4 ± 0.28 9 30 118 48 2.5 ± 0.01 0.14 ± 0.03 5.8 ± 1.13 10 90 118 48 4.5 ± 0.01 0.29 ± 0.11 6.3 ± 2.36 11 30 118 144 3.5 ± 0.00 0.37 ± 0.01 10.7 ± 0.49 12 90 118 144 5.5 ± 0.01 0.13 ± 0.01 2.4 ± 0.26 13 60 118 96 3.3 ± 0.35 0.10 ± 0.01 3.2 ± 0.21 14 60 118 96 3.3 ± 0.35 0.09 ± 0.01 2.7 ± 0.14 15 60 118 96 3.5 ± 0.00 0.08 ± 0.01 2.3 ± 0.14 Lipid (g/L)/ biomass (g/L) dry weight × 100 1414 Ann Microbiol (2016) 66:1409–1418 Table 5 Estimated regression coefficients and analysis of variance for response variables Coefficient Biomass (Y ) Lipid (Y)Lipid Coefficient Biomass (Y)Lipid(Y)Lipid 1 2 1 2 content (Y ) content (Y ) 3 3 Y. lipolytica Metschnikowia sp. Intercept 6.9191* 1.4479* 21.3103* Intercept 3.3462* 0.0918* 2.7149* x 2.2738* 0.1862* – 4.1165* x 0.3515 – 0.0113 – 0.9355 1 1 x – 0.0313 0.1795* 1.1538 x – 0.0938 – 0.0035 – 0.0375 2 2 x 1.3401* 0.6775* 6.3442* x 0.5431* – 0.0339* – 1.4245* 3 3 x x – 0.3227 0.0451 2.9151* x x 0.8700* – 0.0065 – 1.1832 1 2 1 2 x x 0.2500 0.2588* 0.5125 x x 0.0 – 0.0943* – 2.1875* 1 3 1 3 x x 0.5704 0.2114* 1.5569 x x – 0.0691 0.0277 1.1688 2 3 2 3 2 2 x – 0.0521 0.0832 2.0225 x 0.1146 0.0468* 1.3229 1 1 2 2 x 0.0705 – 0.0071 0.3922 x 0.1642 0.0004 0.1246 2 2 2 2 x 0.5104 – 0.3688* – 8.235* x 0.5521* 0.0958* 2.2729* 3 3 Variability Variability 2 2 R of the model 0.806 0.918 0.858 R of the model 0.665 0.705 0.664 2 2 Adjusted R 0.719 0.881 0.794 Adjusted R 0.514 0.573 0.513 F-ratio 9.26 24.87 13.43 F-ratio 4.41 5.32 4.39 Prob > F <0.0001* <0.0001* <0.0001* Prob > F 0.0027* 0.0009* 0.0028* * P-values significant cell mass) using crude glycerol as a carbon source Indeed, Santamauro et al. (2014) obtained the maximum (Rakicka et al. 2015). In contrast, the wild type strain lipid production from M. pulcherrima in raw glycerol Y. lipolytica NS18 achieved lipid production of 12.8 g/ after an incubation at 25 °C for 3 days followed by L with lipid content of 25 % using glucose as a carbon further 12 days at 15 °C. source (Friedlander et al. 2016). Regarding Metschnikowia sp. 271, the linear effect of time Fatty acid composition (x ) was significant for all parameters, whereas other linear terms were not significant for any parameter. The interaction The major fatty acids species identified in Y. lipolytica term x x gave a positive and significant effect on biomass 347 and Metschnikowia sp. 271 are presented in Table 6. 1 2 production, while the interaction term x x was significant for Y. lipolytica is enriched in C16:0, C16:1n7, C18:0, 1 3 lipid production and lipid content. In this regard, C18:1n9, C18:1n7 and C24:0, whereas the sum of others Metschnikowia sp. 271 showed the optimal crude glycerol fatty acids is below 15 %. Metschnikowia sp. is enriched concentration, C/N ratio and fermentation time of 30 g/L, C/ in C16:0, C16:1n7, C18:0, C18:1n9 and C18:2n6, and N 118 and 144 h, respectively, both for lipid production and the sum of remaining acids is below 6 %. Y. lipolytica lipid content (Table 4). Under these conditions, 0.37 g/L lipids exhibited a considerable amount of C18:1n7 (~36 %), was obtained (Fig. 2a), with lipid accumulation in terms of C18:1n9 (~16 %), and C16:0 (~16 %), whereas relative values (lipid content %) of 10.7 % w/w (Fig. 2b). An Metschnikowia sp. produced mainly C18:1n9 (~33 %), increase of glycerol concentration (90 g/L), under the same C16:0 (~21 %), and C16:1n7 (~21 %). Metschnikowia fermentation conditions, was favorable for higher biomass sp. showed a content of C16:1n7, C18:0 and C18:1n9 production (5.5 g/L), as also reported by Santamauro et al. two-fold higher than that of Y. lipolytica. In this respect, (2014)(Fig. 2c). Meng et al. (2009) reported that oils accumulated by Even though the maximum lipid production of yeasts are predominantly oleic (C18:1), linoleic (C18:2), Metschnikowia sp. was 0.37 g/L, a further incubation stearic (C18:0), palmitic (C16:0) or palmitoleic acids for 7 days at 16 °C under static conditions determined (C16:1). There are some differences between the two an increase in lipid production of 32 % (0.49 g/L). strains. In particular, oleic acid (C18:1) is produced more Ann Microbiol (2016) 66:1409–1418 1415 Fig. 1 Response surface and contour plot of the combined effects of glycerol concentration, C/N ratio, time on a lipid production, b biomass and c lipid content of Yarrowia lipolytica 347 abundantly by Y. lipolytica (52 % of total fatty acids), The results of analysis of the fatty acid composition of Y. while in Metschnikowia sp. C18:1 represents a lower lipolytica DISVA 347 are in agreement with previous results percentage (33 %). Moreover, the fatty acid C18:2n6 (Makri et al. 2010;Poli et al. 2014) except for linoleic acid was found only in Metschnikowia sp., whereas C18:1n7 (C18:2), which was not produced by this strain under the and C24:0 were produced only by Y. lipolytica.The sum conditions tested. of saturated fatty acids is almost comparable between the Regarding PUFAs, the fungus strain Mortierella alpina 1S-4 two strains, while Y. lipolytica showed higher content of exhibited PUFA production ranging from 30 % to 70 % of the monounsaturated fatty acids (MUFAs), and, consequent- total fatty acids (Sakuradani and Shimizu 2009). Also, the ge- ly, lower content of polyunsaturated fatty acids (PUFAs) netically engineered Y. lipolitica Y4053producedhighamount with respect to Metschnikowia sp. of PUFAs, up to 56.6 % of the total fatty acids (Xue et al. 2013). 1416 Ann Microbiol (2016) 66:1409–1418 Fig. 2 Response surface and contour plot of the combined effects of glycerol concentration, C/N ratio, time on a lipid production, b lipid content and c biomass by Metschnikowia sp. 271 The high amount of saturated and monounsaturated C16 of use in lipid applications in food, and in pharmaceutical and C18 fatty acids in the lipids produced by both yeast and cosmetic formulations (Carvalho et al. 2015). On the strains, similar to the vegetable oil feedstock commonly other hand, the use of low-cost lipid production by used for biodiesel (rapeseed, soybean, sunflower and palm) Metschnikowia yeast in non-sterile conditions (Santamauro (Leung et al. 2010), indicates the potential use of these et al. 2014), makes the cultivation of this oleaginous yeast lipids for biodiesel production. On the other hand, the in crude glycerol very attractive. However, further investi- ability of Metschnikowia sp. to produce PUFAs at a con- gations are needed to optimize the production of PUFAs centration of > 10 % is an interesting feature that could be by the Metschnikowia strain used. Ann Microbiol (2016) 66:1409–1418 1417 Table 6 Fatty acid profile (%) of lipid content produced by Y. lipolytica Beopoulos A, Cescut J, Haddouche R, Uribelarrea JL, Molina-Jouve C, 347 and Metschnikowia sp. 271 Nicaud JM (2009) Yarrowia lipolytica as a model for bio-oil pro- duction. Prog Lipid Res 48:375–387 Fatty acid Y. lipolytica Metschnikowia sp. Blazeck J, Hill A, Liu L, Knight R, Miller J, Pan A, Otoupal P, Alper HS (2014) Harnessing Yarrowia lipolytica lipogenesis to create a plat- C16:0 15.9 ± 1.30 21.2 ± 0.43 form for lipid and biofuel production. Nat Commun 5:3131 Bligh EG, Dyer WJ (1959) A rapid method of total lipid extraction and C16:1n7 10.5 ± 0.85 20.6 ± 0.17 purification. Can J Biochem Physiol 37:911–917 C18:0 4.3 ± 0.00 9.3 ± 0.17 Carvalho AKF, Rivaldi JD, Barbosa JC, De Castro HF (2015) C18:1n9 15.6 ± 0.40 32.9 ± 0.07 Biosynthesis, characterization and enzymatic transesterification of C18:1n7 36.5 ± 1.02 — single cell oil of Mucor circinelloides—asustainablepathway for biofuel production. Bioresour Technol 181:47–53 C18:2n6 — 10.5 ± 0.09 Chatzifragkou A, Papanikolaou S, Dietz D, Doulgeraki A, Nychas GJE, C18:3n3 1.2 ± 0.01 1.8 ± 0.03 Zeng AP (2011) Production of 1,3-propanediol by Clostridium C24:0 4.0 ± 0.03 — butyricum growing on biodiesel-derived crude glycerol through a Saturated 31.9 33.0 non-sterilized fermentation process. Appl Microbiol Biotechnol 91:101–112 MUFAs 66.7 54.5 Cheirsilp B, Louhasakul Y (2013) Industrial wastes as a promising re- PUFAs 1.4 12.5 newable source for production of microbial lipid and direct transesterification of the lipid into biodiesel. Bioresour Technol Monounsaturated fatty acids 142:329–337 Polyunsaturated fatty acids Dobrowolski A, Mituła P, Rymowicz W, Miro czuk AM (2016) Efficient conversion of crude glycerol from various industrial wastes into single cell oil by yeast Yarrowia lipolytica. Bioresour Technol 207: Conclusions 237–243 Friedlander J, Tsakraklides V, Kamineni A, Greenhagen EH, Consiglio RSM was used to optimize biomass and lipid produc- AL, MacEwen K, Crabtree DV, Afshar J, Nugent RL, Hamilton MA, Joe Shaw A, South CR, Stephanopoulos G, Brevnova EE tion of selected Y. lipolytica 347 and Metschnikowia sp. (2016) Engineering of a high lipid producing Yarrowia lipolytica 271 strains. The maximum lipid production of both strain. Biotechnol Biofuels 9:77 strains was achieved at C/N 118. To promote lipid ac- Leung D, Wu X, Leung MKH (2010) A review on biodiesel production cumulation in Metschnikowia sp.271,a furtherincuba- using catalyzed transesterification. Appl Energy 87:1083–1095 Liu B, Zhao ZK (2007) Biodiesel production by direct methanolysis of ole- tion at low temperature (16 °C) was needed. Both aginous microbial biomass. J Chem Technol Biotechnol 82:775–780 strains exhibited high amounts of C16 and C18 fatty Makri A, Fakas S, Aggelis G (2010) Metabolic activities of biotechno- acids, which indicates the potential use of this lipid logical interest in Yarrowia lipolytica grown on glycerol in repeated for biodiesel production. Metschnikowia sp. 271 strain batch cultures. Bioresour Technol 101:2351–2358 Meng X, Yang J, Xu X, Zhang L, Nie Q, Xian M (2009) Biodiesel produced acceptable amount of PUFAs. This feature production from oleaginous microorganisms. Renew Energy 34:1–5 could be considered a starting point for further investi- Papanikolaou S, Aggelis G (2009) Biotechnological valorization of bio- gations since its lipid could be profitably used in the diesel derived glycerol waste through production of single cell oil food industry, and in pharmaceutical and cosmetic and citric acid by Yarrowia lipolytica. Lipid Technol 21(4):83–87 formulations. Papanikolaou S, Fakas S, Fick M, Chevalot I, Galiotou-Panayotou M, Komaitis M, Marc I, Aggelis G (2008) Biotechnological valoriza- tion of raw glycerol discharged after bio-diesel (fatty acid methyl Acknowledgments This work was supported financially by Ministero esters) manufacturing process: production of 1,3 propanediol, citric delle Politiche Agricole e Forestali (MIPAF) (D.M. 26285/7303/2009) acid and single cell oil. Biomass Bioenergy 32:60–71 project BI lieviti nel recupero e valorizzazione del glicerolo grezzo Poli JS, Da Silva MAN, Siqueira EP, Pasa VMD, Rosa CA, Valente P derivante dalla produzione di biodiesel (LIEBIG). (2014) Microbial lipid produced by Yarrowia lipolytica QU21 using industrial waste: a potential feedstock for biodiesel production. Bioresour Technol 161:320–326 Rakicka M, Lazar Z, Dulermo T, Fickers P, Nicaud JM (2015) Lipid References production by the oleaginous yeast Yarrowia lipolytica using indus- trial by-products under different culture conditions. Biotechnol Ashby RD, Solaiman DKY, Strahan GD (2011) Efficient utilization of Biofuels 8:104 crude glycerol as fermentation substrate in the synthesis of poly(3- Ratledge C (2004) Fatty acid biosynthesis in microorganisms being used hydroxybutyrate) biopolymers. J Am Oil Chem Soc 88:949–959 for Single Cell Oil production. Biochimie 86:807–815 1418 Ann Microbiol (2016) 66:1409–1418 Ratledge C, Evans CT (1984) Influence of nitrogen metabolism on lipid Taccari M, Canonico L, Comitini F, Mannazzu I, Ciani M (2012) Screening of yeasts for growth on crude glycerol and optimization accumulation in oleaginous yeasts. J Gen Microbiol 130:1693–1704 Rymowicz W, Fatykhova AR, Kamzolova SV, Rywinska A, of biomass production. Bioresour Technol 110:488–495 Morgunov IG (2010) Citric acid production from glycerol- Thurnhofer S, Vetter W (2006) Application of ethyl esters and d3-methyl containing waste of biodiesel industry by Yarrowia lipolytica in esters as internal standards for the gas chromatographic quantifica- batch, repeated batch, and cell recycle regimes. Appl Microbiol tion of transesterified fatty acid methyl esters in food. J Agric Food Biotechnol 87(3):971–979 Chem 54:3209–3214 Rywinska A, Juszczyk P, Wojtatowicz M, Robak M, Lazar Z, Uçkun Kiran E, Trzcinski A, Webb C (2013) Microbial oil produced from Tomaszewska L, Rymowicz W (2013) Glycerol as a promising sub- biodiesel by-products could enhance overall production. Bioresour strate for Yarrowia lipolytica biotechnological applications. Biomass Technol 129:650–654 Bioenergy 48:148–166 Xue Z, Sharpe PL, Hong SP, Yadav NS, Xie D, Short DR, Damude HG, Saenge C, Cheirsilp B, Suksaroge TT, Bourtoom T (2011) Potential use Rupert RA, Seip JE, Wang J, Pollak DW, Bostick MW, Bosak MD, of oleaginous red yeast Rhodotorula glutinis for the bioconversion Macool DJ, Hollerbach DH, Zhang H, Arcilla DM, Bledsoe SA, of crude glycerol from biodiesel plant to lipids and carotenoids. Croker K, McCord EF, Tyreus BD, Jackson EN, Zhu Q (2013) Process Biochem 46(1):210–218 Production of omega-3 eicosapentaenoic acid by metabolic engi- Sakuradani E, Shimizu S (2009) Single cell oil production by Mortierella neering of Yarrowia lipolytica. Nat Biotechnol 31:734–740 alpina. J Biotechnol 144:31–36 Yen H-W, Yang Y-C, Yu Y-H (2012) Using crude glycerol and thin Santamauro F, Whiffin FM, Scott RJ, Chuck CJ (2014) Low-cost stillage for the production of microbial lipids through the cultivation lipid production by an oleaginous yeast cultured in non-sterile of Rhodotorula glutinis. J Biosci Bioeng 114:453–456 conditions using model waste resources. Biotechnol Biofuels 7: Zhang X, Shanmugam KT, Ingram LO (2010) Fermentation of glycerol 34–44 to succinate by metabolically engineered strains of Escherichia coli. Sitepu IR, Garay LA, Sestric R, Levin D, Block DE, Bruce German J, Appl Environ Microbiol 76:2397–2401 Boundy-Mills KL (2014) Oleaginous yeasts for biodiesel: current Zhang L, Li P, Sun X, Hu W, Wang X, Zhang Q, Ding X (2014) and future trends in biology and production. Biotechnol Adv 32(7): Untargeted fatty acid profiles based on the selected ion monitoring 1336–1360 mode. Anal Chim Acta 839:44–50

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Annals of Microbiology – Springer Journals

Published: Jun 30, 2016

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