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

Abstract

Nine Metschnikowia spp. strains and six Yarrowia lipolytica strains were tested for lipid production on raw glycerol from a biodiesel plant. Metschnikowia sp. 271 and Y. lipolytica 347 were selected for biomass and lipid production, and optimized using central composite factorial design (CCD) and response surface methodology (RSM). The lipid production (2.60 g/L) of Y. lipolytica 347 was maximized at C/N 118, time 144 h and 90 g/L glycerol, while the maximum lipid production (0.49 g/L) of Metschnikowia sp. 271 was obtained under similar conditions but after a further incubation for 7 days at 16 °C. The fatty acids profile of Y. lipolytica exhibited a considerable amount of C18:1n7 (~36 %), C18:1n9 (~16 %), and C16:0 (~16 %), whereas Metschnikowia sp. produced mainly C18:1n9 (~33 %), C16:0 (~21 %), and C16:1n7 (~21 %). Both yeasts showed similar amounts of unsaturated fatty acids (~70 %). However, considerable amounts of polyunsaturated fatty acid (PUFAs) were exhibited only by Metschnikowia sp. (~12 %).

Introduction

Crude glycerol is the main byproduct of the biodiesel production chain. The recent growth in biodiesel production has resulted in a significant increase in the volume of glycerol produced (Poli et al. 2014). However, the process of refining crude glycerol into a high purity product is expensive and energy consuming (Yen et al. 2012). Hence, the direct use of crude glycerol would help to make biodiesel production more profitable and sustainable (Uçkun Kiran et al. 2013). In this regard, crude glycerol has been used as a sole carbon source by various microalgae, yeasts, molds and bacteria, under both aerobic and anaerobic conditions (Rywinska et al. 2013), for the production of some value-added products such as 1,3-propanediol (Papanikolaou et al. 2008; Chatzifragkou et al. 2011), citric acid (Papanikolaou et al. 2008; Rymowicz et al. 2010), biopolymers (Ashby et al. 2011), succinic acid (Zhang et al. 2010), single cell protein (Taccari et al. 2012) and single cell oils (Papanikolaou and Aggelis 2009; Saenge et al. 2011).

In recent years, growing attention has been paid to the development of single cell oils, and it has been found that many microorganisms, such as algae, yeast, bacteria, and fungi, have the ability to accumulate oils under appropriate cultivation conditions. Microorganisms that can accumulate lipid to more than 20 % of their biomass are defined as oleaginous species. A number of oleaginous yeast species are known, including Cryptococcus curvatus, Lipomyces starkeyi, Rhodosporidium toruloides, Rhodotorula glutinis and Yarrowia lipolytica (Sitepu et al. 2014).

Lipid production in oleaginous yeasts occurs when a nutrient in the medium becomes limited and the carbon source is present in excess. Nitrogen limitation is the most efficient condition for inducing lipid accumulation (Ratledge 2004; 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 and nucleic acid syntheses (Beopoulos et al. 2009).

These lipids usually consist of triacylglycerols (80–90 %) with a fatty acid composition similar to many plant seed oils (Ratledge and Evans 1984). Besides, microbial lipid technology has many advantages, such as the short life cycle of microbes as compared to plants, and lack of competition with agriculture for land use. Thus, microbial lipids are considered as a potential feedstock for biodiesel production (Liu and Zhao 2007; Meng et al. 2009).

The aim of this work was to evaluate lipid accumulation of different Metschnikowia and Y. lipolytica strains using crude glycerol from biodiesel production as the sole carbon source. In selected strains, optimal lipid accumulation conditiond and fatty acid composition were also determined.

Materials and methods

Strains and culture conditions

Fifteen yeast strains were involved in this study: Metschnikowia sp. DiSVA 271 and DiSVA 272; Metschnikowia pulcherrima DiSVA 266, DiSVA 267, DiSVA 268, DiSVA 269, DiSVA 273, DiSVA 274 and DiSVA 275, andY. lipolytica DiSVA 347, DiSVA 352, CBS 599, CBS 2073, CBS 2074 and CBS 2075. These strains were obtained from the Yeast Collection of the Dipartimento di Scienze della Vita e dell’Ambiente (DiSVA), Università Politecnica delle Marche and the Centraal Bureau voor Schimmelcultures, Netherlands (CBS). The yeast strains were grown on YPD medium, which is composed of (g/L): peptone, 20; yeast extract, 10; glucose, 20 and agar, 20, and maintained at 4 °C.

Substrate source

Crude glycerol was obtained from a private biodiesel production company, and had the following characteristics: pH 7.10, purity 88–92 %, water (3–5 %), potassium and sodium salts (4–5 %), non-glycerol organic matter (0.5–1 %), methanol (<0.2 %).

Screening of Metschnikowia and Yarrowia lipolytica strains on crude glycerol

Metschnikowia strains were screened on crude glycerol medium with the following composition (g/L): glycerol, 40; ammonium sulfate, 1 and yeast extract, 0.5 with C/N 60 and pH 6.0, whereas the medium used for Y. lipolytica strains consisted of 30 g/L glycerol, 0.5 g/L ammonium sulfate and yeast extract with C/N 76 and pH 6.0. The experiments were carried out in 250 mL baffled flasks containing 50 mL crude glycerol medium, sterilized at 121 °C/20 min and inoculated from 24 h exponential yeasts pre-culture (2.5 mL). All flasks were incubated at 28 °C under agitation speed 130 rpm and the results were obtained after 144 h (Metschnikowia) and 72 h (Y. lipolytica). All experiments were carried out in duplicate.

Experimental design and process optimization

Response surface methodology (RSM) was conducted using a central composite factorial design (CCD) with three factors and two replicates of the central point. All factors and their respective levels are showed in Table 1. The experiments were performed in baffled flasks (250 mL) containing 50 mL 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, Metschnikowia sp. 271 was incubated for an additional 7 days at 16 °C under static conditions.

Table 1 Experimental range, and levels of the independent variables

Analytical methods

Yeast growth was estimated using a UV-1800 spectrophotometer (Shimadzu, Japan) by measuring the optical density at 600 nm. Biomass concentration was evaluated as cell dry weight. Glycerol concentrations (g/L) were determined using Glycerol kit n° 148270 (Roche, Mannheim, Germany).

Extraction of lipids from dry biomass was performed according to the modified procedure of Bligh and Dyer (1959). In short, lipids were extracted with a mixture of chloroform:methanol (2:1, v/v) for 24 h, centrifuged to obtain a clear supernatant, and the solvent removed by evaporation under vacuum. Total cellular lipids (g/L) were determined gravimetrically.

Lipids were converted to their fatty acid methyl esters (FAMEs) according to a modified method of ISO 12966-2:2011 (revision of ISO 5509:2000). Briefly, ~10 mg lipid extract was dissolved in n-heptane (0.5 mL), then 10 μL 2 N sodium methylate was added. After vortexing for 3 min, the solution was centrifuged at 89 g for 1 min, then a few milligrams of sodium metabisulfite were added. Finally, 100 μL of the solution was transferred to a vial (1 mL), and diluted with 400 μL n-heptane.

Gas chromatography-mass spectrometry (GC-MS) analyses were performed on an Agilent-6890 gas chromatograph with split injection, coupled to an Agilent -5973 N quadrupole mass selective detector. A CPS ANALITICA CC-wax_MS (30 m, 0.25 mm ID, 0.25 μm film thickness) capillary column was used to separate FAMEs. The inlet temperature was 250 °C, with injection volume of 1 μL. The oven temperature was programmed from 100 °C (1 min), to 230 °C, initially at the rate of 25 °C/min from 100 to 150 °C, from 150 °C to 200 °C at the rate of 5 °C/min, and from 200 to 230 °C at the rate of 1 °C/min, and the total run time was 43 min. Helium (6.0, SOL, Italy) (8.0 p.s.i.) was used as the carrier gas. The ion source, transfer line and detector temperatures were 230 °C, 250 °C and 150 °C, respectively. Mass spectra (m/z 50 to m/z 400) were recorded at a rate of three scans per second, with ionization energy of 70 eV. Data were collected under SIM mode. After a solvent delay of 2.0 min, the following fragment ions were recorded: m/z 74 and 87 for saturated, m/z 74 and 55 for monoenoic fatty acids, m/z 67 and 81 for dienoic fatty acids, and m/z 79 and 81 for polyunsaturated fatty acids (Thurnhofer and Vetter 2006; Zhang et al. 2014). Identification of fatty acids was performed using NIST reference mass spectra database (NIST, Mass Spectral Database 02, National Institute of Standards and Technology, Gaithersburg, MD; http://www.nist.gov/srd) MS search 2.0a (NIST 02.L, Ringoes, NJ). The retention time and mass spectra of standard fatty acid methyl esters (Supelco 37-component FAME Mix) were used to confirm the NIST identification of the fatty acids in the sample.

Statistical analysis

The regression and statistical analyses were performed using the statistical software package JMP 12 (http://www.jmp.com). The mathematical relationship of the independent variable was determined by fitting a second order polynomial equation to data obtained from the 15 runs:

$$ Y=\beta 0+\sum \beta ixi+\sum \beta iix{i}^2+\sum \beta ijxixj $$

Where Y is the predicted response, the independent variables (xi and xj), the intercept term (β0), the linear effects (βi), the squared effects (βii) and the first order interaction effect (βij).

Results and discussion

Screening of Metschnikowia and Y. lipolytica strains for lipid production

Carbon and nitrogen source, C/N molar ratio, and culture conditions (temperature and pH) have a significant influence on cell growth and lipid accumulation of oleaginous yeasts. The effects of nitrogen sources (yeast extract, ammonium sulfate) and crude glycerol amounts on biomass and lipid production of Metschnikowia and Y. lipolytica strains are shown in Table 2.

Table 2 Biomass and lipid production from different Metschnikowia and Yarrowia lipolytica strains grown on crude glycerol as carbon source

Metschnikowia and Y. lipolytica strains showed notable differences in biomass production. Generally, Metschnikowia strains showed lower biomass production compared with Y. lipolytica strains. M. pulcherrima 266 and M. pulcherrima 267 are the best producers of lipids [in absolute values (g/L), and relative values (% w/w)] compared with the other Metschnikowia strains, but exhibited low crude glycerol consumption. Strains Y. lipolytica 347 and Y. lipolytica 2074, produced higher lipid amounts [in absolute values (g/L), and relative values (% w/w)] in comparison with the other Y. lipolytica strains. For these reasons, and their ability to metabolize crude glycerol, the strains Metschnikowia sp. 271 and Y. lipolytica 347 were selected for biomass and lipid optimization trials (on the basis of glycerol consumed and biomass production).

Biomass and lipid optimization of Metschnikowia sp. 271 and Y. lipolytica 347 strains by RSM

A Box-Behnken design with three variables (glycerol concentration, C/N ratio and time process) at three levels was carried out to determine the response pattern. The experimental design matrixes and response values are shown in Tables 3 and 4. The second order polynomial equations for biomass (Y₁), lipid production (Y₂) and lipid content (Y₃), as a function of glycerol concentration (x₁), C/N ratio (x₂) and time (x₃) are presented as follows for:

Table 3 Experimental designs and response based on experimental runs of Y. lipolytica 347

Table 4 Experimental designs and response based on experimental runs of Metschnikowia sp. 271

• (1) Yarrowia lipolytica

  $$ \begin{array}{l}\mathrm{Biomass}\left({\mathrm{Y}}_1\right) = 6.9191+2.2738{\mathrm{x}}_1\hbox{--} 0.0313{\mathrm{x}}_2+1.3401{\mathrm{x}}_3\hbox{--} 0{{.0521\mathrm{x}}_1}^2+0{{.0705\mathrm{x}}_2}^2+0{{.5104\mathrm{x}}_3}^2\hbox{--} \hfill \\ {}0.3227{\mathrm{x}}_1{\mathrm{x}}_2+0.2500{\mathrm{x}}_1{\mathrm{x}}_3+0.5704{\mathrm{x}}_2{\mathrm{x}}_3\hfill \\ {}\mathrm{Lipid}\ \mathrm{production}\left({\mathrm{Y}}_2\right)=1.4479+0.1862{\mathrm{x}}_1+0.1795{\mathrm{x}}_2+0.6775{\mathrm{x}}_3+0{{.0832\mathrm{x}}_1}^2\hbox{--} 0{{.0071\mathrm{x}}_2}^2\hbox{--} 0{{.3688\mathrm{x}}_3}^2+\hfill \\ {}0.0451{\mathrm{x}}_1{\mathrm{x}}_2+0.2588{\mathrm{x}}_1{\mathrm{x}}_3+0.2114{\mathrm{x}}_2{\mathrm{x}}_3\hfill \\ {}\mathrm{Lipid}\ \mathrm{content}\left({\mathrm{Y}}_3\right)=21.3103\hbox{--} 4.1165{\mathrm{x}}_1+1.1538{\mathrm{x}}_2+6.3442{\mathrm{x}}_3+2{{.0225\mathrm{x}}_1}^2+0{{.3922\mathrm{x}}_2}^2\hbox{--} 8{{.2350\mathrm{x}}_3}^2+\hfill \\ {}2.9151{\mathrm{x}}_1{\mathrm{x}}_2+0.5125{\mathrm{x}}_1{\mathrm{x}}_3+1.5569{\mathrm{x}}_2{\mathrm{x}}_3\hfill \end{array} $$

• (2) Metschnikowia sp.

  $$ \begin{array}{l}\mathrm{Biomass}\left({\mathrm{Y}}_1\right)=3.3462+0.3515{\mathrm{x}}_1\hbox{--} 0.0938{\mathrm{x}}_2+0.5431{\mathrm{x}}_3+0{{.1146\mathrm{x}}_1}^2+0{{.1642\mathrm{x}}_2}^2+0{{.5521\mathrm{x}}_3}^2+\hfill \\ {}0.8700{\mathrm{x}}_1{\mathrm{x}}_2 + 0.0{\mathrm{x}}_1{\mathrm{x}}_3\hbox{--}\ 0.0691{\mathrm{x}}_2{\mathrm{x}}_3\hfill \\ {}\mathrm{Lipid}\ \mathrm{production}\left({\mathrm{Y}}_2\right)=0.0918\hbox{--} 0.0113{\mathrm{x}}_1\hbox{--} 0.0035{\mathrm{x}}_2\hbox{--} 0.0339{\mathrm{x}}_3+0{{.0468\mathrm{x}}_1}^2+0{{.0004\mathrm{x}}_2}^2+0{{.0958\mathrm{x}}_3}^2\hbox{--} \hfill \\ {}0.0065{\mathrm{x}}_1{\mathrm{x}}_2\hbox{--} 0.0943{\mathrm{x}}_1{\mathrm{x}}_3+0.0277{\mathrm{x}}_2{\mathrm{x}}_3\hfill \\ {}\mathrm{Lipid}\ \mathrm{content}\left({\mathrm{Y}}_3\right)=2.7149\hbox{--} 0.9355{\mathrm{x}}_1\hbox{--} 0.0375{\mathrm{x}}_2\hbox{--} 1.4245{\mathrm{x}}_3+1{{.3229\mathrm{x}}_1}^2+0{{.1246\mathrm{x}}_2}^2+2{{.2729\mathrm{x}}_3}^2\hbox{--} \hfill \\ {}1.1832{\mathrm{x}}_1{\mathrm{x}}_2\hbox{--} 2.1875{\mathrm{x}}_1{\mathrm{x}}_3+1.1688{\mathrm{x}}_2{\mathrm{x}}_3\hfill \end{array} $$

The models fit satisfactorily with the experimental data, as indicated from R ² and P values (Table 5). The values of probability (P) > F for biomass, lipid production and lipid content, indicated that the results of the models were significant. The signs of the factor coefficients in the model equations indicated their relative effects on biomass, lipid production and lipid content, and the significance of each coefficient, as determined by P-values, are illustrated in Table 5. The P-values were used as an instrument to verify the significance of each coefficient, which also shows the interaction strength of each parameter.

Table 5 Estimated regression coefficients and analysis of variance for response variables

For Y. lipolytica, the linear terms glycerol concentration (x₁) and time (x₃) were significant in terms of biomass, lipid production and lipid content. In contrast, the linear term C/N ratio (x₂) was significant only for lipid production. The interaction term x₁x₂ showed a significant effect on lipid content, while other interaction terms were significant for lipid production. The results presented in Table 3 show the effect of glycerol concentration, C/N ratio and time on biomass, lipid production and lipid content of Y. lipolytica. The lipids and biomass were maximized (2.60 g/L, 11.5 g/L) at C/N 118, time 144 h, with 90 g/L glycerol (Fig. 1a,b). The maximum lipid content (32.6 % w/w) was obtained with a glycerol concentration of 30 g/L, C/N 63 and time 96 h (Fig. 1c). Moreover, good results of biomass production, lipid accumulation and lipid content (9.0 g/L, 2.07 g/L, 22.8 % w/w) were achieved with a glycerol concentration of 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 Y. lipolytica QU21 using crude glycerol as a carbon source, and ammonium sulfate as a nitrogen source. Also, the yeast strain Y. lipolytica ACA-DC 50109 achieved lipid production of 1.37 g/L and 0.79 g/L (20.4 % and 13.4 % of the dry cell mass) after a fermentation time of 50 h and 240 h, respectively, with an initial glycerol concentration of 104.9 g/L under bioreactor conditions (Makri et al. 2010). Moreover, Cheirsilp and Louhasakul (2013), using Y. lipolytica TISTR 5151, showed a lipid production of approximately 2 g/L and lipid content of up to 64 % on crude glycerol. In a more recent study, in a flask experiment using Y. lipolytica A101 strain, lipid production of 2.31 g/L and lipid content of 27.3 % were achieved (Dobrowolski et al. 2016).

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

On the other hand, most of the highest lipid contents obtained were from genetically engineered Y. lipolitica strains, while the wild type strains used in this work have a wide margin for improvement in lipid production. In this regard, some genetically engineered Y. lipolitica strains, such as Y. lipolitica PO1f, exhibited lipid accumulation approaching 90 % of cell mass (Blazeck et al. 2014), and the yeast strain Y. lipolitica NS432 achieved lipid production of up to 85 g/L with lipid content of 73 % using glucose as a carbon source (Friedlander et al. 2016). Also, the yeast strain Y. lipolytica JMY4086 produced about 24 g/L lipids (40 % of dry cell mass) using crude glycerol as a carbon source (Rakicka et al. 2015). In contrast, the wild type strain Y. lipolytica NS18 achieved lipid production of 12.8 g/L with lipid content of 25 % using glucose as a carbon source (Friedlander et al. 2016).

Regarding Metschnikowia sp. 271, the linear effect of time (x₃) was significant for all parameters, whereas other linear terms were not significant for any parameter. The interaction term x₁x₂ gave a positive and significant effect on biomass production, while the interaction term x₁x₃ was significant for lipid production and lipid content. In this regard, Metschnikowia sp. 271 showed the optimal crude glycerol concentration, C/N ratio and fermentation time of 30 g/L, C/N 118 and 144 h, respectively, both for lipid production and lipid content (Table 4). Under these conditions, 0.37 g/L lipids was obtained (Fig. 2a), with lipid accumulation in terms of relative values (lipid content %) of 10.7 % w/w (Fig. 2b). An increase of glycerol concentration (90 g/L), under the same fermentation conditions, was favorable for higher biomass production (5.5 g/L), as also reported by Santamauro et al. (2014) (Fig. 2c).

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

Even though the maximum lipid production of Metschnikowia sp. was 0.37 g/L, a further incubation for 7 days at 16 °C under static conditions determined an increase in lipid production of 32 % (0.49 g/L). Indeed, Santamauro et al. (2014) obtained the maximum lipid production from M. pulcherrima in raw glycerol after an incubation at 25 °C for 3 days followed by further 12 days at 15 °C.

Fatty acid composition

The major fatty acids species identified in Y. lipolytica 347 and Metschnikowia sp. 271 are presented in Table 6. Y. lipolytica is enriched in C16:0, C16:1n7, C18:0, C18:1n9, C18:1n7 and C24:0, whereas the sum of others fatty acids is below 15 %. Metschnikowia sp. is enriched in C16:0, C16:1n7, C18:0, C18:1n9 and C18:2n6, and the sum of remaining acids is below 6 %. Y. lipolytica exhibited a considerable amount of C18:1n7 (~36 %), C18:1n9 (~16 %), and C16:0 (~16 %), whereas Metschnikowia sp. produced mainly C18:1n9 (~33 %), C16:0 (~21 %), and C16:1n7 (~21 %). Metschnikowia sp. showed a content of C16:1n7, C18:0 and C18:1n9 two-fold higher than that of Y. lipolytica. In this respect, Meng et al. (2009) reported that oils accumulated by yeasts are predominantly oleic (C18:1), linoleic (C18:2), stearic (C18:0), palmitic (C16:0) or palmitoleic acids (C16:1). There are some differences between the two strains. In particular, oleic acid (C18:1) is produced more abundantly by Y. lipolytica (52 % of total fatty acids), while in Metschnikowia sp. C18:1 represents a lower percentage (33 %). Moreover, the fatty acid C18:2n6 was found only in Metschnikowia sp., whereas C18:1n7 and C24:0 were produced only by Y. lipolytica. The sum of saturated fatty acids is almost comparable between the two strains, while Y. lipolytica showed higher content of monounsaturated fatty acids (MUFAs), and, consequently, lower content of polyunsaturated fatty acids (PUFAs) with respect to Metschnikowia sp.

Table 6 Fatty acid profile (%) of lipid content produced by Y. lipolytica 347 and Metschnikowia sp. 271

The results of analysis of the fatty acid composition of Y. lipolytica DISVA 347 are in agreement with previous results (Makri et al. 2010; Poli et al. 2014) except for linoleic acid (C18:2), which was not produced by this strain under the conditions tested.

Regarding PUFAs, the fungus strain Mortierella alpina 1S-4 exhibited PUFA production ranging from 30 % to 70 % of the total fatty acids (Sakuradani and Shimizu 2009). Also, the genetically engineered Y. lipolitica Y4053 produced high amount of PUFAs, up to 56.6 % of the total fatty acids (Xue et al. 2013).

The high amount of saturated and monounsaturated C16 and C18 fatty acids in the lipids produced by both yeast strains, similar to the vegetable oil feedstock commonly used for biodiesel (rapeseed, soybean, sunflower and palm) (Leung et al. 2010), indicates the potential use of these lipids for biodiesel production. On the other hand, the ability of Metschnikowia sp. to produce PUFAs at a concentration of > 10 % is an interesting feature that could be of use in lipid applications in food, and in pharmaceutical and cosmetic formulations (Carvalho et al. 2015). On the other hand, the use of low-cost lipid production by Metschnikowia yeast in non-sterile conditions (Santamauro et al. 2014), makes the cultivation of this oleaginous yeast in crude glycerol very attractive. However, further investigations are needed to optimize the production of PUFAs by the Metschnikowia strain used.

Conclusions

RSM was used to optimize biomass and lipid production of selected Y. lipolytica 347 and Metschnikowia sp. 271 strains. The maximum lipid production of both strains was achieved at C/N 118. To promote lipid accumulation in Metschnikowia sp. 271, a further incubation at low temperature (16 °C) was needed. Both strains exhibited high amounts of C16 and C18 fatty acids, which indicates the potential use of this lipid for biodiesel production. Metschnikowia sp. 271 strain produced acceptable amount of PUFAs. This feature could be considered a starting point for further investigations since its lipid could be profitably used in the food industry, and in pharmaceutical and cosmetic formulations.