Bioresource Technology 173 (2014) 324–333

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Designing of a ‘‘cheap to run’’ fermentation platform for an enhanced production of single cell oil from Yarrowia lipolytica DSM3286 as a potential feedstock for biodiesel Komi Nambou a, Chen Zhao a, Liujing Wei a, Jun Chen a, Tadayuki Imanaka a, Qiang Hua a,b,⇑ a b

State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road, Shanghai 200237, PR China Shanghai Collaborative Innovation Center for Biomanufacturing Technology (SCICBT), 130 Meilong Road, Shanghai 200237, PR China

h i g h l i g h t s  Different culture media were tested for lipid production.  The feasibility of produced oils for biodiesel production were evaluated.  A cost-effective minimal culture medium for lipid was obtained.  The scale-up of the designed minimal medium led to a lipid content of 65%.  Yarrowia lipolytica’s oils were highly suitable for quality biodiesel production.

a r t i c l e

i n f o

Article history: Received 8 August 2014 Received in revised form 16 September 2014 Accepted 18 September 2014 Available online 28 September 2014 Keywords: Biodiesel Fermentation Lipids Phosphate limitation Yarrowia lipolytica

a b s t r a c t In this study, the culture medium components screening and filtering were undertaken in order to set up efficient and cost effective minimal culture media for lipid production from Yarrowia lipolytica DSM3286. The basal minimal culture medium (S2) designed yielded lipid content up to 35% of the microbial dry cell weight. A set of fermentation strategies based on this minimal medium was developed and the lipid content was raised to 51%. The scale-up under different fermentation conditions based on S2 medium led to a maximum lipid content of 65%. The produced microbial oils displayed interesting properties to be used as a feedstock for high quality biodiesel production. The minimal media and operable cultivation strategies devised in this study, in association with the works done so far by other authors, could enable fast, massive, viable and more economical production of single cell oils and smooth biodiesel manufacture. Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction Yarrowia lipolytica, the archetypical oleaginous yeast of the Yarrowia clade owing to comprehensive information provided by genomic, systems biology, genetic engineering and transcriptomic data (Beopoulos et al., 2009; Michely et al., 2013) achieved these recent years, is talented in the buildup of valuable lipids (Beopoulos et al., 2009; Fontanille et al., 2012; Michely et al., 2013; Papanikolaou and Aggelis, 2002; Tsigie et al., 2011) and is considered as a promising sustainable biocommodity production scheme for biodiesel due to the ‘‘vegetable oils-like’’ profile of fatty

⇑ Corresponding author at: State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road, Shanghai 200237, PR China. Tel./fax: +86 21 64250972. E-mail address: [email protected] (Q. Hua). http://dx.doi.org/10.1016/j.biortech.2014.09.096 0960-8524/Ó 2014 Elsevier Ltd. All rights reserved.

acids accumulated (Katre et al., 2012). Conversely, for a highperformance substantiation of Y. lipolytica for industrial production of biodiesel, a high production rate of lipids able to generate fatty acids leading to high-quality biodiesel is compulsory. Different cultivation modes and conditions developed so far have revealed that engineered or the wild-type strains of Y. lipolytica can only accumulate lipids as up to 62% of its dry mass. The ability of Y. lipolytica to accumulate lipids is intrinsically related to its physiology and biochemistry and occurs via the de novo synthesis or the ex novo accumulation mechanisms. These mechanisms differently affect the amount and the fatty acids profile of lipids produced and subsequently reverberate on the performance of biodiesel fuels. In the de novo synthesis, wild-type cells can only accumulate up to 20% of their dry cell weight as lipids within a period of 3–10 days. Several studies regarding the enhanced oil production from Y. lipolytica either at the microbial system level or by the

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optimization of the fermentation medium composition, cultural conditions and the implementation of an efficient operative fermentation process were envisaged. More recently, the in silico model of Y. lipolytica iYL619_PCP was reconstructed in our research center to survey the would-be improved lipid production from the said species and the odds of employing minimal culture media for cell growth were foretold (Pan and Hua, 2012), suggesting the possibility of making available minimal media for lipid production. The data of its metabolic features also motivated numerous scientists to engineer metabolic pathways of Y. lipolytica to increase oil and biofuels productivity from this species (Beopoulos et al., 2008). More recently, through genotypic and phenotypic optimization, Y. lipolytica’s native metabolism was rewired for superior de novo lipogenesis leading to a lipid content of 90% (Blazeck et al., 2014). Nevertheless, genetic engineering tools encompass a number of shortcomings that are inherent in the fact that these approaches are overpriced and the amounts of oils yielded rarely attain the expected values since the improvement rate is generally too low. In view of these, further studies must be enthused for developing new improvement strategies of the bioprocess routes and substantial attempts have been made to this purpose. For instance, a two-phases fed-batch cultivation of Y. lipolytica for lipid production has been developed with the advantage of providing the possibility of nutrient adjunction in sequential combination and fermentation process control (Fontanille et al., 2012). But still, the development of more cost-effective, efficient and easily operable fermentation strategies needs to be settled. More importantly, components constituting the culture medium need to be screened and filtered in order to eliminate undesirable compounds and subsequently reduce the cost of the fermentation process. Generally, the culture medium components based improvement of lipid accumulation in oleaginous microorganism is carried out by limiting some nutrients such as nitrogen, phosphate and sulfate in the medium, with a carbon source in excess (Beopoulos et al., 2009; Wu et al., 2010). With the ceaselessly increasing development of the mechanization, the rarefaction of natural resources, the more and more increasing demand in term of transport and energy needs, it becomes very essential to set up an effective fermentation platform that will be ready to meet these needs in a permanent and long-lasting way with more productive outcome trough sustainable innovation. Accordingly, the aspiration of this survey is the development of very minimal culture media for an efficacious and reasonably priced production of single cell oils (SCOs) with biodiesel properties from Y. lipolytica DSM3286. Besides, we aim at developing

and scaling-up pure fermentation schemes under precise culture conditions for a maximal production of microbial lipids. 2. Methods 2.1. Media designing Different series of culture media were developed using a stock of chemical compounds consisted of KH2PO4, Na2HPO4, MgSO4 7H2O, Glucose, (NH4)2SO4, Yeast extract, CaCl22H2O, FeCl36H2O, ZnSO47H2O, MnSO4H2O and CoCl26H2O in view of those habitually used in the literature. All designed media were indicated in Table 1. In specific cases, glucose was replaced by other carbon sources such fructose, glycerol, xylose, maltose, trehalose, L-malate, citrate and sucrose in order to explore the effect of these carbon sources. 2.2. Strain and cultivation conditions A wild type strain, Y. lipolytica DSM3286 purchased from the culture collection of the DSMZ (Germany), was used in this study. The pre-culture was obtained by inoculating a separate colony in YPD medium containing (g/L) glucose 20, peptone 20 and yeast extract 10 and incubating it at 30 °C for 24 h prior to cultivation. The lipid production experiments were performed in duplicate, aerobically, in 250 mL Erlenmeyer flasks containing 100 mL of each of the designed media and inoculated with the pre-culture (initial OD600 = 0.01) and incubated at 30 °C in a rotary shaker incubator under agitation conditions (220 rpm). 2.3. Bioreactor scale up and improvement of fermentation conditions The S2 medium was used for improving the aerobic fermentation conditions through the monitoring of dissolved oxygen (DO). Cultivations were operated in batch mode in a 1.5 L stirred-tank fermenter (Shanghai Biotech, Shanghai, China) with a working volume of 1 L at 30 °C without pH control. The aeration rate was fixed at 1 vvm (volume air per volume per minute) and the agitation rate was set automatically in function of dissolved oxygen (DO) concentration desired. Sterile air filters with 0.2 lm pores were used for air transfer into bioreactor. The dissolved oxygen concentration (DO) in the culture broth was measured using a pO2 electrode. Silicone was periodically added as an antifoam agent. The aeration and agitation conditions that gave best productivity were used in fed-batch mode which was carried out in conformity

Table 1 Composition and characteristics of designed culture media (g/L).

K1 K2 K3 K4 E1 E2 E3 E4 E5 E6 E7 E8 S1 S2 S3 S4

KH2PO4

Na2HPO4

MgSO47H2O

Glucose

(NH4)2SO4

Yeast extract

CaCl22H2O

FeCl36H2O

ZnSO47H2O

MnSO4H2O

CoCl2 6H2O

pH without glucose

7 1.75 0.007 0.0007 7 3.5 1.75 0.875 0.07 0.007 0.0007 0 0 0 0 0

2.5 2.5 2.5 2.5 0 0 0 0 0 0 0 0 0 0 0 0

1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 15 1.5 0.1 0

30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30

0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5

0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5

0.15 0.15 0.15 0.15 0.15 0.15 0.15 0.15 0.15 0.15 0.15 0.15 0 0 0 0

0.15 0.15 0.15 0.15 0.15 0.15 0.15 0.15 0.15 0.15 0.15 0.15 0 0 0 0

0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0 0 0 0

0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.06 0 0 0 0

0.0001 0.0001 0.0001 0.0001 0.0001 0.0001 0.0001 0.0001 0.0001 0.0001 0.0001 0.0001 0 0 0 0

5.78 6.37 7.32 7.30 4.14 4.11 4.09 4.10 4.10 4.09 4.09 4.37 6.05 6.24 6.42 6.75

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with strategy 1 as described previously in the preliminary cultivation strategies section. 2.4. Analytical methods Glucose consumption was determined by spectrophotometric method using a Glucose Reagent Kit (Kexin Biotech Co., Ltd, Shanghai, China) according to the manufacturer’s instructions. The dry cell weight (DCW) was determined by gravimetric method. 5 mL of culture broth was centrifuged for 5 min in preweighed test tubes at 4 °C and 10,000 g, washed once with water, and dried for 24 h at 105 °C until constant weight. Citric acid was measured by HPLC using a BIORAD 30 cm  7.8 mm Aminex HPX37H organic acids separation column. The mobile phase used was 0.01 N of KH2PO4. The pH of the mobile phase was adjusted to 2.5 with a solution of HPO3. The concentration was determined using a standard curve of pure citric acid.

the flow rate of carrier gas was set at 1 mL/min. Other settings were as follows: 250 °C of interface temperature, 230 °C of ion source temperature, and electron impact ionization (EI) at 70 (or 70) eV with a full scan ranging from 70 to 560 m/z and a solvent delay of 1.5 min. 2.6. Data analysis statistics and similarity test Graphs and statistical analysis of data were operated using the software GraphPad prism version 5.0 (GraphPad Software, Inc., La Jolla, CA 92037 USA). Two-way analysis of variance (ANOVA) with Tukey’s multiple comparisons, with a < 0.01 as significant effect, was applied to the data. Further cluster analysis test between obtained products was carried out using PAST software version 2.03. 3. Results and discussion

2.5. Lipid extraction, biodiesel production and GC–MS analysis

3.1. Lipid and biomass production in designed media

Lipid extraction was carried out as follows. Samples were collected after 72 h of cultivation by centrifugation at 4000g for 10 min. The resulting pellet was washed once and re-suspended in 7 mL of a 60:80 (volume HCl: volume H2O) HCl solution and then heated to boiling in order to disrupt yeast cells. After cooling in ice, 30 mL of chloroform–methanol 3:2 (v:v) solution was added and the whole mixture incubated at 37 °C in a shaker incubator for almost 1 h. After this time, the lower phase was recuperated and purified by distillation under vacuum at 80 °C, then dried at 105 °C for 1 h, cooled under vacuum and weighed. The extracted oil was derivatized using a solution of boron trifluoride methanol. The extracted oil was dissolved in 3 mL of 0.5 mol/L potassium hydroxide–methanol solution and heated in the 75 °C water for 20 min. Subsequently, 3 mL of 14% boron trifluoride methanol solution were added and incubated in 75 °C water for additional 20 min in term of which 1 mL of saturated sodium chloride solution was added. After centrifugation, the supernatant oil layer was gently and carefully pipetted and mixed with 0.5 ml of hexane and diluted 10 times. The microbial oils were transesterified to fatty acid methyl esters (FAMEs or Biodiesel) and 0.2 lL of the resulting derivatized sample was injected into the Agilent 6890–5975 (GC–MS) (Agilent Technologies, Santa Clara, CA). The HP-5 ms column (30 m  0.25 mm  0.25 lm) (Agilent Technologies) was used for GC–MS analysis. GC oven temperature was programmed from 180 °C (2 min) to 250 °C at 5 °C/min, and

The results of lipid production together with biomass yielded after 72 h of cultivation are depicted in Table 2. It can be seen that for media in which both phosphate sources were used, the biomass yielded was proportional to the phosphate concentration in designed media. The highest biomass in term of dry cell weight was obtained in the K1 medium (7.31 g/L) and decreased respectively for K2 (6.46 g/L), K3 (5.44 g/L) and K4 (5 g/L). The lipid production in these media ranged from 0.74 g/L in K7 to 1.13 g/L in K3 while the lipid content in the biomass varied from 14.11% to 17.57% of the dry cell weight. For media with KH2PO4 as single phosphate source, the biomass similarly had the tendency to decrease, except for E6, probably due to the culture medium osmolarity. The biomass concentration decreased from 6.94 g/L in E1 to 1.35 g/L in E8. Meanwhile, the lipid amounts obtained were above 1 g/L of culture broth except for E8 which was hostile for lipid synthesis. The lipid content was between 20% and 26%. From the aforesaid, it is deducible that phosphate is important for biomass production. The nature of phosphate sources differently affected lipid biosynthesis. Indeed, the non-adjunction of Na2HPO4 conducted to an increase in lipid synthesis. Phosphate limitation was found to be lipid production stimulator in various studies. The observation in the present study showed that media deprived of phosphate gave high lipid content. This was corroborated by the work claiming that lipid accumulation by Rhodosporidium toruloides Y4 was directly linked to the

Table 2 Lipid and biomass production and Glucose consumption by Y. lipolytica on different designed media after 72 h of cultivation in flasks. Values represent the means of two separate repeated experiments.

K1 K2 K3 K4 E1 E2 E3 E4 E5 E6 E7 E8 S1 S2 S3 S4

Biomass X (g/L)

Lipid (g/L)

Lipid-free biomass (g/L)

Lipid content (%)

Consumed glucose (g/L)

Lipid yield (g/g glucose)

Lipid productivity (g/h/L)

Final pH

7.31 6.46 5.44 5.00 6.94 5.75 5.16 5.3 4.89 5.82 4.50 1.35 6.23 3.99 2.46 1.09

1.05 1.14 0.77 0.74 1.00 1.27 1.33 1.27 1.06 1.42 1.04 0.08 0.51 1.37 0.59 0.13

6.26 5.32 4.67 4.26 5.95 4.48 3.83 4.03 3.83 4.40 3.46 1.27 5.72 2.62 1.87 0.96

14.34 14.34 14.34 14.34 14.34 14.34 14.34 14.34 14.34 14.34 14.34 14.34 14.34 14.34 14.34 14.34

17.69 20.75 18.05 14.09 17.50 18.99 17.90 16.80 16.58 16.74 18.00 4.54 17.50 18.99 17.90 16.80

0.06 0.05 0.04 0.05 0.06 0.07 0.07 0.08 0.06 0.08 0.06 0.02 0.03 0.07 0.03 0.01

0.15 0.16 0.11 0.10 0.14 0.18 0.19 0.18 0.15 0.20 0.14 0.01 0.07 0.19 0.08 0.02

2.76 2.44 2.66 2.63 2.5 2.26 2.17 2.07 2 2.03 1.96 2.07 2.08 2.03 1.93 2.21

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carbon to phosphorus (C/P) molar ratios of the culture media (Wu et al., 2010). The present study showed that under phosphate limited conditions, other factors are likely to guide lipid synthesis. The effect of sulfate and mineral elements limitation were investigated and the result showed that the simultaneous non-adjunction of phosphate and mineral solution increased the lipid production (E8 compared to S2). This can be explained by the fact that phosphate may intervene in the regulation of the chemical oxidative stress provoked by mineral elements which delayed the cell growth and lipid accumulation. The increase of MgSO47H2O concentration led to greater biomass production while its decrease was followed by low biomass production. The greatest amount of lipid was obtained with the S2 medium in which cells were able to accumulate up to 35% of their dry weight. However, a complete depletion of MgSO4 was followed by the slowdown of lipid production. In S4 which was composed only of glucose and nitrogen sources, no production of lipid was observed. Given that the addition of MgSO47H2O to this medium in different proportions permitted lipid biosynthesis, it can be concluded that MgSO47H2O governs lipid synthesis in Y. lipolytica undoubtedly through the activity of its constitutive ions (Mg2+, SO2 4 ). Indeed, several studies reported that higher Mg2+ concentration favors biomass production. Here, we remarked the same effect. This is explainable by the fact that Mg might be involved in a great assortment of living functions and enzymatic reactions as it may interact with phospholipids, proteins and nucleic acids (Cowan, 2002). Groundbreaking work has connected Mg as a crucial factor of the ostensible ‘‘coordinated response of growth and metabolism’’. The mainstream enzymes involved on one hand in glycolysis, the citric acid cycle and the respiratory chain, which embody the mainstay of energy metabolic rate, and on the other hand in replication, transcription and translation reactions, are Mg-dependent and Mg acts either as an allosteric modulator or as a cofactor in the form of Mg-ATP2 (Cowan, 2002). In yeasts, the PAH1-encoded PAP1 is reported as an Mg2+-dependent enzyme that generates the lipid synthesis precursor DAG (Kosa and Ragauskas, 2011). This could enlighten the highest lipid accumulation in media made with MgSO47H2O. 3.2. Glucose consumption The measurement of residual glucose after 72 h of cultivation (Table 2) showed that less than 20 g/L of glucose was consumed in designed media by yeast cells either for cell growth or metabolites production such as lipids. The greatest glucose uptake (20.75 g/L) was recorded in K3 medium while only 5 g/L of glucose was consumed in E8 medium. In k7 medium, less than 15 g/L of glucose was consumed. In the rest of media, the consumed glucose varied between 17 and 20 g/L. The residual amount of glucose showed an excess of glucose in culture media. Katre et al. (2012) found the same tendency with wild type strains of Y. lipolytica even after 96 h of cultivation. This implies that the correct monitoring of glucose could be more cost effective for lipid production from Y. lipolytica. In their study, Papanikolaou et al. (2006)) cultivated a strain of Y. lipolytica in 34, 42 and 52 g/L of glucose media and the remaining glucose concentrations were respectively 1.1, 4.0 and 2.5 g/L, which were lower than the residual glucose measured in our designed media. In addition to the possible weak ability of the used strain to uptake glucose, we can also stipulate the economical assets of media designed here. The maximum lipid yield calculation showed that in E6 (lipid yield = 0.085 g/g), the consumed glucose was actively channeled for lipid biosynthesis, followed by E4 (0.076 g/g), E3 (0.074 g/g) and S2 (0.072 g/g). The lowest maximum yields were obtained in media E8 (0.018), S1 (0.029) and S4 (0.008). In their review,

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Papanikolaou and Aggelis reported that, stoichiometrically, about 1.1 mol of acetyl-CoA are generated from 100 g of glucose (0.56 mol) catabolized. Therefore, in case of total channelization of total produced acetyl-CoA towards lipid synthesis, the maximum theoretical lipid yield is approximately 0.32 g/g but experimentally, even under ideal conditions for SCO production (e.g. highly aerated chemostat cultures) lipid yield can rarely be higher than 0.22 g/g and is situated in the level of 0.20 g/g or even lower. In this study, the lipid yields obtained from different glucose culture media were far lower, suggesting the probable diversion of consumed glucose for other metabolic functions apart from lipids production. Especially, the consumed glucose may be channeled towards organic acids production namely citric acid. 3.3. GC–MS analysis of produced oils and their quality assessment as biodiesel feedstock The fatty acids composition of oils produced from different designed media was assayed by GC–MS and the fatty acid profile is summarized in Table 3A). These fatty acids were eluted as fatty acids methyl esters (FAMEs or biodiesel). The result showed the presence of 5 distinct fatty acids methyl esters namely palmitic acid methylester, palmitoleic acid methylester, linoleic acid methylester, oleic acid methylester and stearic acid methylester in all designed media. In addition, 15-tetracosenoic acid methylester was found in small extent in E3 and S2. Oleic acid methyl ester occupied 61.60–78.42% of the total FAMEs, followed by palmitic acid methyl ester, linoleic acid methyl ester, stearic acid methyl ester and 15-tetracosenoic acid methyl ester. The fatty composition was rather different from previous reports where, all the time, linoleic acid level is higher that this report and stearic acid quite low. High relative oleic acid concentrations (up to 58.5%) have also been observed in the wild-type or engineered strains of Y. lipolytica, and is more similar to profiles of other oleaginous yeasts that accumulate more than 50% lipid content (Beopoulos et al., 2009). In conditions of rapid lipid production, oleic acid might be more rapidly stored and easier to accumulate, as DGA1p is known to have varying specificities for different acyl-CoA. Despite the huge number of oleaginous microorganisms, they have dissimilar prospects for biodiesel manufacture. The potential of produced oils as feedstocks for biodiesel production was evaluated by comparing their fatty acid composition to that of oils reported in biodiesel production. The survey revealed that oils produced in this study contained fatty acids usually found in plant oils used for biodiesel production. In addition, palmitoleic acid (C16:1) was recorded in the present study, which is contributive to the augmentation of the degree of unsaturation, a feature that is appreciated for biodiesel production. The unsaturated FAMEs represented 73.66–85.12% of the total esters. The fraction of monounsaturated FAMEs was comprised between 69.34% and 83.27%. Other parameters such the saponification number SN, the Iodine value IV and the cetane number CN that characterize biodiesel were calculated with the help of Eqs. 1–3, respectively (Khot et al., 2012):

SN ¼ IV ¼

X

X

560  Ai=MWi

ð1Þ

254  D  Ai=MWi

ð2Þ

wherein, Ai is the percentage, D is the number of double bonds and MWi is the molecular mass of each component. CN of FAMEs was calculated from Eq. (3) (Khot et al., 2012):

CN ¼ 46:3 þ 5458=ðSN  0:225  IVÞ:

ð3Þ

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Table 3 Fatty acids methyl esters (FAMEs) profile of oils obtained from A) different designed media, B) different carbon sources and C) after scale up under different fermentation conditions. ME: methyl ester. 15-Tetracosenoic acid ME

Palmitoleic acid C16:1 ME

Palmitic acid C16:0 ME

Linoleic acid C18:2 ME

Oleic acid C18:1 ME

Stearic acid C18:0 ME

Linolenic acid ME

(A) Designed media

K1 K2 K3 K4 E1 E2 E3 E4 E5 E6 E7 S2

0 0 0 0 0 0 0.49 0 0 0 0 0.31

8.07 7.8 8.4 8.18 4.84 5.52 6.62 6.45 6.62 7.74 13 14.34

13.05 12.2 11.83 12.67 10.38 11.48 12.39 14.63 13.44 18.59 19.7 12.15

2.71 2.68 3.02 2.14 1.86 2.44 3.18 2.52 3.55 4.32 0.86 1.06

70.32 71.51 71.06 72.38 78.42 74.88 69.82 70.5 69.89 61.6 62.7 68.93

5.86 5.8 5.68 4.63 4.51 5.68 6.14 5.9 6.5 7.75 3.75 3.2

0 0 0 0 0 0 0 0 0 0 0 0

(B) Carbon sources

Glucose Fructose Glycerol Sucrose

1.13 0.91 1.54 0

15.04 13.93 13.78 11.01

12.6 14.67 12.91 13.71

1.65 2.06 0 0

65.75 63.63 67.81 68.01

3.83 4.82 3.96 7.26

0 0 0 0

(C) Fermentation conditions

DO = 20%-48 h DO = 20%-72 h DO = 50%-48 h DO = 50%-72 h DO = 50%-MgSO4 7H2O-48 h DO = 50%MgSO47H2O-72 h

0 0 0 0 0

14.32 14.39 11.21 12.63 12.85

16.04 13.58 15.96 14.05 17.61

1.25 2.05 2.58 0 2.88

60 65.83 65.3 68.04 58.45

4.31 4.15 4.95 5.55 5.45

3.26 0 0 0 2.72

0

13.12

14.44

2.48

64.6

5.38

0

The CN is the ability of fuel to ignite quickly after being injected. Oils with higher CN value are of better ignition quality fuel. It is one of the imperative considerations taken in account during the selection of FAMEs for use as biodiesel. Biodiesel standards of USA (ASTM D 6751), Germany (DIN 51606) and European Organization (EN 14214) have fixed this value at 47, 49 and 51, respectively. All the FAMEs of oils produced by Y. lipolytica on the designed media had CN value of 75.08–75.95, which were higher than 51, the highest minimum value among the three biodiesel standards. Furthermore, the CN of produced oils was over than that of vegetable oils such rapeseed oil, coconut oil, jatropha oil, linseed, peanut oil, etc. . . and some oleaginous microorganisms such fungi (Khot et al., 2012; Puhan et al., 2010), this potentiates these microbial oils as a suitable feedstock for biodiesel production and shows its importance in the reduction of the utilization of vegetable oils in biodiesel production with its associated competition with food crops. Another imperative norm for choosing FAMEs is the degree of unsaturation, which is measured as IV. The occurrence of unsaturated fatty acids in FAMEs is essential for limiting the FAMEs from solidification. All the produced oils meet the specification of IV. Their IV was comprised between 70.91 and 78.71, beneath 115, the lowest maximum limit within the three biodiesel standards. Another benchmark recommends that the concentration of linolenic acid and acid containing four double bonds in FAMEs should not exceed the limit of 12% and 1%, respectively. None of the produced oils contains fatty acids with more than three double bonds and the percentage of linoleic acid recorded here varied from 0.86% to 4.32%. Other properties of produced biodiesels such as high heating value (HHV), viscosity (VS) at 313.2 K and flash point (FP) were determined using mathematical Eqs. 4–6 established by (Demirbas (2008) in this way:

The HHV of biodiesels produced from different vegetable oils ranges from 37.27 to 40.12 MJ/kg with flash points (FP) of 415–463 K, viscosity values of 2.83–5.12 cSt and densities of 848–885 g/L (Puhan et al., 2010). The HHV of biodiesels produced here had values situated between 42.63 to 42.75 MJ/kg, which is slightly higher than that mentioned above and in coherence with the HHV of diesel fuel (40–45 MJ/kg), then characterizing the energetic value and the fuel property of microbial oils produced by Y. lipolytica. The HHVs of biodiesels (39–41 MJ/kg) are, to some extent, beneath those of gasoline (46 MJ/kg), petrodiesel (43 MJ/ kg) or petroleum (42 MJ/kg), but above those of coal (32–37 MJ/ kg. It obvious that biodiesels produced herein had HHVs similar to those of petroleum or petrodiesel. According to the biodiesel standards of USA (ASTM D 6751) and European Organization (EN 14214), a minimum flash point of 130 K and a viscosity value between 1.9 and 6.0 cSt are necessary. The flash point (FP) and viscosity values calculated in the present study were somewhat higher than that of plant oils however the FP met the requirement whilst the viscosity values of 6.53–7.15 were a little bit over the range. The higher viscosity array of biodiesel facilitates the reduction of container/plunger outflow and improves injector efficacy in engines. For that reason, the viscosity qualifications recommended are approximately identical to that of the diesel fuel. Therefore, the viscosity values obtained above could be of great contribution on the performance of biodiesels produced with Y. lipolytica’s lipids. Otherwise, as the gap is so minute, upgrading work needs to be done so as to shift the viscosity in the entailed interval. The properties of produced biodiesels are recapitulated in Table 4. Along these lines, oils produced by Y. lipolytica DSM3286 respect the conditions of US biodiesel standard, Germany and European Standard Organization and are then found to be a suitable feedstock for biodiesel production.

VS ¼ 49:43  0:041SN þ 0:015IV

ð4Þ

FP ¼ 0:4625VS þ 39:450

3.4. Test of similarity regarding the most relevant medium for biodiesel production

ð5Þ

HHV ¼ 0:021FP þ 32:12

ð6Þ

The test of similarity was carried out taking into account, on a global scale, test parameters including the fatty acid composition, the

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CN

HHV

VS

FP

72.31 73.03 73.8 73.19 74.72 73.34 71.65 70.61 72.03 67.33 67.27 74.41

77.03 77.1 77.12 77.07 77.33 77.21 77.57 76.97 77.06 76.67 76.46 76.95

42.64 42.66 42.66 42.65 42.72 42.68 42.76 42.61 42.64 42.52 42.47 42.63

6.89 6.93 6.95 6.92 7.07 6.99 7.15 6.84 6.9 6.64 6.53 6.87

500.78 501.7 502.12 501.47 504.66 503.03 506.5 499.62 500.98 495.39 492.92 500.41

0.9998

0.9999

Similarity 0.9994

0.9995

0.9996

0.9997

1.0000

S2 E6 E7 E2 E3 E4 E5 E1 K3 K4 K1 K2 Fig. 1. Test of similarity (cluster analysis) regarding the suitability of designed media for biodiesel production from Y. lipolytica.

biomass, the lipid concentration, the lipid content and the biodiesel properties of the produced SCOs such CN, IV, SN, HHV, VS and FP. The resulting cluster is shown in Fig. 1. The results showed that oils produced on different designed media shared more than 99.94% of similarity between obtained products. This result was substantiated by two-way ANOVA statistical analysis which showed no significant difference amongst biodiesels produced from designed media. Bearing in mind the above statement, it is noticeable that S2 medium is the most suitable for lipid production using Y. lipolytica DSM3286 because of the minimalism of its composition. Indeed, in spite of its simple composition, Y. lipolytica DSM3286 is able to grow on S2 medium and accumulate lipid up to more than 35% of its dry cell weight, which is more than the lipid content of other media. Furthermore, the SCOs produced in S2 present almost the same biodiesel properties as those obtained with other media. Therefore, S2 is found to have cost effective intrinsic worth and its scale up could help to promote the industrial production of biodiesel. 3.5. Malleability of S2 medium based on the replacement of the carbon sources To make microbial oils low-priced, the exploration of other carbon sources rather than glucose is incredibly imperative specif-

8

40

6

30

4

20

2

10

0

0

Lipid content (%)

IV

192.15 191.95 192.01 192.12 191.05 191.38 188.98 192.13 191.95 193.09 194.33 193.11

Biomass X (g/L) Lipid (g/L)

SN

-G lu co S2 se -F ru cto S2 se -G lyc er S2 ol -S uc ro se S2 -X yl o se S2 -M al to S2 se -T re ha lo S2 se -L -m al at e S2 -C itr at e

K1 K2 K3 K4 E1 E2 E3 E4 E5 E6 E7 S2

ically for oils harnessed to biodiesel production. With the goal of broadening lipid production, a set of eight carbon sources were used as substitutes to glucose (Fig. 2). The strain used grew weakly and was unable to produce lipids when glucose was replaced by maltose, xylose, trehalose, L-malate and citrate probably because of enzymatic deficiency or enzymatic inhibition effect. The results showed that instead of glucose, fructose, sucrose and glycerol can be used for oil production from Y. lipolytica. Glycerol, in the form of pure glycerol or industrial waste glycerol has been used for many applications with Y. lipolytica among which microbial oil production has been the main topic (Papanikolaou and Aggelis, 2002; Yang et al., 2014). Numerous studies have also reported the ability of several wild type or engineered strains of Y. lipolytica to grow on sucrose (Praphailong and Fleet, 1997) but none has reported lipid production from this species on this carbohydrate. This may constitute a controversial point since it is generally admitted that the wild-type strains of Y. lipolytica are incapable to grow on sucrose. Nevertheless, it was previously reported that wild-type strains of Y. lipolytica was able to grow on sucrose to concentrations up to 50% under different pH values varying from 2 to 7 (Praphailong and Fleet, 1997). This was in corroboration with the findings of the present study which demonstrated the double capacity of Y. lipolytica DSM3286 to grow and produce lipids on sucrose. This might be explained by the fact that there are different wild-type strains of Y. lipolytica and those wild-type strains might have similar or different physiological and adaptation responses to the medium environment. Moreover, lipid production by this species on fructose has not yet been reported. This paper is the first to convey the capability of a wild type strain of Y. lipolytica to accumulate lipid on both carbon sources. Undeniably, Y. lipolytica DSM3286 exhibited alike cell growth and lipid biosynthesis behavior when fructose as well as glycerol and sucrose were used as carbon sources. This behavior was close to that unveiled in S2 medium. Nevertheless, as shown in Table 3B, the FAMEs profiles of oils obtained from fructose and glucose were similar. The profile obtained on glycerol was different from the aforementioned by the absence of linoleic acid while the profile achieved on sucrose differed from that of oil produced on glycerol by the nonappearance of 15-tetracosenoic acid methylester. On a global point of view, the FAMEs profiles got from different carbon sources diverge in term of fatty acid composition and their relative concentrations; this led to different biodiesels with particular properties.

S2

Table 4 Calculated properties of produced biodiesels from Y. lipolytica on different designed media. SN, saponification number; IV, Iodine value; CN, cetane number; HHV, high heating value; VS, viscosity; FP, flash point.

Carbon sources Biomass X (g/L)

Lipid (g/L)

Lipid content (%)

Fig. 2. Growth and lipid production abilities of Y. lipolytica on different carbon sources. Error bars represent standard deviation between the means of two separate repeated experiments.

K. Nambou et al. / Bioresource Technology 173 (2014) 324–333

The S2 medium was used to produce lipid in a 1.5 L fermenter in batch mode. The dissolved oxygen (DO) concentration was fixed respectively at 20% and 50%. Lipid and biomass production in these conditions are depicted on Fig. 3A. At DO 20%, the dry cell weight of 4.09 and 4.46 g/L was obtained at 48 h and 72 h, respectively. At these time points, lipid yield was 1.66 g/L and 2.02 g/L corresponding respectively to lipid content of 40.70% and 45.51%. At DO 50%, lipid content of 45.83% (DCW = 3.1 g/L; lipid yield = 1.41 g/L) and 65.19% (DCW = 3.03 g/L; lipid yield = 1.88 g/L) were obtained at 48 h and 72 h of fermentation, respectively. Dissolved oxygen concentration (DOC) is one of the major parameters affecting yeast physiological and biochemical characteristics, and in consequence fermentation performance. In the bioreactor trials, it has been demonstrated that cultures presenting higher dissolved oxygen tension (DOT) values store higher quantities of lipids in DCW than in trials with lower DOT values. Especially, at lower DOC, Y. lipolytica cells cultivated in glucose medium are grown in the form of mycelia and produce low quantities of lipids. On the contrary, at higher DOC, the formation of single cells and lipogenesis, as a result of the up-regulation of enzymes implicated in lipid biosynthesis such as ATP-citrate lyase and malic enzyme, are favored (Bellou et al., 2014). This observation was correlated with the high lipid content obtained at DO 50% which was over that obtained at DO 20% in this study. With another Y. lipolytica strain cultivated on a fatty substrate (thus, ex novo lipid accumulation mechanism had been performed), exactly the inverse trend had been seen; fat accumulation was, indeed, restricted when high DOT values were imposed (Papanikolaou et al., 2007). This implies that for the de novo synthesis of lipid with Y. lipolytica, high dissolved oxygen concentration is required. The time course of glucose consumption and biomass production (OD), and the pH evolution during fermentation are represented on Fig. 3B The obtained result showed that at DO 20%, the biomass production was slower than that at DO 50% during the first 16 h and these trends was inverted thereafter. The glucose uptake at DO 20% was greater than that at DO 50% over the whole fermentation process whereas the pH evolution showed contrary tendencies. This could help to explain the biomass yielded in both fermentation conditions. Moreover, the fact that the pH was not controlled made it clear the conditions that can promote lipids production by Y. lipolytica. Specifically, the medium acidification seems to be the physiologic stress initiating lipid accumulation in this strain. An attempt was also made in order to scale up the strategy 1 developed in preliminary designing studies in fed–batch mode by

6

4 40 2

20

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Lipid content (%)

60

0 48h

72h

DO=20%

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DO=50%

48h

72h

DO=50% MgSO4*7H2O

Fermentation conditions X (g/L)

B

Lipid (g/L)

Lipid content (%)

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OD, pH

3.6. Scale-up in 1.5 L fermenter

A Biomass X (g/L) Lipid (g/L)

The exploration of these carbon sources showed the malleability of S2 medium and its broad range adaptability and applicability as a biodiesel fermentation platform. Y. lipolytica is well-known for its ability to assimilate a wide range of carbon sources such hydrocarbons, carbohydrates, oils and their derived waste materials (Fontanille et al., 2012; Tsigie et al., 2012, 2011). The adequate monitoring of these compounds using the mineral solution of S2 (S2 without glucose) could give a variety of minimal media and enable a large scale and worldwide production of microbial oil from Y. lipolytica and strengthen its capacity as an enthralling podium for biodiesel production. On the other hand, this could help to produce SCOs with specific characteristics for the manufacture of biodiesel with specific quality properties. Even though there still are some enhancement works such as process optimization and scaling up that need to be realized supplementarily, the use of cheap carbon sources for SCOs production from Y. lipolytica is an alternative way for oil cost reduction and biodiesel production.

Glucose (g/L)

330

0 0

20

40

60

Fermentation time (hours) Glucose-DO20% Glucose-DO50%

OD-DO20% OD-DO50%

pH-DO20% pH-DO50%

Fig. 3. Scale-up of S2 medium under different fermentation conditions. (a) Lipid and biomass production after 72 h of fermentation and (b) Time course of biomass, glucose and pH during 72 h of fermentation in a fermenter at DO 20% and DO 50%. Error bars represent standard deviation between the means of two separate repeated experiments.

periodic addition of 1.5 g/L of MgSO47H2O at DO 50%. The result (Fig. 3A) revealed that the feeding of MgSO47H2O was accompanied by biomass increase. Nevertheless, lipid yield of 1.87 g/L and 2.05 g/L corresponding to a lipid content of 45.37% and 45.98% were achieved respectively after 48 h and 72 h of fermentation. The lipid content was slightly lesser than that obtained in shake flasks possibly due to the fermentation conditions. Overall, the basic minimal medium (S2) was found to be scalable and this might be applicable to other S2-derived minimal media using a multiplicity of carbon sources that are assimilable by Y. lipolytica especially fructose, glycerol and sucrose as it is demonstrated in this work. In addition, as the gap between lipid amount at 48 h and 72 h of fermentation is not too big, the fermentation time can be reduced to 48 h and subsequently diminish the cost of fermentation process. Table 3C depicts the biodiesel profiles displayed during the scale-up process. These profiles were affected by the time of fermentation and cultural conditions. The profile obtained at DO20% was different from that at DO50%. In addition, at DO50%, the fatty acids composition was affected by the addition of MgSO47H2O. Similarly, the biodiesel profiles obtained at 48 h differed to some extent from that at 72 h. Therefore, by playing on the fermentation conditions and the fermentation duration, diverse biodiesels with

K. Nambou et al. / Bioresource Technology 173 (2014) 324–333

particular features could be generated by way of adequate cultivation of Y. lipolytica DSM3286. 3.7. Production of citric acid by Y. lipolytica grown in S2 medium As stated, many Y. lipolytica strains, when cultivated batch-wise on glucose or similarly metabolized compounds (e.g. glycerol) under nitrogen-limited conditions produce besides lipid also citric acid. The above-mentioned complex regulation makes it difficult in many instances to obtain high rates of lipid accumulation in batch cultures of Y. lipolytica, since lipid accumulation and citric acid biosynthesis are antagonistic. In this study, citric acid production by Y. lipolytica in S2 medium was carried out in a fermenter at DO 50% since these conditions led to a high lipid content. The result reported in Fig. 4 showed three important phases. During the first 36 h of fermentation corresponding to the cell growth phase, the citric acid production was low and reached a value of 0.55 g/L at 36 h. Thereafter, citric acid began to rapidly accumulate in the fermentation environment and reached concentrations up to 1.09, 2.23, 3.85 g/L respectively at 48 h, 60 h and 72 h of fermentation when the cell growth entered the stationary phase with increasing lipid accumulation. Following this phase, the citric acid production was slowed and lipid content decreased after 144 h of fermentation corresponding obviously to a lipid turnover phenomenon. During growth on glucose or similarly metabolized compounds in many Y. lipolytica strains, three distinct phases of growth have been described: (A) Balanced growth phase; (B) Lipid accumulation phase; (C) Citric acid production phase, accompanied by storage lipid turnover. These phases were found in the present study. In batch nitrogen-limited experiments, only a restricted number of Y. lipolytica strains manages to perform ‘‘real’’ lipid accumulation (i.e. lipid in DCW > 25%, w/w) since in most cases, citric acid is secreted into the medium. Y. lipolytica is a producer of notable quantities of citric acid as it was reported in several studies. For instance, in diluted O.M.Ws enriched with high glucose amounts Y. lipolytica produced citric acid to concentration up to 28.9 g/L while at low pO2 value and a high iron concentration, citric acid amount of 120 g/L was obtained (Kamzolova et al., 2003; Papanikolaou et al., 2008). In the present study, Y. lipolytica showed higher lipid contents and a lower maximum citric acid concentration (4.69 g/L) which was lower than aforementioned productions. Presumably, this was in relation with the fermentation conditions and the fermentation medium S2 used. This observation validates the designed S2 medium to be suitable for lipid production.

80

60 4 40 2 20

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6

0

Fermentation time (hours) Biomass (g/L) Lipid (g/L)

Lipid content (%) Citric acid (g/L)

Fig. 4. Kinetic of citric acid production on S2 medium at DO 50% correlated with biomass and lipid production. Error bars represent standard deviation between the means of two separate repeated experiments.

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3.8. Comparative analysis The work carried out herein was compared to those reported in the literature in order to state its relative significance. Some results are summarized in Table 5. Throughout this study, we have achieved a lipid content of 51% (g lipid per g dry cell weight) in shake flasks and further scale-up experiments in a fermenter conducted to a lipid content of up to 65% (g lipid per g dry cell weight). The biomass yield of Y. lipolytica on different designed media was between 1.09 g/L and 7.31 g/L and was in line with previous works. Basically, wild type strains of Y. lipolytica were found to grow poorly. Our work is in correlation with a previous work of Katre and his colleagues in which 5 wild type strains of Y. lipolytica grown for 96 h on glucose showcased a biomass production ranging from 3.77 to 7.42 g/L with lipid content of 22–31% DCW (Katre et al., 2012). At pH 6 and a temperature of 28–33 °C, Y. lipolytica, grown on a complex medium with higher concentration of nitrogen sources generated 9–12 g/L of dry biomass with lipid content of 44–54% (Papanikolaou et al., 2002). In a different research, a minor biomass concentration (4.2–8.2 g/L) was registered when Y. lipolytica strains were cultivated in media made with glycerol stemming from biodiesel (André et al., 2009). Additionally, it was demonstrated that the increase of crude glycerol concentration (from 30 to 90 g/L) did not have a positive effect on yeast growth, as the biomass level reached 6.6 and 5.9 g/L, respectively (Chatzifragkou et al., 2011). In a recent study, the biomass and lipid production by Y. lipolytica QU21 in glucose medium were respectively 3.28 g/L and 0.75 g/L while the use of glycerol as the sole carbon source led to a decreased biomass of 2.22 g/L (Poli et al., 2014). The biomass reported in the above glucose medium was in corroboration with that produced in S2 medium except that the lipid concentration with S2 was higher than that reported. According to above references, the biomass produced during this study on different culture media was in the range of that generally reported. Nevertheless the biomass produced in S2, S3 and S4 was low undoubtedly because of their simple composition whereas lipid composition was the highest in S2. Differences concerning the present study and published findings rely on the point that in the latter the culture media are multifaceted in terms of their composition, the concentrations of their components and their nature. In earlier or more recent studies, Y. lipolytica strains have been considered as perfect candidates amenable to produce biomass rich in single cell oil during growth on hydrophobic substrates. This is the so-called ex novo lipid synthesis, the biochemical mechanism of which is completely different from the one of the de novo lipid synthesis, employed in the current study. Most of the time, media used for lipid production from Y. lipolytica are originated from waste materials (e.g. tallow derivatives, vegetable oils, industrial fats) containing in some cases inhibitors; therefore additional detoxification preprocessing will be required (Tsigie et al., 2012, 2011) and subsequently increase the production steps and cost. Furthermore, in spite of its simplest composition and the wild type strain of Y. lipolytica used, the S2 medium designed herein gave a lipid content (65%) above that reported so far with the exception of a recent publication where through genetic manipulations a lipid content of 90% was achieved (Blazeck et al., 2014), then proving its commercial asset. In batch nitrogen-limited experiments, only a restricted number of Y. lipolytica strains manages to perform ‘‘real’’ lipid accumulation (i.e. lipid in DCW > 25%, w/w) since in most cases, citric acid is secreted into the medium. Significant lipid accumulation from sugars in batch experiments was reported only in few research works (Fontanille et al., 2012; Katre et al., 2012; Sarris et al., 2011; Tsigie et al., 2012, 2011). The citric acid production observed in our study

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Table 5 Lipid and biomass production from oleaginous microorganisms from diverse culture media. NA, not available. Strain

Carbon source

Culture mode

Biomass (g/L)

Lipid Content (%)

Lipid (g/L)

References

C. C. C. Y. Y. Y. Y. Y. Y. Y. Y. Y. Y. Y. Y. Y. Y. Y. Y. Y.

1% Methanol Mixtures of stearin, glucose, and technical glycerol 8% Molasses Bio-diesel derived glycerol Bio-diesel derived glycerol Bio-diesel derived glycerol Glycerol Glucose + acetic acid Industrial fats Commercial glucose Glucose Teucrium polium L. aqueous extract Industrial derivative of tallow Industrial derivative of tallow Glucose and olive mill wastewater-based media Glucose and olive mill wastewater-based media Glucose and olive mill wastewater-based media Sugarcane bagasse hydrolysate Glucose Glucose

Flasks Flasks Flasks Flasks Flasks Flasks Flasks Fed-batch Flasks Flasks Chemostat Chemostat Flasks Flasks Flasks Flasks Flasks Flasks Flasks Batch

NA 11.40 NA 8.1 8.2 6.5 5.2 30.83 8. 70 5.50 9.30 9.30 8.00 15.19 7.00 6.80 4.20 11.42 3.99 3.09

4.90 30 59.90 10 7.43 30.76 9.62 12.36 44 14 250 33 48.75 52 17.14 27.94 11.90 58.50 34.41 65.19

NA 3.40 NA 0.81 0.61 2 0.5 40.69 3.80 NA 2.30 3.12 3.90 7.90 1.20 1.90 0.50 6.68 1.37 1.88

Rupcic et al. (1996) Papanikolaou et al. (2003) Karatay and Donmez (2010) André et al. (2009)

lipolytica 33 M lipolytica lipolytica lipolytica LFMB 19 lipolytica LFMB 20 lipolytica ACA-YC 5033 lipolytica LFMB 19 lipolytica MUCL 28849 lipolytica lipolytica ACA-YC 5033 lipolytica LGAM (7)1 lipolytica LGAM (7)1 lipolytica LGAM S(7)1 lipolytica LGAM S(7)1 lipolytica ACA-YC 5028 lipolytica W9 lipolytica ACA-YC 5033 lipolytica Po1g lipolytica DSM3286 lipolytica DSM3286

was low whilst the lipid content as high as 65% was achieved. Thus, the minimal media designed in this study could be efficient in lipid production through the fermentation using different Y. lipolytica strains and close oleaginous yeasts. In general, during growth on fatty substrates, the microorganism can substantially ‘‘upgrade’’ the fatty acid composition of the fat used [e.g. production of ‘‘tailor-made’’ intra-cellular lipids presenting composition similarities with the cocoa-butter (Papanikolaou and Aggelis, 2011). On the contrary, the de novo synthesis, as achieved here, generates specific fatty acids based on the cultivation conditions. Despite the fact that Y. lipolytica’s fatty acid composition is strain-dependent, the cultivation conditions such as the nature of the carbon source used, its initial or inlet concentration, the dissolved oxygen concentration, the culture mode used and specifically the fermentation time impact profoundly on the profile of the biosynthesized fatty acids. Similarly to this study, several research works indicated oleic acid to be the predominant fatty acid (>60% w/w of total lipids) produced when Y. lipolytica strains are cultivated on glucose (André et al., 2009; Chatzifragkou et al., 2011; Sarris et al., 2011). The great proportion of unsaturated fatty acids observed in this study revealed pronounced metabolic rate, as a response of the microbial cell to stressed environmental conditions. With the S2 minimal medium, the wild-type strain of Y. lipolytica exhibited an interesting fatty acids profile that is suitable for high quality biodiesel production. This profile was different from those reported previously considering the presence or absence of some fatty acids and their relative proportions. 4. Conclusions The current study imparts a novel point of view of a kind of pure fermentation formula that takes in consideration economical aspect of the fermentation route using Y. lipolytica. This type of fermentation is apt to make available consistent feedstock for biodiesel production and will show undeniable contribution in the resolution of energy matters. The media designed through this study are cost effective and could potentiate Y. lipolytica and other oleaginous yeasts as robust cell factories for biodiesel and other biodiesel-like biofuels manufacture. Supplementary material concerning preliminary fermentation strategies related to this work is available on the journal website.

Chatzifragkou et al. (2011) Fontanille et al. (2012) Papanikolaou et al. (2001) Papanikolaou et al. (2009) Aggelis and Komaitis (1999) Aggelis and Komaitis (1999) Papanikolaou et al. (2007) Papanikolaou et al. (2007) Sarris et al. (2011)

Tsigie et al. (2011) This study

Acknowledgements This work was financially supported by National Basic Research Program of China (973 Program) (2012CB721101), National Special Fund for State Key Laboratory of Bioreactor Engineering (2060204). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.biortech.2014. 09.096. References Aggelis, G., Komaitis, M., 1999. Enhancement of single cell oil production by Yarrowia lipolytica growing in the presence of Teucrium polium L. aqueous extract. Biotechnol. Lett. 21, 747–749. André, A., Chatzifragkou, A., Diamantopoulou, P., Sarris, D., Philippoussis, A., Galiotou-Panayotou, M., Komaitis, M., Papanikolaou, S., 2009. Biotechnological conversions of bio-diesel-derived crude glycerol by Yarrowia lipolytica strains. Eng. Life Sci. 9, 468–478. Bellou, S., Makri, A., Triantaphyllidou, I.E., Papanikolaou, S., Aggelis, G., 2014. Morphological and metabolic shifts of Yarrowia lipolytica induced by alteration of the dissolved oxygen concentration in the growth environment. Microbiology 160, 807–817. Beopoulos, A., Mrozova, Z., Thevenieau, F., Le Dall, M.T., Hapala, I., Papanikolaou, S., Chardot, T., Nicaud, J.M., 2008. Control of lipid accumulation in the yeast Yarrowia lipolytica. Appl. Environ. Microbiol. 74, 7779–7789. Beopoulos, A., Cescut, J., Haddouche, R., Uribelarrea, J.L., Molina-Jouve, C., Nicaud, J.M., 2009. Yarrowia lipolytica as a model for bio-oil production. Prog. Lipid Res. 48, 375–387. Blazeck, J., Hill, A., Liu, L., Knight, R., Miller, J., Pan, A., Otoupal, P., Alper, H.S., 2014. Harnessing Yarrowia lipolytica lipogenesis to create a platform for lipid and biofuel production. Nat. Commun. 5, 3131. Chatzifragkou, A., Makri, A., Belka, A., Bellou, S., Mavrou, M., Mastoridou, M., Mystrioti, P., Onjaro, G., Aggelis, G., Papanikolaou, S., 2011. Biotechnological conversions of biodiesel derived waste glycerol by yeast and fungal species. Energy 36, 1097–1108. Cowan, J.A., 2002. Structural and catalytic chemistry of magnesium-dependent enzymes. Biometals 15, 225–235. Demirbas, A., 2008. Relationships derived from physical properties of vegetable oil and biodiesel fuels. Fuel 87, 1743–1748. Fontanille, P., Kumar, V., Christophe, G., Nouaille, R., Larroche, C., 2012. Bioconversion of volatile fatty acids into lipids by the oleaginous yeast Yarrowia lipolytica. Bioresour. Technol. 114, 443–449. Kamzolova, S.V., Shishkanova, N.V., Morgunov, I.G., Finogenova, T.V., 2003. Oxygen requirements for growth and citric acid production of Yarrowia lipolytica. FEMS Yeast Res. 3, 217–222. Karatay, S.E., Donmez, G., 2010. Improving the lipid accumulation properties of the yeast cells for biodiesel production using molasses. Bioresour. Technol. 101, 7988–7990.

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