Research Paper

Biosynthesis of lipids and organic acids by Yarrowia lipolyticastrains cultivated on glucose

Seraphim Papanikolaou1, Afroditi Chatzifragkou1, Stylianos Fakas1, 2, Maria Galiotou-Panayotou1,Michael Komaitis1, Jean-Marc Nicaud3 and George Aggelis2

1 Department of Food Science and Technology, Agricultural University of Athens, Athens, Greece2 Group of Microbiology, Department of Biology, Division of Genetics, Cell and Development Biology, University

of Patras, Patras, Greece3 AgroParisTech, U.M.R. 2585, C.N.R.S., Centre de Grignon, Thiverval-Grignon, France

The biochemical behavior of wild-type or genetically modified (presenting decreased expression of intra-cellular acyl-CoA oxidases) Yarrowia lipolytica strains cultivated on commercial glucose was studied. Flasknitrogen-limited cultures were performed favoring the production of organic acids (and potentially theaccumulation of lipid). Nitrogen depletion induced secretion of citric acid, while intracellular lipid was notproduced in high quantities. Maximum total citric acid up to 49 g/L (yield 0.85 g/g glucose) was pro-duced. In some of the wild-type strains, an increase of glucose in the medium also induced noticeableproduction of acetic acid. Increasing the amount of added glucose led to an increase in the total lipidquantity (%) produced, although in the stationary growth phase the concentration of lipid declined, indi-cating lipid degradation even for the genetically modified strains. Total lipid amount did not exceed thevalue of 14%, while neutral fractions increased with increase in glucose concentration. In all cases, the totalmicrobial lipids and major lipid fractions were composed of C16 and C18 (principally unsaturated) fattyacids. Finally, in several of the strains cultured in media containing a low glucose concentration, unicellularmorphology was observed, while at high glucose concentrations mycelia were predominant.

Keywords: Cellular lipids / Citric acid / Kinetics / Yarrowia lipolytica

Received: March 11, 2009; accepted: June 4, 2009

DOI 10.1002/ejlt.200900055

Eur. J. Lipid Sci. Technol. 2009, 111, 1221–1232 1221

1 Introduction

Citric acid, with an annual demand of more than 1.4 milliontons, represents 70% of all food acidulates, and is producedvia fermentations principally by strains of the mold Aspergillusniger [1–5]. However, during the last few years numerousYarrowia (Candida) lipolytica strains (natural or mutant) havebeen shown to be capable of producing citric acid in signifi-cant quantities during cultivation on various hydrophilic orhydrophobic substrates and fermentation configurations [6–19]. Due to the easier handling of yeast compared to moldculture and the potential of growing yeasts on various carbonsubstrates, citric acid production has became one of the most

important biotechnological applications of Y. lipolytica yeast[20]. Studies on a biochemical level indicate that onset of citricacid accumulation inside the microbial cell is due to decreasedactivity of enzymes implicated in the primary anabolic growthbecause of nitrogen-limited conditions [9, 21–24]. The bio-process has also been successfully simulated and optimizedwith the aid of various models [6, 11, 12, 25–27]. Productionof extracellular citric acid or intracellular lipid when sugars orsimilarly metabolized compounds are used as the sole sub-strate are, in their first steps, biochemically identical, andoccur after intracellular nitrogen exhaustion [21–23, 28]. Theonly difference in these processes is in the level of the enzymeATP-citrate lyase, which catalyzes the cleavage of intracellularcitric acid into oxaloacetate and acetyl-CoA, the latter beingused as base-unit for the synthesis of intracellular fatty acids,by a quasi-inverted b-oxidation process [21, 22, 29]. In theabsence of this enzyme, the carbon flow is directed towardsthe synthesis of citric acid that is secreted to the growth me-dium; yeast strains, hence, are classified into citric acid-pro-

Correspondence: Dr. Seraphim Papanikolaou, Laboratory of Food Micro-biology and Biotechnology, Department of Food Science and Technology,Agricultural University of Athens, 75 Iera Odos, 11855 Athens, GreeceE-mail: spapanik@aua.grFax: 130-210-5294700

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1222 S. Papanikolaou et al. Eur. J. Lipid Sci. Technol. 2009, 111, 1221–1232

ducing or lipid-producing strains [23, 28]. In the case of Y.lipolytica yeast, various strains of this species have the cap-ability of producing large quantities of lipids that are locatedinto lipid bodies when growth is conducted on hydrophobicsubstrates. On the other hand, growth of these strains undernitrogen-limited culture conditions, with glucose or similarlymetabolized compounds used as substrates, is not accom-panied by significant lipid accumulation [11, 30–36]. Con-cerning the case of prokaryotic microbial strains, the mech-anism of lipid accumulation is similar to that of eukaryoticstrains (e g., onset of lipid accumulation under nitrogen-lim-ited conditions) [21, 22]; however, bacteria can also storeother types of lipophilic compounds besides triacylglycerolsinto their cells, such as poly-hydroxyalkanoate-type molecules[21].

The aim of the present investigation was to assess the bio-chemical behavior (substrate assimilation, biomass production,biosynthesis of intracellular lipid, production of extracellularcitric acid) of various Y. lipolytica strains grown on commercialglucose (a low-cost renewable material utilized in confectionaryindustries). Growth was carried out in nitrogen-limited mediathat favored citric acid production (and potentially accumula-tion of reserve lipid). The strains used were either natural orgenetically modified. The genetically modified strains are cap-able of accumulating high lipid quantities during growth onhydrophobic substrates. This is due to decreased expression ofvarious acyl-CoA oxidases [35, 37]. However, given that theaccumulation of lipid from hydrophobic substances has funda-mental differences in comparison with the de novo accumulationof lipid from glucose [21, 22, 32, 34], the genetically modifiedstrains would not be capable of producing higher quantities oflipid compared with the wild-type (wt) strain from which theyare produced, on glucose-based nitrogen-limited media. Dif-ferences in the fatty acid composition of the cellular lipids aswellas in the degradation rate of microbial lipids produced, in com-parison with the wt strain from which they derived, were foundin cultures carried out on sugar-based media. Therefore, thegoal of the current investigation was the detailed study of thebiochemical behavior of Y. lipolytica strains during growth inglucose-based nitrogen-limited media, with emphasis focusedupon the biosynthesis of organic acids and lipids.

2 Materials and methods

2.1 Microorganism and culture conditions

Y. lipolytica strains ACA-YC 5028, ACA-YC 5029, ACA-YC5030, ACA-YC 5031, ACA-YC 5032 and ACA-YC 5033isolated from various types of sourdoughs [38] were kindlyoffered by the Laboratory of Dairy Science, AgriculturalUniversity of Athens, Greece. Strain LFMB 15 was isolatedfrom fish products and characterized in the Laboratory ofFood Microbiology and Biotechnology. Strains W29, JMY798 (MTLY 36-2P) and JMY 794 (MTLY 40-2P) came from

the culture collection of AgroPrisTech, Paris-Grignon,France. The genetically modified strains JMY 798 and 794were subjected to disruptions of the genes implicated inencoding various acyl-CoA oxidases (AOX) [35, 37], whilestrain W29 was the wt strain from which JMY 798 and 794were derived. Strains JMY 798 and 794, therefore, eitherunderexpressed or did not express various AOX enzymes thatare implicated in the catabolism (b-oxidation) of aliphaticstrains. When cultures were performed using oleic acid as thesole substrate, the genetically engineered strains showed al-most equivalent microbial growth compared with the wt strain(W29) from which they derived. However, in contrast to theW29 strain, they presented significantly higher formation oflipid bodies and, hence, increased lipid accumulation [35, 37].

Strains were kept on potato dextrose agar at 47C. Saltcomposition of culture medium was similar to that describedin Papanikolaou et al. [33]. Initial pH after sterilization was6.2 6 0.1 in all trials. The nitrogen sources used in the presentstudy were (NH4)2SO4 and yeast extract (concentration ofeach at 0.5 g/L). In all experiments it was desirable to maintaina pH value greater than 5.2. Therefore, a volume (500–600 mL) of 5 m KOH was periodically and aseptically addedinto the flasks to maintain pH value above 5.2 (see Papaniko-laou et al. [12]). The carbon source used was commercialglucose utilized in various confectionary industries (HellenicIndustry of Sugar S.A., Thessaloniki, Greece; purity 95%,impurities comprised 2% maltose, 0.5% maltodextrins, 1.5%water and 0.5% inorganic salts).

Experiments were performed in liquid submerged batchcultures in 250-mL non-baffled flasks containing 50 6 1 mLmedium in an orbital shaker (New Brunswick Scientific, NJ,USA) at an agitation rate of 180 6 5 rpm and incubationtemperature of 28 6 17C. Flasks were inoculated with 1 mL ofa 24-h exponential pre-culture (around 16107–36107 cells,initial biomass concentration 0.12 6 0.02 g/L). The pre-cul-ture was carried out in the above-mentioned synthetic medi-um containing 30 g/L glucose.

2.2 Analytical methods

Biomass concentration, pH measurement, glucose con-centration and organic acids determinations were carried outas previously described [12]. Dissolved oxygen (D.O.) con-centration (%) was measured with the aid of a selective elec-trode (oxi200 Sensodirect, Lovinbod) as follows: before har-vesting, the shaker was stopped and the probe was placed intothe flask. The shaker was then switched on and the measure-ment was taken after D.O. equilibration (usually within10 min). All trials were conducted in fully aerobic conditions(D.O. .20%, for all growth steps). Given that the separationof citric and iso-citric acid under the chromatographic condi-tions used was not satisfactory, citric acid amount representsthe sum of citric and iso-citric acids (total citric acid) in allexperiments. Moreover, iso-citric acid was determined insome of the trials with the use of an enzymatic method [33].

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Total cellular lipid was extracted from the dry biomasswith a mixture of chloroform/methanol 2:1 and was deter-mined gravimetrically. In some trials, cellular lipids were frac-tionated into their lipid fractions. In brief, a known weight ofextracted lipid (approximately 300 mg) was dissolved inchloroform (3 mL) and was fractionated on a column(256100 mm) of silicic acid, activated by heating overnight at1107C [39]. Successive applications of 1,1,1-trichloroethane,acetone and methanol produced fractions containing neutrallipids (NL), glycolipids plus sphingolipids (G1S) and phos-pholipids (P), respectively [39]. The weight of each fractionwas determined after evaporation of the respective solvent.Total or individual lipid fractions were converted to methylesters in a two-step reaction with methanolic sodium andhydrochloric methanol and were analyzed by GLC asdescribed in Fakas et al. [39]. Ammonium was determined inthe culture medium by an ammonia-selective electrode(Hach 95-12, Germany). Yeast morphology was checked inall experiments by light microscopic observations by a Zeiss(Axioscop 40, Germany) microscope.

2.3 Notation

The following notations have been used: X, biomass (g/L);S, glucose (g/L); Cit, total citric acid (citric plus iso-citricacid) (g/L); Ac, acetic acid (g/L); L, total lipid (g/L); YL/

X, total lipid in biomass produced (g formed/g biomass pro-duced); YX/S, biomass yield on glucose consumed (g/g sugarconsumed); YCit/S, total citric acid yield on glucose (g/g glu-cose consumed); YAc/S, acetic acid yield on glucose (g/g glu-cose consumed); Subscripts 0, c and max indicate the initial,consumed and maximum quantity, respectively, of the com-ponents.

3 Results

3.1 Kinetics in nitrogen-limited media with initialglucose at 30 g/L

In the first series of experiments a screening of the natural orgenetically engineered Y. lipolytica strains used in the presentstudy was carried out under high carbon excess conditions(initial molar C/N ratio 100 mol/mol) and initial glucose con-centration (S0) adjusted at 30 g/L. Thus, these kinetic studieswere carried out in culture conditions that favor extracellularcitric acid production (and potentially intracellular lipid accu-mulation). In all experiments, microorganisms consumed, withcomparable rates, the available extracellular nitrogen (initialNH4

1 concentration at 110 6 15 ppm, exhaustion within55 6 10 h after inoculation with final NH4

1 concentration at5 6 3 ppm) and almost simultaneously reached a biomassconcentration plateau, in which X concentration was around5.4 6 0.5 g/L (kinetics not shown). Total cellular lipids wereextracted in all growth phases, with total lipid in dry biomass

(YL/X, in g/g) values being in the range of 0.03–0.06 g/g. Lmax

corresponded in all cultures to values around 0.20–0.33 g/L,and it was achieved 80–100 h after inoculation. Furthermore,in all fermentations, a remarkable drop of the culture mediumpH was observed, suggesting accumulation of organic acidsinto the culture medium. The pH was always maintained withinthe range 5.2–6.2, since in previous investigations it had beendemonstrated that the maintenance of pH in this range was as avery important factor for the success of the fermentation [28,33]. Cit was the major organic acid accumulated into the cul-ture medium, while other organic acids (e. g., a-ketoglutaric,acetic, pyruvic acid) were produced in negligible quantities(sum of organic acids produced was around 10% of Cit).Likewise, the enzymatic analysis carried out showed that iso-citric acid quantity represented around 8–12% of Cit pro-duced, regardless of the strains used.

The results of the screening study regarding biomass andcitric acid synthesis and glucose consumption are illustrated inTab. 1. From the obtained result it can be seen that some of thestrains utilized in the present investigation (strains ACA-YC5031, 5032 and LFMB 15) produced insignificant quantitiesof Cit (Citmax ranging from 1.9 to 2.8 g/L). These particularstrains consumed some glucose during the first growth step,before nitrogen had been depleted from the culture medium,and principally microbial mass was produced with conversionyields (YX/S) ranging from 0.31 to 0.39 g/g (Tab. 1). Afternitrogen exhaustion, small quantities of glucose were furtherconsumed (kinetics not presented). Other strains (includingW29 and both genetically engineered strains) consumed sig-nificant glucose amounts during all growth phases and pro-duced noticeable quantities of citric acid (Citmax ranging from8.2 to 18.0 g/L with respective yield YCit/S values from 0.29 to0.62 g/g; Tab. 1). In the case of citric acid-producing yeasts, theyield YX/S was lower compared with that of non-citric acid-producing ones (YX/S=0.20–0.22 compared to 0.31–0.39 g/g);in the first case carbon was channeled mainly into the glucose-citric acid pathway. Furthermore, substrate uptake rate (rS),expressed as rs=-DS/Dt and estimated during the “productive”phase of the culture (phase in which glucose was virtually con-sumed by the microorganism), is shown in Tab. 1; it can be seenthat rS presented rather small values for the cases in whichinsignificant citric acid quantities were produced (see case ofACA-YC 5031, 5032 and LFMB 15 strains in Tab. 1). For theother wt strains that produced non-negligible citric acid quan-tities, glucose uptake was uninterrupted and different rS valueswere obtained. As for the wt W29 strain and the geneticallymodified derivatives JMY 798 and 794, although they pro-duced Cit in almost equal quantities, rS was somehow reducedin the genetically engineered strains.

3.2 Kinetics in nitrogen-limited media with initialglucose at 60 g/L

In the first screening test, natural or the genetically engineeredY. lipolytica strains were used at high carbon excess conditions

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Table 1. Data of natural and genetically engineered Y. lipolytica strains originating from kinetics in media containing commercial glucose(30 g/L) and nitrogen-limited conditions (initial molar ratio C/N=100). Representation of biomass produced (X, g/L), consumed glucose (Sc,g/L), biomass produced per glucose consumed (YX/S, g/g) and total citric acid produced per glucose consumed (YCit/S, g/g) at fermentationtime in which the maximum concentration of citric acid (Citmax, g/L) was achieved. Culture conditions: incubation temperature 287C,growth on flasks, initial pH 6.2 6 0.2, pH in the medium ranging from 5.2 to 6.2, D.O. .45%, for all growth phases. Each point is the meanvalue of two independent measurements.

Strain Fermentation time (h) X g/L Sc g/L Citmax g/L YX/S g/g YCit/S g/g rS§ g/L?h

AC-YC 5028 98 5.5 28.2 8.2 0.20 0.29 0.29AC-YC 5029 119 4.9 23.9 14.5 0.20 0.61 0.20AC-YC 5030 119 5.9 28.9 15.5 0.20 0.54 0.21AC-YC 5031 72 5.6 16.9 1.9 0.33 0.11 0.15AC-YC 5032 96 5.1 13.5 2.8 0.38 0.21 0.16AC-YC 5033 94 5.1 24.8 11.1 0.20 0.45 0.26LFMB 15 95 5.2 13.2 2.1 0.39 0.16 0.16wt W29 142 5.8 29.2 18.0 0.20 0.62 0.23JMY 798 160 5.9 29.7 17.2 0.20 0.58 0.20JMY 794 160 5.9 29.5 17.1 0.20 0.58 0.19

§ rS was estimated by the formula rs ¼ �DS=Dt during the “productive” phase of the culture (phase in which glucose was virtually consumed bythe microorganism).

(initial molar C/N ratio 100 mol/mol) to determine the ca-pacity of these microorganisms to produce citric acid (orpotentially lipid). In a second series of experiments, glucoseconcentration was doubled (S0=60 g/L) and all other cultureconditions remained constant, to study the effects of highernitrogen limitation and relatively increased glucose con-centration upon the biochemical behavior of the most prom-ising of the yeast strains screened above. A higher productionof citric acid was expected in terms of both Cit concentrationand YCit/S, given that carbon would have been more efficientlychanneled towards the glucose-citric acid pathway due to thehigher nitrogen limitation employed (“over-flow metabolism”phenomenon, see Anastassiadis et al. [23], Kamzolova et al.[15]).

As previously mentioned (see Section 3.1) intracellularlipids were not produced in high concentrations. Nevertheless,in the second set of experiments, somewhat higher quantitiesof lipids were produced, since Lmax values were around 0.40–0.59 g/L in all cultures, with lipid in dry weight (YL/X) beingwithin the range of 0.06–0.14 g/g. Additionally, Lmax con-centrations were obtained rather later compared with growthon S0=30 g/L, and were achieved around 150–200 h afterinoculation. The pH value of the culture was again maintainedin the range 5.2–6.2. The four natural strains, ACA-YC 5028,5029, 5030 and 5033, produced significant amounts of bio-mass (Tab. 2). However, strains ACA-YC 5029 and 5030presented lower final X concentration compared with that ofthe trial in S0=30 g/L (Tab. 1). This can be attributed to slightinhibition exerted by the relatively high initial sugar con-centration in the culture medium. Glucose was consumed insignificant quantities, while a continuous and noticeable dropof the culture pH (followed by correction) occurred, suggest-

ing triggering of organic acids biosynthesis due to carbonexcess conditions. Glucose consumption was uninterrupted,but substrate uptake rate (rS) presented, in all cases, lowervalues compared with the experiment at S0=30 g/L (Tabs. 1and 2). Total citric acid was produced in some cases in notablequantities (e.g., Cit concentration up to 38.2 g/L). The quan-tity of iso-citric acid represented around 8–10% of total citricacid produced by the strains. Nevertheless, under the presentculture conditions (increase of glucose concentration in themedium) total citric acid was not the sole organic acid pro-duced in remarkable quantities; acetic acid (Ac) was alsoaccumulated into the medium in significant amounts (Acmax

from 11.5 to 24.1 g/L). Yield YAc/S presented remarkably highvalues (from 0.21 to 0.48 g/g). Other organic acids produced,in low quantities (e.g., 1.0–2.5 g/L) were pyruvic and a-keto-glutaric acids. Although in all of the natural strains studied aproduction of acetic acid was observed, suggesting carbonflow losses from the glucose–citric acid pathway, in the threeout of four studied strains (ACA-YC 5028, 5030 and 5033)the increment of glucose concentration increased both Citmax

and yield YCit/S values compared with the trial at low glucoseconcentration media (Tabs. 1 and 2). In contrast, for theACA-YC 5029 strain, the trial with S0=60 g/L was accom-panied by a final concentration of acetic acid higher than ofcitrate. Additionally, the same strain produced citric acid inhigher concentrations and yields at the lower S0 concentration(Tabs. 1 and 2).

The wt W29 strain and genetically engineered JMY 798and 794 strains presented significant cell growth at S0=60 g/L(Tab. 3) with final biomass concentration reaching slightlylower values compared with the trial at 30 g/L (Tabs. 1 and 3).As previously observed, increments of glucose concentration

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Table 2. Data of natural Y. lipolytica strains originating from kinetics in media containing commercial glucose (60 g/L) and nitrogen-limitedconditions (initial molar ratio C/N=200). Representation of biomass produced (X, g/L), consumed glucose (Sc, g/L), biomass produced perglucose consumed (YX/S, g/g), acetic acid produced (Ac, g/L), acetic acid produced per glucose consumed (YAc/S, g/g) and total citric acidproduced per glucose consumed (YCit/S, g/g) at fermentation time in which the maximum concentration of citric acid (Citmax, g/L) wasachieved. Culture conditions: incubation temperature 287C, growth on flasks, initial pH 6.2 6 0.2, pH in the medium ranging from 5.2 to6.2, D.O. .20%, for all growth phases. Each point is the mean value of two independent measurements.

Strain Fermentation time (h) X g/L Sc g/L Citmax g/L Ac g/L YX/S g/g YCit/S g/g YAc/S g/g rS§ g/L?h

ACA-YC 5028 356 5.8 59.8 31.1 21.5 0.10 0.52 0.36 0.15ACA-YC 5029 219 3.9 50.8 13.2 24.1 0.08 0.26 0.48 0.17ACA-YC 5030 262 4.8 53.4 33.1 11.5 0.09 0.62 0.22 0.17ACA-YC 5033 309 5.5 58.8 38.2 12.3 0.10 0.65 0.21 0.17

§ rS was estimated by the formula rs ¼ �DS=Dt during the “productive” phase of the culture (phase in which glucose was virtually consumed bythe microorganism).

Table 3. Data of the natural Y. lipolytica W29 strain and its genetically engineered derivatives originating from kinetics in media containingcommercial glucose (60 g/L) and nitrogen-limited conditions (initial molar ratio C/N=250). Representation of biomass produced (X, g/L),remaining unconsumed glucose (Sc, g/L), biomass produced per glucose consumed (YX/S, g/g) and total citric acid produced per glucoseconsumed (YCit/S, g/g) at fermentation time in which the maximum concentration of citric acid (Citmax, g/L) was achieved. Culture condi-tions as in Tab. 2.

Strain Fermentation time (h) X g/L Sc g/L Citmax g/L YX/S g/g YCit/S g/g rS§ g/L?h

wt W29 315 5.0 57.7 49.0 0.09 0.85 0.18JMY 798 330 5.5 43.6 36.2 0.13 0.83 0.13JMY 794 330 5.3 45.5 32.3 0.12 0.71 0.13

§ rS was estimated by the formula rs ¼ �DS=Dt during the “productive” phase of the culture (phase in which glucose was virtually consumed bythe microorganism).

were accompanied by a decrease of rS value, while at S0=60 g/LW29 strain consumed higher glucose quantities (in terms ofboth absolute value and uptake rate rS) in comparison with itsgenetically engineered derivatives. In contrast with the resultsachieved with the wt strains ACA-YC 5028, 5029, 5030 and5033 at S0=60 g/L, biosynthesis of other organic acids(including Ac) was negligible, and Cit was the principalorganic acid produced, with its synthesis being enhanced dueto high carbon excess conditions (Citmax=49 g/L, YCit/S=0.71–0.85 g/g; Tab. 3). Iso-citric acid quantity represented around9–13% of Cit produced by the strains. A characteristic kineticsof biomass and organic acids production and glucose con-sumption by the strain Y. lipolytica ACA-YC 5033 is pre-sented in Fig. 1.

3.3 Biosynthesis and composition of cellular lipids byY. lipolytica

In all cases cellular lipids were not produced in significantconcentrations (YL/X from 0.03 to 0.14 g/g), indicating thatcultivation of Y. lipolytica strains on flask glucose-based nitro-gen-limited cultures was not accompanied by a “real” de novoaccumulation of storage lipid inside the microbial cells (i.e.,

production of lipids in quantities higher than 0.2 g lipid/g drymatter). Therefore, for all strains the metabolic network atcarbon-excess conditions was mainly directed towards thesecretion of extracellular organic acids instead of lipid accu-mulation. Moreover, increasing the glucose concentration ofthe culture medium induced a remarkably higher biosynthesisof microbial lipid, in terms of total lipid in dry matter (YL/X, ing/g) for all strains tested (see evolution of YL/X for the strainsACA-YC 5029, 5033, W29, JMY 794 and 798 in Figs. 2a andb). For S0=30 g/L, the maximum yield YL/X was around0.04 6 0.02 g/g. The respective value for S0=60 g/L was0.12 6 0.02 g/g, and similar results were achieved for all test-ed strains. The highest quantity of cellular lipid produced wasfor the strain ACA-YC 5033 and corresponded to a value of0.14 g total lipid/g biomass (see Fig. 2a). Although as it wasstated that no “real” lipid accumulation occurred, the max-imum quantity of lipid produced by the genetically engineeredstrains JMY 794 and 798 was somewhat higher comparedwith that of the wt W29 strain, specifically at S0=60 g/L(Fig. 2b). It is also of importance to state that some mobiliza-tion (degradation – turnover) of cellular lipids was observedprincipally at the high S0 concentration media. This degrada-tion, accompanied by a noticeable reduction of the value of

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Figure 1. Changes of biomass (X, g/L), glucose (S, g/L), citric acid(Cit, g/L) and acetic acid (Ac, g/L) during growth of Yarrowia lipoly-tica ACA YC 5033 on nitrogen-limited glucose-based media. Cul-ture conditions: initial glucose concentration 60 g/L, initial molar C/N ratio 200 mol/mol, incubation temperature 287C, growth onflasks, initial pH 6.2 6 0.2, pH in the medium ranging from 5.2 to6.2, D.O. .20% for all growth phases. Each point is the mean valueof two independent measurements.

YL/X (see Figs. 2a and b), occurred at the stationary growthphase and was observed for all of the tested strains. A quitesurprising and interesting result was that microbial lipidturnover was a dominant event even for the geneticallymodified JMY 798 and 794 strains (Fig. 2b). These strainsunderexpress, or do not express, various acyl-CoA oxidasesimplicated in the catabolism (b-oxidation) of intracellularfatty acids and, theoretically, the value of YL/X should haveremained more or less constant in these strains at the sta-tionary phase; a fact that, finally, did not happen. Analysisof microbial oil at the early stationary growth phase (theperiod in which lipids were synthesized occurred with vir-tually no turnover) revealed that increasing the glucoseconcentration in the medium significantly increased theconcentration of neutral fraction (N) and reduced the con-centration of glycolipids plus sphingolipids (G1S) andphospholipids (P) (Tab. 4).

Lipids of both wt and genetically engineered strains wereanalyzed at the early stationary phase (incubation time 100–150 h, the period in which lipid concentration increased) andthe late stationary phase (incubation time 200–260 h, theperiodin which lipid turnover occurred). In all cases, the cellular fattyacids detected were of C16 and C18 chains. The main fatty acidproduced in all trials was oleic acid (D918:1). Other cellular fattyacids produced in lower quantities were palmitic (16:0), palmi-toleic (D916:1), stearic (18:0) and linoleic (D9,1218:2) acids(Tabs. 5 and 6). Analysis of cellular lipids indicated that dis-tribution of theabove fattyacids into thecellular lipidswas strain

Figure 2. Changes of total lipid in dry biomass (YL/X, g/g) duringgrowth of Yarrowia lipolytica strains ACA-YC 5029 and ACA-YC5033 (a) and wt W29, JMY 794 and JMY 798 (b) in nitrogen-limitedglucose-based media. Culture conditions: initial glucose con-centration 30 g/L (open symbols) or 60 g/L (filled symbols), incu-bation temperature 287C, growth on flasks, initial pH 6.2 6 0.2, pHin the medium ranging from 5.2 to 6.2, D.O. .20% for all growthphases. Each point is the mean value of two independent meas-urements.

dependent. Furthermore, it appears that increase of glucoseconcentration in the growth medium induced some smallmodifications in the cellular lipid composition; for the naturalstrains ACA-YC 5028, 5029, 5030 and 5033 it appears that

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Table 4. Distribution of lipid fractions as function of the initialquantity of glucose (S0, g/L) in the fermentation medium for Y.lipolytica strains ACA-YC 5033 and JMY 798. N: Neutral lipids;G1S: glycolipids plus sphingolipids; P: phospholipids. Cultureconditions as in Tabs. 1 and 2, lipids were analyzed at the earlystationary growth phase (100–150 h).

Lipid fraction %

N 79.0ACA-YC 5033, S0=30 g/L G1S 12.5

P 8.5

N 90.0ACA-YC 5033, S0=60 g/L G1S 2.2

P 7.8

N 70.1wt W29, S0=30 g/L G1S 23.9

P 6.0

N 80.2wt W29, S0=60 g/L G1S 13.8

P 6.0

N 73.1JMY 798, S0=30 g/L G1S 19.9

P 7.0

N 83.1JMY 798, S0=60 g/L G1S 10.0

P 6.9

growth in high initial glucose concentration media induced adecrease in the concentration of the cellular D918:1 fatty acid,while the fatty acids D9,1218:2 and D916:1 tended to increase(Tab. 5). Similar observations principally concerning thetrend of D918:1 in relation with the concentration of glucose inthe medium were found for the wt strain W29 and the geneti-cally modified JMY 794 and 798 strains (Tab. 6). Moreover,comparison in the total fatty acid composition between W29,JMY 794 and 798 showed that, in general, the geneticallymodified strains presented more saturated total cellular lipids.When intracellular lipids were subjected to degradation at thelate stationary phase, D918:1 appeared to be preferentiallyconsumed and subsequently the concentration of 18:0 wasraised (Tab. 6). Finally, analysis of the various lipid fractions(N, G1S and P fractions) showed that fatty acid compositionof these fractions was unaffected by the increment of S0 con-centration in the culture medium, whereas in general thefraction of phospholipids was greater in polyunsaturated fattyacids (principally D9,1218:2) compared with total and neutrallipids (Tab. 7). As for the glycolipids plus sphingolipids, dis-tribution of fatty acids within this fraction was strain depend-ent and also presented differences when compared with thefatty acid composition of total and neutral lipids (Tab. 7). Forinstance, for the strains W29 and JMY 798, the G1S fractionwas more or less richer in D918:1 compared with the totallipids, while the opposite was seen for the wt strain ACA-YC5033 (Tab. 7).

Table 5. Fatty acid composition of total cellular lipids produced by wt Y. lipolytica strains found at stationary growth phase during growthon nitrogen-limited media containing commercial glucose at two initial concentrations (30 and 60 g/L). Culture conditions as in Tabs. 1and 2.

Fatty acids (%)

Strain S0 (g/L) Growth phase 16:0 D916:1 18:0 D918:1 D9,1218:2

ACA-YC 5028 30 g/L Early stationary 13.3 11.1 7.2 56.2 12.1Late stationary 10.1 9.1 8.2 63.4 9.2

60 g/L Early stationary 15.3 14.2 9.1 46.3 15.1Late stationary 17.1 12.3 9.2 48.1 17.3

ACA-YC 5029 30 g/L Early stationary 9.1 11.3 8.2 64.1 7.3Late stationary 9.2 9.3 4.1 67.2 10.2

60 g/L Early stationary 8.3 12.1 9.1 60.2 9.1Late stationary 9.2 15.2 5.2 56.3 13.1

ACA-YC 5030 30 g/L Early stationary 11.3 8.1 5.1 70.2 5.3Late stationary 10.1 7.1 4.5 71.1 7.2

60 g/L Early stationary 12.2 8.2 7.3 61.2 11.1Late stationary 13.2 6.1 6.3 58.2 16.2

ACA-YC 5033 30 g/L Early stationary 16.3 12.1 6.3 54.2 11.1Late stationary 11.1 10.2 7.4 59.1 12.2

60 g/L Early stationary 13.2 11.2 7.2 53.3 11.2Late stationary 13.2 13.1 5.5 52.1 14.1

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1228 S. Papanikolaou et al. Eur. J. Lipid Sci. Technol. 2009, 111, 1221–1232

Table 6. Total cellular fatty acid composition of wt Y. lipolytica W29 strain and its genetically engineered derivatives JMY 794 and 798found at stationary growth phase during growth on nitrogen-limited media containing commercial glucose at two initial concentrations (30and 60 g/L). Culture conditions as in Tabs. 1 and 3.

Fatty acids (%)

Strain S0 (g/L) Growth phase 16:0 D916:1 18:0 D918:1 D9,1218:2

wt W29 30 g/L Early stationary 14.3 7.1 6.1 55.2 17.3Late stationary 19.1 8.2 8.2 46.3 18.2

60 g/L Early stationary 20.1 8.2 2.3 51.3 18.1Late stationary 18.3 8.1 8.3 47.1 16.2

JMY 798 30 g/L Early stationary 18.1 8.2 9.3 47.2 17.2Late stationary 19.2 8.1 10.3 42.2 20.2

60 g/L Early stationary 20.1 10.3 6.0 42.2 19.0Late stationary 18.3 13.2 12.2 37.1 19.2

JMY 794 30 g/L Early stationary 17.2 9.2 8.2 51.1 14.3Late stationary 18.1 8.3 9.3 44.2 20.1

60 g/L Early stationary 19.3 11.3 10.2 41.1 18.1Late stationary 19.2 11.1 12.2 37.4 20.1

Table 7. Fatty acid composition of total lipids, neutral lipids (N), glycolipids plus sphingolipids (G1S) and phospholipids (P) during growthof Y. lipolytica strains ACA-YC 5033 and JMY 798 on glucose-based media. Culture conditions as in Tabs. 1–3, lipids were analyzed at theearly stationary growth phase (100–150 h after inoculation).

Fatty acids (%)

16:0 D916:1 18:0 D918:1 D9,1218:2

ACA-YC 5033, S0=30 g/L Total lipids 15.4 11.2 6.1 54.2 10.1N 14.4 10.0 5.7 56.3 11.6G1S 19.2 9.1 9.9 47.5 10.5P 19.9 6.9 7.7 50.4 13.5

ACA-YC 5033, S0=60 g/L Total lipids 12.9 10.4 6.8 56.5 10.9N 13.8 9.6 5.6 57.5 12.7G1S 20.1 7.9 10.8 49.2 11.7P 15.7 8.1 10.1 48.1 12.6

wt W29, S0=30 g/L Total lipids 19.1 8.2 7.9 46.3 18.2N 18.2 7.5 10.3 47.1 15.8G1S 22.3 3.2 8.9 52.1 12.2P 14.6 11.2 4.9 45.9 22.3

wt W29, S0=60 g/L Total lipids 18.3 8.3 8.5 47.4 16.4N 18.5 7.7 12.1 47.2 14.1G1S 15.4 12.4 1.0 49.3 21.4P 15.8 9.1 3.1 48.1 23.2

JMY 798, S0=30 g/L Total lipids 16.2 8.2 9.1 47.8 18.3N 17.2 7.5 11.3 48.2 15.1G1S 23.9 3.5 8.4 51.3 11.2P 14.2 10.7 5.3 45.7 23.8

JMY 798, S0=60 g/L Total lipids 19.1 10.3 5.1 45.1 19.6N 20.2 9.1 5.5 49.5 14.4G1S 23.5 2.1 12.8 48.9 10.1P 17.4 8.9 6.8 39.8 23.3

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Eur. J. Lipid Sci. Technol. 2009, 111, 1221–1232 Biochemistry of Yarrowia lipolytica yeasts 1229

3.4 Microbial morphology

All of the wt Y. lipolytica strains were present in unicellularform when growth was carried out at S0=30 g/L at all growthsteps. In contrast, when S0 increased, all of the strains duringthe first growth step (0–40 h after inoculation) were presentin a true mycelium form. After 40 h of inoculation, a gradualmycelium-to-yeast transition was observed. This transitionoccurred simultaneously with the decrease of S concentrationin the culture medium. Yeast-like morphology became pro-gressively predominant. After 120–140 h, the strains werealmost completely present in their unicellular morphology.For the genetically engineered strains, growth always fol-lowed a unicellular morphology, without the presence ofmycelium, regardless of substrate concentration in thegrowth medium.

4 Discussion

Aim of the present study was to assess the biochemical be-havior of Y. lipolytica strains grown on commercial glucoseunder conditions of carbon excess to trigger biosynthesis oforganic acids and/or cellular lipids. The strains utilized wereeither natural or genetically modified to present decreasedexpression of various acyl-CoA oxidases (capable of accu-mulating fat from hydrophobic substrates [35, 37]). However,given that fat production from hydrophobic materials is acompletely different process from de novo lipid accumulation[21, 22, 32, 34, 40], the genetically modified strains should notbe capable of producing higher amounts of lipid from sugarsutilized as substrate compared with the wt strain from whichthey were derived. However, these microorganisms couldpotentially present variations in the fatty acid composition oftheir lipids in relation with the wt W29 strain, or even withother wt strains; they could thus become “tools” by which wecould study the potential effect of glucose concentration onthe biosynthesis and the degradation rate of intracellular fattyacids by Y. lipolytica strains.

Despite the high initial C/N molar ratio and the remark-able oxygen saturation imposed (prerequisites for satisfactoryintracellular lipid accumulation [21, 22]), none of thescreened strains were able to accumulate significant quan-tities of fat. On the contrary, carbon-excess conditionsfavored the secretion of organic acids (principally citric acidand to lesser extent acetic acid) into the growth medium.Nevertheless, it is important to state that increasing the S0

concentration in the medium clearly increased the yield (YL/

X). Interestingly, the genetically modified strains JMY 794and 798 produced somewhat higher quantities of total lipidcompared with the wt W29 strain (Fig. 2b). Moreover, rais-ing the glucose concentration in the medium, increased theamount of the neutral fraction of microbial lipids produced.Neutral lipids are the main fraction of accumulated lipids inthe oleaginous microorganisms [21, 36, 39, 41]. However,

none of the tested yeast strains were able to carry out a “real”accumulation of microbial lipid under the given culture con-ditions. In general, various Y. lipolytica strains, althoughbeing incapable of producing lipid via the de novo lipidaccumulation mechanism, were able to accumulate hugeamounts of lipid when growth was carried out on varioushydrophobic carbon sources as substrates [30, 32–34, 36],since, as stated above, accumulation of lipid from fat materi-als is a completely different process from that from sugars.Moreover, some cellular lipid consumption (turnover) wasobserved in the stationary phase of microbial growth, espe-cially in the high S0 concentration cultures (which in generalpresented higher YL/X values). Surprisingly, microbial oilturnover occurred even in the genetically modified JMY 798and 794 strains that underexpress or do not express variousacyl-CoA oxidases. These strains should theoretically notdegrade their cellular lipids, or degrade them only at slowrate. In general, microbial lipid turnover is a phenomenonobserved regardless of the carbon source utilized for lipidbiosynthesis [32, 34, 41, 42], and is followed by differentuptake rates of the various intracellular fatty acids subjectedto degradation [32, 41].

The main cellular fatty acid of Y. lipolytica lipid producedduring growth on glucose-based media was D918:1. The 16:0,D9,1218:2, D916:1 and 18:0 fatty acids were produced in lowerquantities. The distribution of intracellular fatty acids seemedto be strain dependent. It seems that S0 concentration increaseresulted in a lower concentration of the D918:1 cellular fattyacid. Moreover, during the lipid degradation period, lipidswere richer in 18:0, suggesting selective uptake of the D918:1fatty acid (Tab. 6). In cultures performed with Y. lipolyticaACA-DC 50109, this strain was shown to preferentially con-sume unsaturated intracellular fatty acids, even when theywere present in significantly lower quantities inside the cellscompared with the saturated ones [32]. Likewise, growth ofthe ACA-DC 50109 strain on glucose-based nitrogen-limitedflask experiments was accompanied by a slightly lower pro-duction of cellular unsaturated fatty acids when comparedwith that on cultures with lower S0 [11]. In contrast, in thesame strain, concentrations of cellular D916:1 and D918:1increased when the culture medium was enriched with olive-mill waste water, and this was attributed to microbial adapta-tion to the high-phenol concentration in the medium, sincehigher unsaturation of cellular lipids increases the selectivityand the fluidity of the microbial membrane in non-favor-able environmental conditions [12]. Additionally, Finoge-nova et al. [9] reported significant decrease of cellularD916:1 and D9,1218:2 and concomitant noticeable increase ofcellular D918:1 concentrations during growth of Y. lipolyticastrain N1 in continuous cultures on ethanol used as sub-strate. The quantity of total cellular lipids slightly increasedwhen the concentration of ethanol in the feed mediumincreased.

In the present investigation, the impact of S0 concentrationupon the fatty acid composition of the various fractions (N,

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1230 S. Papanikolaou et al. Eur. J. Lipid Sci. Technol. 2009, 111, 1221–1232

G1S, P) of the cellular lipid was studied. The results showedthat the distribution of fatty acids within the main lipid frac-tions was less sensitive in relation to S0 concentration increasein the medium (see Tab. 7). In general, it seems that the Pfraction was noticeably richer in polyunsaturated fatty acids(principally D9,1218:2) compared with total lipids and the Nfraction, while the distribution of fatty acids (and mainly thatof D918:1) in G1S fraction was strain dependent. Cultivationsof the mold Cunninghamella echinulata on glucose-enrichedtomato waste hydrolysate in both carbon- and nitrogen-lim-ited experiments showed that the quantity of polyunsaturatedfatty acids (e.g., D9,1218:2 and D6,9,1218:3) was remarkablyhigher in the P fraction in comparison with the total lipids andthe N fraction [39, 41].

The S0 concentration substantially influenced the mor-phology of several strains. Some strains were present in atrue mycelium form at the beginning of cultures in whichmedia with a high S0 concentration were employed. Thesestrains underwent a progressive transition from the myce-lium to a single-cell morphology. In contrast, with low S0

media, strains showed a single-cell morphology. In general,the nature of the carbon source employed (and not its con-centration) is considered as a factor of importance for themorphology of Y. lipolytica; however, the results obtainedare rather equivocal. For instance, in some reports it is stat-ed that development of Y. lipolytica strains principally infilamentous form was enhanced by the presence of fat in themedium [34, 43]. In contrast, cultivation of Y. lipolyticastrains on hydrophobic materials other that fats (e.g.,alkanes) or glycerol seemed to favor the development of themicroorganism in its yeast form [33, 44]. In other reports,different Y. lipolytica strains grown on hydrophilic materials(e.g., glucose, citric acid) showed a significant myceliumformation [45, 46].

In the present study, growth of various Y. lipolytica strainsin glucose-based media was accompanied by accumulation oforganic acids in the growth medium. Total citric acid was themajor organic acid produced, with a maximum level of 38.2–49.0 g/L. This Citmax quantity can be considered as satisfac-tory and comparable with the values reported for other Y.lipolytica or Candida oleophila strains. In most cases, Citmax

quantities of 29–79 g/L have been reported regardless of thecarbon source used and the fermentation configurationemployed [6, 8, 11–13, 18, 23, 28, 29, 47, 48]. Finally, ex-tremely high Cit concentrations (100–195 g/L) have beenachieved using mutant Y. lipolytica strains cultivated onethanol [14], vegetable oils [15] or raw glycerol [16, 17] inbatch, fed-batch or continuous bioreactor operations. YCit/S

significantly increased with higher S0 concentrations in themedium, since biosynthesis of citric acid is a non-growthassociated process occurring after nitrogen had been deplet-ed from the culture medium, and the more carbon substrateavailable in the medium the more the microbial metabolism isdirected towards the biosynthesis of citric acid, due to“metabolic over-flow” under carbon excess conditions [8, 14,

15, 23, 29]. The highest YCit/S achieved in the present studywas 0.85 g/g (see Tab. 3). This value was significantly highand comparable with the highest YCit/S values achieved in theinternational literature for glucose or similarly metabolizedsubstrates. The YCit/S values routinely observed were alsowithin the range of 0.65–0.83 g/g (see Tabs. 2 and 3). To thebest of our knowledge, the highest YCit/S value for glucose orsimilarly metabolized compounds was reported by Wojtato-wicz et al. [7], who found a YCit/S of 0.93 g/g in experimentswith a strain of Y. lipolytica grown on hydrolyzed potatostarch containing 15% glucose. In contrast, in most reportslower yields were obtained compared with the present study(e.g., 0.45–0.60 g/g) [6, 11, 12, 16–18, 23, 28, 29, 33, 47].Yields are significantly dependent on the carbon source uti-lized since substances with higher carbon and reductancedegree (i.e., fats, ethanol) compared with sugars can lead tonotably higher conversion yield values (e.g., 0.87–1.20 g/g)[13–15, 48].

In some of the fermentations carried out in the presentstudy, principally at high S0 media, non-negligible quantitiesof acetate were produced. In at least one case (ACA-YC5029) the Acmax concentration obtained was higher com-pared with Citmax at high S0 concentrations (see Tab. 2).Given that insignificant quantities of Ac were produced intrials with lower S0 media (see Tab. 1), it may be assumedthat at high S0 concentrations (in which higher intracellularcarbon flow occurs), the glucose–citric acid pathway seemedto be saturated, possibly due to a bottleneck in the level of thecitrate synthase (CS). Although we did not carry out an assayof the intracellular enzymes, saturation of CS could result insuch a carbon flow bottleneck in the level of conversion ofacetyl-CoA to citric acid with concomitant intracellularaccumulation of acetyl-CoA and subsequent secretion ofacetic acid. Acetic acid production by Y. lipolytica has rarelybeen reported, and in every case this production was not high[9, 33]. In general, production of acetic acid by the means offermentation technology is carried out using bacteria [49,50].

The present study showed the effect of glucose con-centration on the biosynthesis of organic acids and cellularlipids by Y. lipolytica strains. The level of Citmax achieved(49 g/L) was somewhat lower than the highest valuesreported in the literature, whereas the conversion yieldachieved was excellent (0.85 g/g). In some cases, increasingthe S0 concentration induced secretion of acetic acid,besides the production of citrate. Accumulation of storagelipid was not a dominant event; nevertheless, as glucoseconcentration increased, non-negligible increases in theamount of lipid in dry matter and neutral fractions of totallipid were observed. Cellular lipid was mainly composed ofunsaturated C16 and C18 fatty acids. The distribution ofthese acids was strain dependent. At low S0 concentrations,unicellular morphology was observed in several of thestrains, while at high glucose concentrations mycelia werepredominant.

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Eur. J. Lipid Sci. Technol. 2009, 111, 1221–1232 Biochemistry of Yarrowia lipolytica yeasts 1231

Conflict of interest statement

The authors have declared no conflict of interest.

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