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Lipidsofoleaginousyeasts.PartII:Technologyandpotentialapplications

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DOI:10.1002/ejlt.201100015

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Review Article

Lipids of oleaginous yeasts. Part II: Technology andpotential applications

Seraphim Papanikolaou1 and George Aggelis2

1 Laboratory of Food Microbiology and Biotechnology, Department of Food Science and Technology,

Agricultural University of Athens, Iera Odos, Athens, Greece2 Unit of Microbiology, Division of Genetics, Department of Biology, Cell and Development Biology,

University of Patras, Patras, Greece

The process of lipid accumulation in the oleaginous yeasts cultivated in various fermentation configurations

when either sugars and related compounds or hydrophobic substances are used as substrates is presented

and kinetic models describing both de novo and ex novo lipid accumulation are analyzed. Technological

aspects related with single cell oil (SCO) produced by oleaginous yeasts are depicted. The influence of

culture parameters upon lipid production process is presented. Lipid production has been studied in batch,

fed-batch, and continuous cultivation systems using yeasts belonging to the species Lipomyces starkeyi,

Rhodosporidium toruloides, Apiotrichum curvatum, Candida curvata, Cryptococcus curvatus, Trichosporon

fermentans, and Yarrowia lipolytica. The potentiality of yeasts to produce SCO as starting material of

2nd generation biodiesel is indicated and discussed. Of significant importance is also the utilization of yeast

lipids as substitutes of high added value exotic fats (e.g., cocoa butter). Lipid produced by the various yeasts

presents, in general, similar composition with that of common vegetable oils being composed of

unsaturated fatty acids, whereas cocoa butter is principally composed of saturated fatty acids,

consequently the various strategies that are followed in order to increase the cellular saturated fatty

acid content of the yeast lipid are presented and comprehensively discussed.

Keywords: 2nd generation biodiesel / Cocoa butter substitute / Lipid biotechnology / Modeling / Oleaginous yeasts /

Single cell oil

Received: January 10, 2011 / Revised: March 19, 2011 / Accepted: April 12, 2011

DOI: 10.1002/ejlt.201100015

1 Introduction

Two major sectors of lipid biotechnology, the non-conven-

tional biocatalysis and the production of lipid deriving from

microbial sources (the so-called ‘‘single cell oil, SCO’’)

present a continuous expansion in the last years. In the former

sector, hydrolytic enzymes (e.g., lipases or other esterases,

glycosidases, etc.) in free and/or immobilized form are used in

media presenting a feeble water concentration (organic or

non-conventional media), in order to synthesize novel bio-

molecules of industrial, technological, and medical interest

(for reviews see: Buchholz and Bornscheuer [1]; Adlercreutz

[2]; Metzger and Bornscheuer [3]; Bottcher et al. [4]). A

significant part of this work refers to the synthesis of specific

types of structured TAGs that either present composition

similarities with various high-added value exotic fats like

cocoa butter, or have medical significance (i.e., medium-

chain TAGs) through reactions of trans- or inter-esterifica-

tion, in which in several cases low added value fatty materials

(e.g., lard, pomace olive oil, etc.) are used as substrates [3, 5–11].

Likewise, by using such types of reactions it is possible to

concentrate polyunsaturated fatty acids (PUFAs) of medical

importance, found in various fatty materials [12]. Moreover,

in recent developments, hydrolytic enzymes (principally

lipases) have been used in non-conventional media in order

to synthesize with an eco-friendly strategy biofuels (inmost cases

Correspondence: Dr. Seraphim Papanikolaou, Laboratory of Food

Microbiology and Biotechnology Department of Food Science and

Technology, Agricultural University of Athens, Athens, Greece

E-mail: spapanik@aua.gr

Fax: þ30-210-5294700

Abbreviations: CBS, cocoa butter substitute; FAMEs, fatty acid methyl-

esters; g, of A substance formed per g of B substance consumed; Glc,

Glucose (in g/L); K, saturation constant (in g/L); L, total cellular lipid

(in g/L); q, specific rate of product formed (g/g/h); S, substrate (in g/L);

SCO, single cell oil; TSFAs, total saturated fatty acids; X, total biomass (in

g/L); Xf, lipid-free biomass (in g/L); YA/B, conversion yield; m, biomass

specific growth rate (h�1), subscript max indicates the maximum quantity

and O the initial quantity of the components

1052 Eur. J. Lipid Sci. Technol. 2011, 113, 1052–1073

� 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.ejlst.com

biodiesel that principally refers to fatty acid methyl-esters –

FAMEs) (for reviews see: Adamczak et al. [13]; Szczesna-

Antczak et al. [14]).

The second sector of lipid biotechnology refers to the

production of SCOs. This sector is of particular interest

due to the capacity of various microorganisms (principally

yeasts, molds, and algae and to lesser extent bacteria) to

synthesize lipids with specific structure and/or composition

[15–22]. The continuously increasing demand of the 1st

generation biodiesel (FAMEs deriving from trans-esterifica-

tion of principally plant oils) has increased the cost of various

food-stuffs, and this situation has led to the necessity of

discovery of non-conventional sources of oils, that could

be subsequently converted into biodiesel. The oleaginous

microorganisms are considered as potential candidates for

the production of this lipid that would result in the generation

of the ‘‘2nd generation’’ biodiesel deriving from lipid pro-

duced by oleaginous microorganisms growing on wastes or

agro-industrial residues like sewage sludge, hemicelluloses

hydrolysates, waste glycerol, cheese whey, etc. [23–31], or

the 3rd generation biodiesel deriving from lipid produced by

oleaginous micro-algae, with carbon being offered by atmos-

pheric CO2 sequestration [22, 26, 32].

In general, the oleaginous yeasts produce lipid containing

unsaturated fatty acids similar to that found in common plant

oils [18, 20–22, 33]. Although the production cost of the

microbial lipids generally remains higher than that of the

conventional vegetable oils [21], the production of yeast

lipids with composition similarities with high added-value

specialty fatty materials (like the cocoa butter or other exotic

fats) has been considered as a process potentially economi-

cally viable [15, 17, 18, 20, 22, 34], specifically if various low-

or negative cost raw materials (e.g., whey, industrial-crude

fatty acids, waste glycerol, xylose, etc.) are utilized as sub-

strates [35–41]. Yeast lipid presenting composition sim-

ilarities with the cocoa butter has been produced in

industrial scale at the end of 1980s [42]. Furthermore, due

to the last crisis in the production and price of the various

comestible products, the cost of the various plant oils (e.g.,

rapeseed oil, soybean oil, etc.) has considerably increased the

last years [21]; this event has resulted in a non-negligible

increase of conventional biodiesel production cost. However,

the application of biofuels in a large commercial scale is

strongly recommended by various authorities; with the EU

directive 2003/30/EC, a quantity of 5.75% w/w of biofuel

was planned to be introduced in the conventional fuel by

2010 [26, 28]. Thus, discovery of novel sources for a massive

production of lipid presents significant importance, with the

oleaginous microorganisms being considered as potential

candidates for this purpose (for reviews see: Chisti [32];

Luque et al. [26]; Meng et al. [43]). Specifically, the yeasts

have the remarkable capability to be cultivated on a plethora

of renewable- or waste-type materials in various fermentation

configurations (e.g., xylose-based materials, hemi-cellulose

hydrolysates, (raw) glycerol, sewage sludge, various types of

whey permeate, etc.) [15, 24, 27, 33, 44–48] while they

present a significantly maximum higher specific growth rate

compared to molds and algae [17, 21, 34], therefore yeast

lipids can be considered as potential starting materials for the

synthesis of this 2nd generation biodiesel [28].

In the part I [151] of this work, we were interested in the

biochemical events related with the accumulation of storage

lipid during growth of oleaginous yeasts on hydrophilic or

hydrophobic substances utilized as substrates. Fundamental

differences on biochemical level exist between the two proc-

esses, since in the former case de novo lipid accumulation

occurs, while lipid is synthesized during the secondarymetab-

olism performed usually after nitrogen (or to lesser extent

after other essential nutrient like phosphorus or sulfate)

depletion from the medium, whilst in the later case (ex novo

lipid accumulation), lipid is synthesized together with the

production of lipid-free material, irrespective of the nitrogen

presence into the medium. Moreover, after carbon source

depletion from the medium, cellular lipid is subjected into

degradation, regardless of the mechanism that had been

previously used in order for its formation. In the present part

II of this review-article, modeling approaches of lipid

accumulation during growth of oleaginous microorganisms

on several substrates will be presented. Also, the principal

industrial applications of the utilization of oleaginous yeasts

for the production of lipid suitable for biodiesel production,

or the production of substitutes of the cocoa butter will be

discussed.

2 Biochemical engineering and potentialapplications of lipid produced by oleaginousyeasts

2.1 Technology and modeling of lipid accumulationin oleaginous yeasts

2.1.1 De novo lipid accumulation

A large variety of substrates have been used as carbon sources

for the oleaginous microorganisms performing de novo lipid

accumulation, in shake-flask, batch-bioreactor, fed-batch

bioreactor, and continuous culture modes. The substrates

used include analytical-grade or industrially derived sugars

[15, 23, 25, 29, 49–59], molasses [27, 58, 59], cheese whey

[31, 33, 36, 37, 44, 60], glucose-enriched tomato waste

hydrolysate [61–64], glucose-enriched sewage sludge [24],

polysaccharides [65–67], (raw) glycerol [45–47, 57, 68–73],

glycerol-enriched tomato waste hydrolysate [64], prickly pear

juice [74, 75], N-acetyl-glucosamine [30], starch hydroly-

sates [76], inulin or Jerusalem artichoke hydrolysates [77–

79], rice straw hydrolysates [48], sweet sorghum extracts

[80], organic acids [51, 81–84], and ethanol [85–87].

In general onset of lipid accumulation through the de

novo anabolic pathway is given after an essential nutrient

Eur. J. Lipid Sci. Technol. 2011, 113, 1052–1073 The biotechnology of oleaginous yeasts 1053

� 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.ejlst.com

(in most cases nitrogen) depletion into the growth medium,

while in some cases (specifically through utilization of the

yeast Yarrowia lipolytica), instead of or simultaneously with

lipid accumulation, nitrogen depletion into the medium in

batch or fed-batch experiments leads also to the secretion of

citric acid into the culture medium [28, 68, 70]. Only in few

investigations so far, batch cultivations of Y. lipolytica yeasts

in nitrogen-limited sugar-based media have resulted in

noticeable accumulation of lipid inside the yeast cells (i.e.,

lipid >40% w/w, in dry yeast mass), without simultaneous

production of organic acids into the medium [78]. While the

number of investigations concerning lipid accumulation from

sugars or similarly metabolized substrates is indeed high,

kinetic modeling approaches related with this bioconversion

are relatively restricted. Moreover, in most of the case studies

performed, turnover of accumulated lipid was not taken into

consideration in the models proposed. In a first approach

appeared in early investigations, Glatz et al. [60] have pro-

posed a model describing cell growth and lipid synthesis of

the yeast Apiotrichum curvatum growing on whey-permeate in

batch-bioreactor trials. A similar approach has been estab-

lished by Ykema et al. [88] in which the same strain was

cultivated in continuous experiments with glucose utilized as

the sole substrate. It has been considered that lipid accumu-

Table 1. Representation of predicted parameter values estimated from numerical or analytical models describing de novo lipid accumulation

process (or the similar bioprocess of production of extra-cellular citric acid fromyeasts) or ex novo lipid accumulation process from oleaginous

microorganisms

Parameter Reference

Lmax (g/L) 2.1 (Aggelis et al. [96])a), 3.2 (Papanikolaou and Aggelis [107])b), 2.7 (Papanikolaou and Aggelis [107])c)

mXf ðSÞmax(h�1) 0.20 (Ykema et al. [88])d), 0.19 (Glatz et al. [60])e), 0.23 (Papanikolaou and Aggelis [107])b),c),

0.18–0.36 (Papanikolaou and Aggelis [93])f), 0.09 (Fakas et al. [69])g), 0.32 (Papanikolaou et al. [90])h),

0.24 (Anastassiadis et al. [92])i), 0.566 (Economou et al. [80])j)

qLmax (g/g/h) 0.06 (Aggelis and Sourdis [89])a), 0.025 (Glatz et al. [60])e), 0.16 (Papanikolaou and Aggelis [107])b),

0.10 (Papanikolaou and Aggelis [107])c), 0.028 (Fakas et al. [69])g), 2.73 � 10�3 (Papanikolaou et al. [90])h),

0.785 (Economou et al. [80])j)

mXf ðLÞmax(h�1) 1.4 � 10�2 (Aggelis and Sourdis [89])a), 5.1 � 10�3 (Papanikolaou and Aggelis [107])b),

4.0 � 10�3 (Papanikolaou and Aggelis [107])c)

YXf/S (g/g) 0.55 (Ykema et al. [88])d), 0.69 (Glatz et al. [60])e), 0.86 (Papanikolaou and Aggelis [107])b),

0.78 (Papanikolaou and Aggelis [107])c), 2.22 (Papanikolaou et al. [90])h), 0.08 (Fakas et al. [69])g),

0.345 (Economou et al. [80])j)

YL/S (g/g) 0.58–0.60 (Aggelis and Sourdis [89])a), 0.41 (Ykema et al. [88])d), 0.30 (Glatz et al. [60])e),

0.63 (Papanikolaou and Aggelis [107])b), 0.86 (Papanikolaou and Aggelis [107])c), 0.43 (Fakas et al. [69])g),

0.52 (Papanikolaou et al. [90])h), 0.242 (Economou et al. [80])j)

YXf/N (g/g) 38.6 (Glatz et al. [60])e), 60.4 (Papanikolaou et al. [90])h), 31.9–33.6 (Papanikolaou and Aggelis [93])f),

39.3 (Fakas et al. [60])g), 18.21 (Economou et al. [80])j)

YXf/L (g/g) 0.63–0.66 (Aggelis and Sourdis [89])a), 1.37 (Papanikolaou and Aggelis [107])b),

0.70 (Papanikolaou and Aggelis [107])c)

KN (g/L) 0.12 (Glatz et al. [60])e), 0.196 (Papanikolaou and Aggelis [93])f), 0.15 (Fakas et al. [69])g),

0.047 (Anastassiadis et al. [92])i), 0.085 (Economou et al. [80])j), 0.179 (Arzumanov et al. [91])k)

KS (g/L) 59 (Glatz et al. [60])e), 20 (Fakas et al. [69])g), 1.256 (Economou et al. [80])j)

KLS (g/L) 69.27 (Economou et al. [80])j)

Lmax, maximum concentration of SCO produced; mXf ðSÞmax, maximum specific growth rate attributed to the extra-cellular carbon source;

qLmax , maximum specific rate of storage lipids production; mXf ðLÞmax, maximum specific growth rate attributed to the consumption of storage

lipids; YXf/S, conversion yield of lipid-free biomass formed per carbon substrate consumed; YL/S, conversion yield of storage lipid formed per

carbon substrate consumed; YXf/N, conversion yield of lipid-free biomass formed per nitrogen consumed; YXf/L, conversion yield of lipid-free

biomass formed per storage lipid consumed; KN, KLS, and KS are saturation constants.a) Mucor circinelloides on sunflower oil, batch flask culture, SCO production.b) Y. lipolytica on stearin, batch flask culture, SCO production.c) Y. lipolytica on stearin/hydrolyzed rapeseed oil 50/50, batch flask culture, SCO production.d) A. curvatum on glucose, single-stage continuous culture, SCO production.e) A. curvatum on whey permeate, batch bioreactor culture, SCO production.f) Y. lipolytica on biodiesel-derived glycerol, batch flask culture, citric acid production.g) T. elegans on biodiesel-derived glycerol, batch flask culture, SCO production.h) Y. lipolytica on mixture of commercial-industrial glucose and stearin, batch flask culture, simultaneous SCO and citric acid production.i) Candida oleophila on analytical-grade glucose, batch flask culture, citric acid production.j) M. isabellina on sweet sorghum extract, batch flask culture, SCO production.k) Y. lipolytica on ethanol, fed-batch bioreactor culture, citric acid production.

1054 S. Papanikolaou and G. Aggelis Eur. J. Lipid Sci. Technol. 2011, 113, 1052–1073

� 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.ejlst.com

lation process was a combination of two different

mechanisms:

The first mechanism corresponds principally at balanced

cell growth phase, in which lipid accumulation is proportional

to the production of non-lipid cell mass production. In the

second phase, lipid biosynthesis, being independent from the

production of non-lipid cell mass, was performed through the

formation of a rate-controlling intermediate of lipids I,

between sugar and storage lipid [60, 88], describing, thus,

the nitrogen-limited phase (unbalanced growth phase). Not

any accumulation of storage materials other than lipophilic

compounds was assumed in the above approach during the

nitrogen-limited period.

The process of lipid accumulation is performed

when extra-cellular nitrogen is the limiting factor of cell

growth. Therefore, the specific growth rate of lipid-free

material (mXf) was illustrated by the following formula:

mXf ¼ mXfmaxN=ðKN þ NÞð Þ, where KN was the saturation

constant for nitrogen (in g/L) [60]. In this assumption is

considered that lipid-free material is the ‘‘active’’ biomass

portion [60, 88–90].

It should be stressed that similar Monod-type models

have also been used in order to simulate biomass production

during citric acid fermentation using sugars or alcohols [91–

93], or the production of fatty and lipid-free material during

growth of the fungus Thamnidium elegans CCF-1465 on

glycerol-based nitrogen-limited cultures [69]. On the other

hand, in similar types of nitrogen-limited growth of the fun-

gus Mortierella isabellina ATHUM 2935 on sweet sorghum

extracts in shake-flask experiments, the specific growth rate of

lipid-free material synthesized was considered to be influ-

enced by both the sugar and the nitrogen, being described by

a type of double-substrate limitation with sugar inhibition,

according to Andrews’ equation:

mXf ¼ mXfmax

N

KN þ N

� �S

KS þ S þ S2=Ki1

� �;

where KN and KS are the saturation constants for sugar and

nitrogen, respectively (in g/L), and Ki1 is the inhibition con-

stant [80].

A power function f ¼ mXf =mXfmaxpresented the transition

of the microbial metabolism toward the accumulation of

cellular lipids. Specifically, a type function ð1�fÞm was uti-

lized for this purpose [60], therefore, the more the extra-

cellular nitrogen concentration decreased, the more the value

of the function f became lower. The specific formation rate of

the intermediate qI (in g/g/h), related to extra-cellular nitro-

gen exhaustion was presented by the following formula:

qI ¼ ð1�fÞmqImax [60].

The consumption of sugar (substrate – S) contributed to

generation of Xf and I, while the specific formation rate of the

intermediate qI was expressed by aMichaelis–Menten expres-

sion [qI ¼ qImax S=ðKS þ SÞð Þ], where KS was the saturation

constant for sugar substrate (in g/L) [60]. Under the same

optics, the accumulated fat was generated from the inter-

mediate product I through its specific formation rate (qL),

that, again, was expressed by aMichaelis–Menten expression

[qL ¼ qLmax I=ðKI þ IÞð Þ], where I was the intermediate con-

centration (g/L), and KI was the saturation constant for I (in

g/L) [60].

Although intra-cellular lipid storage through de novo lipid

accumulation process is a non-growth associated process, the

specific production rate of cellular lipids is not expressed as

constant. In a similar type of modeling approach recently

appeared in the literature (nitrogen-limited cultures of

T. elegans on glycerol), qL was expressed as combination of

Blackman- and Michaelis–Menten-type expressions:

qL ¼ qLmax

S

KS þ S

� �N0�N

N0

� �;

where KS was the saturation constant for glycerol (in g/L) and

N0 was the initial quantity of assimilable nitrogen [69].

Economou et al. [80] while performing shake-flask cultures

of the fungus M. isabellina on sweet sorghum extracts con-

taining variable initial quantities of assimilable nitrogen and

sugars, proposed the expression

qL ¼ qLmax

S

KLS þ S þ S2=Ki2

� �k2

k2 þ N

� �;

where KLS is the saturation constant (in g/L), Ki2 the inhi-

bition constant (in g/L), and k2 is a constant ensuring that at

high nitrogen concentration lipid production is low [80].

Nevertheless, in similar types of processes (e.g., production

of citric acid from glucose or glycerol), the specific production

rate of citric acid was considered to be a constant (qCit ¼ a)

[90, 92, 93].

2.1.2 Ex novo lipid accumulation

A remarkable plethora of hydrophobic carbon sources have

been used as substrates for ex novo lipid accumulation. These

substrates are various vegetable oils like olive oil, corn oil,

sunflower oil, etc. [60, 89, 94–99], fatty by-products or

wastes such as crude fish oils, soap-stocks, ‘‘stearin’’ (a

low-cost derivative of tallow composed of saturated free-fatty

acids), hydrolyzed rapeseed oil, etc. [39, 100–109], pure free-

fatty acids [110–112], fatty esters [113], and n-alkanes [114,

115]. In some investigations utilization of mixtures of hydro-

(i) Sugar (substrate) Lipid-free biomass (Xf) þ accumulated lipid (L)

(ii) Sugar (substrate) Intermediate (I) Accumulated lipid (L).

Eur. J. Lipid Sci. Technol. 2011, 113, 1052–1073 The biotechnology of oleaginous yeasts 1055

� 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.ejlst.com

philic substrates (glucose and/or glycerol) with various fatty

materials has been employed [40, 90, 113, 116–120],

whereas with some exceptions in which batch bioreactor

experiments were performed [94, 105, 106, 108, 113,

116], in most trials shake-flask trials have been carried out.

As in the case of de novo lipid accumulation process, a

scarce number of modeling studies of growth and lipid

accumulation in oleaginous microorganisms growing on

hydrophobic materials have been realized so far. Storage lipid

accumulation occurs during primary metabolic growth when

fatty materials are used as substrate and therefore after the

exhaustion of the substrate fat the culture environment is still

favorable for growth [96, 97, 105]. For this reason, in all cases

in which kinetic studies have been performed on fatty sub-

strates lipid accumulated was always re-consumed in favor of

lipid-free material generation regardless of the fatty acid

composition of the fat used as substrate [39, 89, 104–

108]. Therefore, storage lipid degradation should obligatorily

be taken into consideration in order to correctly simulate the

bioprocess [89, 107]. It is considered that the microbial dry

matter (total biomass – X) corresponds to the sum of total

cellular accumulated lipids (L) and lipid-free biomass (Xf).

Likewise, Xf should be capable of consuming from the two

available lipid pools, the extra- as well as the intra-cellular one

[89, 107]. Under this optics, it is considered that the micro-

organism grows with two specific growth rates (diauxic

growth-type model), with mXf(S) being the specific rate of

lipid-free biomass generation from the extra-cellular lipid

substrate (in h�1) and mXf(L) being the specific rate of

lipid-free biomass formation deriving from turnover of the

intra-cellular reserve lipid (in h�1). The lipid-free biomass

formation, thus, is the result of the utilization of two carbon

sources, the extra-cellular lipid substrate as well as the reserve

lipids as follows: dXf =dt ¼ mXf ðSÞXf þ mXf ðLÞXf , where

mXf(S) and mXf(L) are the specific rates of lipid-free biomass

formation [89, 107].

The equation describing the specific formation rate of

lipid-free biomass from the extra-cellular lipid pool, inspired

by the models proposed by Aggelis and Sourdis [89] and

Galiotou-Panayotou et al. [121], is illustrated by the follow-

ing formula: mXf ðSÞ ¼ mXf ðSÞmaxðS=S0Þ, where mXf(S) is the

specific rate of lipid-free biomass formation from the extra-

cellular lipid, and S0 is the initial concentration of the limiting

substrate (in g/L) that is a Blackman-type equation. It may be

assumed that this rate presents its maximum value at the

beginning of the fermentation and it decreases linearly with

the decrease of the carbon substrate. A Verhust-typemodel in

which biomass formation is governed by the culture density

[m ¼ mmax 1�ðX=XmaxÞð Þ], is also applied in Papanikolaou

et al. [106]. Of course, the above mentioned types of models

(Blackman- or Verhust-type) can also perfectly simulate the

microbial growth in fermentations in which carbon is not

the limiting factor [90, 93], while, in general, Michaelian-

type expressions of specific growth rate (m) seem to be more

suitable for description of models in the steady-stages of

single-stage continuous cultures [122]. Moreover, the

specific formation rate of lipid-free material performed

through the degradation of intra-cellular lipid is presented

by the following formula: mXf ðLÞ ¼ mXf ðLÞmaxðL=LmaxÞ [89]. It

should be stressed that in general the optimized values of

mXf ðLÞmaxwere significantly lower compared with those of

mXf ðSÞmaxsuggesting that lipid turnover was a process less

rapid than that the consumption of extra-cellular aliphatic

chains [107]. Likewise, consumption of both lipid pools

(extra- and intra-cellular lipid) resulted in the formation of

lipid-free biomass that was characterized from its yield per

lipid (extra-cellular – YXf/S and intra-cellular – YXf/L) con-

sumed [89, 107]. Storage lipid turnover was accompanied by

high YXf/L values, sometimes higher than YXf/S. This can be

explained by the fact that transport of extra-cellular aliphatic

chains inside the cell, a phenomenon energetically costly, is

missing during reserve lipid degradation period, while reserve

lipid consumption is used almost exclusively for the synthesis

of lipid-free material. In contrast, at the first growth steps,

when substrate fat was presented in significant amounts, the

microorganism grew with high specific rates, but part of the

energy is consumed in the incorporation of fatty acids and

the formation of storage lipids [107].

As far as the evolution of intra-cellular lipids through

the ex novo process is concerned, it is consisted of the

result of two inverted processes: that of cellular lipid

formation from the extra-cellular pool, and the one of cellular

lipid uptake for lipid-free biomass formation: dL=dt ¼qLXf�mXf ðLÞXf 1

�YXf =L

� �, where qL is the specific formation

rate of accumulated fat (in g/g/h). Taking into consideration

that in the case of the ex novo lipid accumulation process, fat

is produced during primary metabolic growth simultaneously

with lipid-free material, the expression of qL is of the type

qL ¼ amXf ðSÞ and specifically qL ¼ qLmaxðS=S0Þ [89, 107]. It

should also be stated that in one case, simulation of intra-

cellular lipid (and extra-cellular citric acid) production was

performed during growth of Y. lipolytica ACA-DC 50109 on

mixtures of hydrophobic substrates (a low-cost industrial

derivative of tallow composed of fully saturated free-fatty

acids called ‘‘stearin’’) with commercial glucose in nitro-

gen-limited flask experiments, and at that study taking into

consideration that nitrogen had been already exhausted from

the medium, production of lipid was considered as non-

growth coupled process with qL ¼ a [90].

In Table 1, a representation of the various predicted

parameter values that have been obtained in the literature

are presented. As it can be observed, the maximum concen-

tration of lipids inside the cells (Lmax, g/L) largely depends on

the microbial strain used. Furthermore, parameter mXf ðSÞmax

presents similar values regardless of the carbon source (fat or

sugar) and the microorganism (yeast or mold) used.

Likewise, KN values present similarities regardless of the

non-growth associatedmetabolite produced (de novo derived

intra-cellular SCO or extra-cellular citric acid), while YXf/S

and YL/S values present differences related with the carbon

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substrate used. Finally, qLmax values are influenced by de novo

or ex novo lipid accumulation process with higher values

reported during growth on fatty media, compared with that

obtained on sugar-based ones. As previously stated growth of

oleaginous microorganisms on fatty substrates was accom-

panied by amXf ðSÞmaxvalue higher that the respectivemXf ðLÞmax

indicating that lipid turnover was a less rapid process com-

pared with the fat uptake from the extra-cellular medium.

2.2 Yeast lipid suitable for biodiesel production

Current industrialization and decrease of petroleum stock

have raised the worldwide need for energy generation deriv-

ing from various alternative and renewable resources (e.g.,

biodiesel, bio-hydrogen, and/or bio-ethanol) with biodiesel

being considered as one of the most important renewable

energy sources due to its economic and environmental

benefits [123]. Biodiesel is prepared through trans-esterifi-

cation of vegetable oils or animal fats with short chain alco-

hols (principally methanol and to lesser extent ethanol or

butanol). Specifically, according to the ‘‘US Standard

Specification for biodiesel’’ (ASTM 6751-02), biodiesel is

defined as ‘‘a fuel that is composed of mono-alkyl esters of

long-chain fatty acids deriving from vegetable oils or animal

fats’’ [124]. This definition is also acceptable in the European

Union specification concerning biodiesel (EN 14214).

Stricter regulations define biodiesel as FAMEs, but current

considerations could likely extend this definition also to fatty

acid ethyl-esters, which can be obtained using bio-ethanol as

alcohol donor (see also: Lois [124]; Adamczak et al. [13]).

Synthesis of biodiesel is performed principally by chemi-

cal catalysis, but it can also be performed via enzyme-cata-

lyzed methods, that although are still hampered by the high

costs of the biocatalyst, significant progress has recently been

made leading to the first industrial enzymatic biodiesel pro-

duction [13]. Although the production cost of the enzyme-

catalyzed and produced biodiesel is around one order of

magnitude higher than the conventional chemical production

[125], enzymatic production of FAMEs is considerably

attractive taking into consideration that starting materials

like low or negative cost waste frying oils, oils with high water

content, etc., for which conventional chemical trans-esteri-

fication can hardly be applied, can perfectly be used as sub-

strates for the enzymes [13]. Moreover, despite the

continuous need for biodiesel production, the last years lack

of oil feedstock has created problems related with the pro-

duction of conventional or so-called ‘‘1st generation biodie-

sel,’’ and for this reason alternative fatty media were used

(e.g., utilization of waste lipids). Specifically in Europe, it has

been realized that the use of conventional plant oils would

rapidly increase their current prices (a situation that has

already been realized in the case of rapeseed oil), allocate

vast areas of cultivable land from food to biodiesel production

and jeopardize the biodiversity in these areas [57, 126]. In

addition, if the need for biodiesel is to be fulfilled by rapeseed

oil it is estimated that the current total volume of European

rapeseed production is not sufficient [126].On the other hand,

the production cost of SCO remains always much higher than

that of plant oils [21]. A representative value of yeast SCO

produced on 2008 was around 3.0 US $ per kg (excluding cost

of feedstock used for SCO production) while the ones of

rapeseed oil, soybean oil, and sunflower oil are 1.4–1.5, 1.2–

1.3, and 1.8–1.9 US $ per kg, respectively [21]. However, the

previously described situation with the increment of biodiesel

needs has resulted in the fact that between 2007 and 2008

there has been a twofold increase of the price of conventional

plant commodity oils [21]. Therefore, the necessity of dis-

covery of novel (non-conventional) sources of oils, which

could be subsequently converted into biodiesel is of crucial

importance, with the oleaginous microorganisms being con-

sidered as potential candidates for the production of this

‘‘2nd generation biodiesel’’ [23]. It should also be taken into

considerations that the economics of SCO bio-processes can

be further ameliorated by using wastes as substrates because

most of them have a negative value and their discharge causes

environmental problems (nevertheless, in this case a well-

established network providing the waste material into the

fermentation plant is of extreme importance for the economic

viability of the process – see: Ratledge andCohen [21]). Then

the produced biomass rich in lipid can be directly trans-

esterified to yield the biodiesel [57, 127], thus avoiding the

oil extraction step, which is one of the most costly steps of the

SCO production procedure [16]. Autotrophic algae present

also advantages with the principal ones being the utilization of

sunlight and CO2 sequestration for SCO production amend-

able for biodiesel production (the so-called ‘‘3rd generation’’

biodiesel) factors that are considered to be as very crucial that

make these microorganisms as the most appropriate for an

environmentally friendly production of biodiesel [21, 32].

However, the small growth rates, the decreased quantity of

CO2 found into the atmosphere that results in carbon-limited

culture conditions, the difficulty to carry out high cell density

cultures and the current increased cost of photo-bioreactors,

pose problems that need to be solved in the future [32], and

therefore the cost of autotrophically produced algal SCO is

significantly higher than the previous categories, being in the

range of 5.6–21.0 US $ per kg, without taking into consider-

ation the cost of oil extraction [21].

The oleaginous yeasts, with their unicellular form and

their higher specific growth rate compared with molds and

algae, can be considered as appropriate organisms that could

be used for the production of this 2nd generation biodiesel

[23]. Moreover, yeasts can be cultured in various carbon

sources including waste materials presenting simultaneously

rapid and significant biomass and oil production. Likewise,

the potential of several oleaginous yeasts to present

efficient growth and lipid accumulation under nitrogen-

excess conditions provided that another nutrient (e.g., phos-

phorus) is limited [29], can be exploited in industrial level for

the valorization of agro-industrial residues and surpluses that

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are carbon- and nitrogen-rich, like media composed of N-

acetylglucosamine [30] (it is known that this compound is the

base-unit of chitin, whilst various chitin-based residues are

currently generated in enormous quantities from crustacean-

fabricating facilities and these wastes are disposed by either

burning or land filling, both of which are harmful to the

environment).

It is evident that considering the production of yeast lipid

that will be subsequently converted into biodiesel, only the

process of de novo lipid accumulation is concerned, since

cultivation in fatty materials is principally performed in order

to add value into the fatty material converted, and, therefore,

ex novo lipid accumulation is principally performed in order

to produce specialty ‘‘tailor-made’’ lipids, like substitutes of

cocoa butter of other high-added value exotic fats. In the next

chapter the influence of cultivation parameters on yeast lipid

accumulation will be discussed.

2.2.1 Factors influencing the accumulation of lipid byoleaginous microorganisms

In the process of de novo accumulation of storage lipid the

concentration of the limiting nutrient (nitrogen) frequently

determines the quantity of the biomass produced, whilst the

concentration of the carbon source (e.g., glucose) found in

excess in the growth environment largely determines the

amount of accumulated lipid. Therefore, the molar ratio

C/N plays a key-role in determining the oil content and

biomass density of the oleaginous microorganisms [17,

18]. Generally, it is considered that the process of lipid

accumulation is induced at molar ratio C/N > 20. In some

cases cultures in media in which very high initial C/N ratios

were imposed (e.g., higher than 70) resulted in decreased fat

accumulation, suggesting that optimum initial C/N molar

ratios are required for the conversion conducted (case of

R. toruloides yeast – see Moreton [15]). In contrast, in other

cases (e.g., culture of L. starkeyi on glucose), in trials per-

formed with constant initial glucose and decreasing initial

nitrogen concentrations, accumulation of storage lipid inside

the cells constantly increased even though in some cases high

initial C/N ratios (around 150 moles/moles) were used, for

the range of initial nitrogen concentrations tested [24]. In

similar types of experiments performed withR. glutinisNRRL

Y 1091, the production of total microbial lipids constantly

increased with increase of the C/N molar ration imposed, for

the range of the initial nitrogen concentrations tested [128].

Likewise, in similar trials (utilization of M. isabellina fungus

growing at constant initial nitrogen and increasing initial

glucose concentrations into the medium), for the range of

glucose concentrations tested, despite high initial sugar

quantities (e.g., 100 g/L) and initial C/N ratios (e.g., initial

C/N � 340 moles/moles), constantly increasing SCO

quantities were produced, while also other lipid-free storage

materials were accumulated (Fig. 1 – Papanikolaou et al.

[54]). Moreover, in some cases of lipid accumulating micro-

organisms (e.g., strains belonging to C. echinulata,

M. ramanniana, etc.), cultivations elaborated in high initial

C/N ratio media can be accomplished with significant

quantities of sugar remaining unconsumed into the culture

medium without tendency of finally being consumed [57, 64,

67, 129]. Considering that, in general, both intra-cellular

NADþ- and NADPþ-isocitrate dehydrogenases during

lipid-accumulating conditions show a very limited activity

[55, 130, 131], the key-enzyme regulating the intra-cellular

carbon flow is the one NADPþ-malic enzyme [131], the low

activity of which could down-regulate and cease the uptake of

sugar during the period of accumulation of lipid constituting

the limiting step of the process [21].

Recent investigations have indicated that the process of

storage lipid accumulation is critically influenced by the

specific sugar uptake rate (qS) inside the cells or the fungal

mycelia; in cultures of the oleaginous mold C. echinulata on

nitrogen-limited glucose-based tomato waste hydrolysate

media or starch-based media, decreased qS values resulted

in increased total biomass (X, in g/L) and total biomass yield

(YX/S, in g/g) values, with concomitant lower lipid production

[64, 67]. It appears that in high qS values reported, carbon

flow was channeled more rapidly and in higher quantities

inside themycelia, suggesting higher intra-cellular C/Nmolar

ratio compared with cultures presenting low qS values. For

Figure 1. Cultures of the oleaginous fungus M. isabellina on glu-

cose used as the sole carbon source at increasing initial glucose

concentrations (Glc0 concentrations 45, 50, 60, 70, and 100 g/L)

and constant nitrogen concentration (ammonium sulfate and yeast

extract adjusted at 0.5 g/L of each). Representation of lipid-free

material (Xf, g/L), lipid (L, g/L) and lipid in dry weight (g per 10 g of

total biomass) when the maximum concentration of lipid was

obtained (data from Papanikolaou et al. [54]).

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this reason, the carbon flow in the later case seemed of having

been directed principally toward the synthesis of lipid-free

material rather than the storage lipid formation [67].

Furthermore, in the case of C. echinulata, the presence of

proteins in the culture medium seemed to critically influence

the uptake rate of sugar, and, hence, the process of lipid

accumulation, since organic nitrogen of tomato waste hydro-

lysate clearly enhanced glucose uptake and storage lipid pro-

duction, while cultures in the above waste in which proteins

had been removed was accompanied by drastically lower

uptake rate of glucose and production of lipid [64].

Apparently, this is the main reason for which although in

various cases trials have been performed with sugars that

present very high similarity in biochemical level (e.g., glucose,

maltose, lactose, soluble starch, etc.) non-negligible differ-

ences in lipid accumulation have been observed [48, 67, 132].

The nitrogen source has been also reported to be of

importance for the process of de novo lipid accumulation

in yeasts and molds; for instance, in the case of C. albidus

strain CBS 4715, addition of inorganic nitrogen sources

(ammonium sulphate or ammonium chloride) favored the

accumulation of lipid the yeast cells in comparison with

organic nitrogen source (e.g., urea, L-arginine, etc.) [132].

In contrast, in other cases addition of organic nitrogen sour-

ces into the medium has substantially increased the quantity

of SCO produced [15, 133]. In R. toruloides CBS 14 addition

of asparagine instead of ammonium chloride increased the

lipid content in dry yeast mass from 18 to 51%, w/w [133].

Further study of this strain in flask or bioreactor trials showed

the same trend with a wide range of organic N sources

used [15].

The pH and the incubation temperature have been

reported as factors that equally have importance in relation

with the process of SCO accumulation. Specifically, the

incubation temperature critically influenced lipid accumu-

lation in the yeasts C. albidus, A. curvatum, R. minuta, and

Y. lipolytica [16, 105, 132, 134]. For the case of Y. lipolytica

(ex novo lipid accumulation with low-cost saturated free-fatty

acids used as substrate), temperatures of 19 and 398C did not

allow high growth, whilst significant growth was observed

within the range of 24–338C (Xmax ¼ 7.5–8.7 g/L,

mmax ¼ 0.26 h�1). However, fat accumulation was favored

only at T ¼ 288C [105]. Similar trend has been observed for

R. minuta [134], while the optimum temperature for biomass

and lipid production by C. albidus was 208C [132]. Finally,

during continuous cultures of A. curvatum on whey at con-

stant dilution rate (D ¼ 0.04 h�1) and variations in the incu-

bation temperature (from 30 to 358C) at the steady-states

achieved microbial growth and SCO production were clearly

favored when T ¼ 308C [33]. Likewise, it should be noted

that besides the quantity of fat accumulated into the cells or

the mycelia, the incubation temperature has also significant

effect upon the composition of fatty acids of total cellular

lipids produced. For instance, Ferrante et al. [135] have

demonstrated that in C. lipolytica growing on glucose, the

activity of the enzyme catalyzing the transformation of oleoyl-

CoA to linoleoyl-CoA (D12-desaturase) at T ¼ 108C was

doubled compared with that at T ¼ 258C, with correspond-

ing significant rise of the concentration of intra-cellularD9;12C18 : 2 fatty acid in the later case. Similar observations

have been done by Granger et al. [128] during growth of

R. glutinis on glucose. In similar batch bioreactor experiments

in which growth of R. glutinis on glucose was performed, rise

of the incubation temperature resulted in the increment of

total saturated fatty acids (TSFAs), and principally the ones

of medium aliphatic chain (C12:0, C14:0) [134]. On the

other hand, continuous cultures of A. curvatum at constant

D and variations in the incubation temperature did not reveal

noticeable differentiations in the composition of intra-cellular

fatty acids produced [33]. Finally, in contrast with the results

reported by Ferrante et al. [135] and Granger et al. [128], in

batch shake-flask experiments performed by the yeast

C. curvatus ATCC 20509 on N-acetyl-glucosamine utilized

as the sole substrate, decrease of the incubation temperature

(e.g., T ¼ 228C) was accompanied by synthesis of a fat that

was remarkably enriched in saturated fatty acids (principally

C16:0 and C18:0), in comparison with growth in higher

temperatures (e.g., T ¼ 26–308C) [30].

As far as the pH of the medium is concerned, for the case

of Y. lipolytica growing on stearin, medium pH was con-

sidered as crucial factor for SCO production, since cultures

were performed in initial pH values of 5.0–7.0, and substan-

tial growth was observed only at pH 6.0–6.5 whereas fat

accumulation was favored only at pH ¼ 6.0 [105]. On the

other hand, continuous cultures of A. curvatum growing on

whey at constant D and variations of the pH of the medium

resulted in significant and almost unaffected from the pH

accumulation of fat for a pH range between 3.5 and 5.5 [33,

42]. Apparently continuous fermentation at pH ¼ 3.5 pro-

vides considerable advantages since this operation can be

feasible with a pasteurized whey feed in industrial scale [33].

Various yeast strains have been tested in relation with the

potential growth and SCO production on the sugars deriving

from hemicelluloses hydrolysis [23, 25, 48]. It is known that

lignocelluloses biomass hydrolysis results in generation of

various by-products that may have affect on down-stream

SCO biotransformation. Therefore, such types of products

like acetic acid, syringaldehyde, vanillin, furfural, etc. were

added into a glucose-based medium and the production and

study of SCO by R. toruloides Y4 was assessed in the presence

of these inhibitors [136]; it was found that some products like

hydroxymethylfurfural (HMF) and acetic acid had a very

slight inhibitory effect upon growth (e.g., addition of

100 mM of acetic acid or 16 mM of HMF did almost not

at all negatively influence biomass production). In contrast,

addition of other compounds like furfural or vanillin in

quantities ranging between 8 and 12 mM, almost completely

ceased microbial growth [136]. The addition of these inhibi-

tors influenced also the fatty acid composition of total

microbial lipids produced [136].

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A significant number of investigators have studied the

synthesis of microbial lipid in nitrogen-limited batch, fed-

batch (flask or bioreactor) and single-stage continuous or

continuous with biomass recycling cultures. In most of the

cases, renewable-or waste-type carbon sources have been

used and in almost all cases, SCO produced can potentially

be used for 2nd generation biodiesel synthesis. In some

reports, investigations deal with SCO production in single-

stage or cell-recycling continuous yeast cultures [33, 44, 47,

49, 137, 138]. It has been demonstrated that lipid accumu-

lation was strongly depended on the dilution rate imposed

[17, 52, 137] as well as of the molar ratio C/N of the growth

medium [17, 88]. D of less than 0.06 h�1 were normally

required for optimum conversions [17, 49], since the

microbial cells need to remain within the chemostat for at

least 12–24 h in order to consume the available nitrogen and

then convert the remaining sugar to oil and ‘‘fatten’’ [17, 47].

Moreover, in a restricted number of reports fed-batch exper-

iments have been performed, remarkably high biomass pro-

duction (�100 g/L) containing various lipid quantities has

been observed [23]. Finally, concerning strains of the non-

conventional yeast Y. lipolytica, cultivation of this microor-

ganism in nitrogen-limited glucose- (or glycerol-) based

media resulted in equivocal results; in highly aerated batch

bioreactors, quantities of lipids were accumulated inside the

yeast cells, whereas nitrogen limitation led also to citric acid

biosynthesis [72]. In contrast, in highly aerated chemostat

cultures and low dilution rates imposed (e.g., D < 0.04 h�1),

lipids were produced in high quantities (e.g., >25% w/w)

inside the yeast cells [47, 138], whereas in at least one case, a

strain of the above-mentioned species produced simul-

taneously SCO and citric acid in shake-flask nitrogen-limited

cultures [70]. It should finally be stressed that in a recent

development, the well-studied lipid- and citric acid-producing

Y. lipolytica strain ACA-DC 50109, was subjected to genetic

manipulation and was revealed capable to secrete significant

inulinase quantities (it was the transformant Z31), that was

revealed capable to simultaneously break-down inulin-based

compounds and produce significant SCO quantities in shake-

flask experiments, e.g., L up to 7.4 g/L with lipid in dry weight

of more than 50% w/w, on nitrogen-limited media containing

the extract of Jerusalem artichoke tubers [78]. The most

representative examples of de novo SCO production by ole-

aginous yeasts are depicted in Table 2.

2.3 Production of cocoa butter substitutes byoleaginous yeasts

One major industrial application referred to yeast lipid pro-

duction is that of the synthesis of microbial substitutes of

cocoa butter [15, 17, 18, 20, 22]. Cocoa butter is commonly

used in the food technology and principally in chocolate

fabrication process, whereas it is also used in various cosme-

tology applications. It is principally produced in some of the

African and Central American countries such as Ivory Coast,

Nigeria, Jamaıca, etc. This lipid is mainly composed of TAGs

of the type P–O–S and S–O–S (P: palmitic acid, O: oleic acid,

S: stearic acid). Oleic acid, hence, is always found esterified

in the position sn-2 of glycerol. Cocoa butter contains

55-67% w/w saturated fatty acid while its composition is

dependent of the plant variety and the culture conditions.

An average fatty acid profile of this fat is: C16:0 23–30%w/w;D9C18:1 30–37%w/w; C18:0 32–37%w/w; D9;12C18:2 2–4%

w/w [17, 22, 140].

The production of cocoa butter substitutes (CBSs), econ-

omically viable during the years 1980–1990 (at that time the

price of the cocoa butter was >8.0 US $ per kg), is depend-

able of the price of this fat. During the years 1990–1994, a

significant fall of cocoa butter price (<2.5 US $ per kg) has

constituted an enormous disadvantage for the production of

various substitutes of this fat [17, 20]. However, the years

after 2000, there has been increment again of its price, due to

the prevalence of harmful insects and viruses that have been

reported to create several problems on cocoa butter pro-

duction [20, 30, 39]. Currently, the price of cocoa butter

is around 5.0 US $ per Kg, though the tendency of this price,

is to present (a remarkably significant) increase in the near

future [22, 30]. An ‘‘extreme’’ scenario recently presented in

both the written and the electronic international press

indicates that cocoa butter risks to disappear the next years

due to general failure of the cultivation techniques of the

cocoa plant, and, thus, the utilization and application of the

various CBSs will be generalized for the food industry.

Numerous approaches have been conducted, in order to

produce lipids having composition similarities with those of

the cocoa butter. The first strategy performed referred to the

preparation of mixtures of different fats of exotic plants (e.g.,

illipe butter, mango fat, kokum butter, sal fat) with fractions

of palm oil [140–142] in order to create fatty materials with

composition and technological properties relatively close to

that of cocoa butter. It is noted however, that already the price

of some of these exotic fats is remarkably high [22]. In

parallel, different biotechnological approaches, either enzy-

matic- or fermentative-ones have been already carried out

for the production of CBSs (for reviews see: Ratledge [17];

Lipp and Anklam [140]; Papanikolaou and Aggelis [22]).

Concerning the utilization of enzymes, it is, in fact, one of the

first (and of primordial importance) axes of lipid biotechnol-

ogy that has been developed in the late 80s. This approach

was related with the utilization of hydrolytic enzymes (prin-

cipally lipases) in media presenting a feeble water concen-

tration in order to synthesize specified TAGs by reactions of

trans- and/or inter-esterification of various plant lipids (e.g.,

trans-esterification of palm oil from stearic acid resulting

in the synthesis of TAGs of the types P–O–S and S–O–S)

[3, 5, 6]. The last years, reactions of the above type have been

ameliorated, optimized, and carried out in larger scale, with,

principally, utilization of various low-cost fatty materials

(e.g., lard, pomace olive oil, etc.) as substrates in order to

create various CBSs [8–11].

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Table 2. Lipid production through de novo fatty acid biosynthesis pathwayduring growth of oleaginous yeasts on various carbon sources and

fermentation configurations

Microorganism Culture mode Carbon source Xa) (g/L) Lipid (% w/w) Reference

Candida sp. 107 Single-stage continuous Glucose 18.1 37.3 Gill et al. [137]

C. curvatus ’’ Glucose 13.5 29.0 Evans and Ratledge [49]

’’ ’’ Sucrose 16.0 28.0 ’’

’’ ’’ Lactose 18.0 31.0 ’’

’’ ’’ Xylose 15.0 37.0 ’’

Cryptococcus albidus Shake flasks Xylose 7.3 33.0 Hansson and Dostalek [132]

’’ ’’ Glucose 8.2 40.1 ’’

’’ ’’ Maltose 8.2 37.7 ’’

’’ ’’ Lactose 6.5 26.3 ’’

’’ ’’ Glycerol (pure) 1.4 43.8 ’’

Rhodosporidium toruloides ’’ Glucose 8.0 42.5 Moreton [15]

’’ ’’ Fructose 7.0 27.0 ’’

’’ ’’ Glycerol (pure) 5.8 34.4 ’’

’’ Batch bioreactor Glucose 12.5 42.9 ’’

’’ ’’ Fructose 8.7 39.8 ’’

’’ ’’ Xylose 8.3 42.2 ’’

A. curvatumb) Single-stage continuous Whey 24.1 37.4 Davies [33]

’’ ’’ ’’ 20.8 44.3 ’’

’’ ’’ ’’ 21.2 43.4 ’’

C. curvatus Batch bioreactor ’’ 21.6 36.0 Ykema et al. [44]

’’ Continuous-recycling ’’ 85.0 35.0 ’’

C. curvata Single-stage continuous Lactose 20.0 40.0 Brown et al. [52]

A. curvatum Batch bioreactor Glucose n.r.c) 35.5 Hassan et al. [38]

’’ Single-stage continuous ’’ 14.5 45.6 ’’

C. curvatus Batch bioreactor Prickly pear juice 10.9 45.8 Hassan et al. [74]

’’ ’’ ’’ 11.1 43.2 Hassan et al. [75]

’’ Fed-batch bioreactor Glycerol (pure) 118.0 25.0 Meesters et al. [45]

’’ Fed-batch air-lift bioreactor ’’ 91.0 32.0 Meesters et al. [46]

Y. lipolytica Single-stage continuous Glucose 9.2 25.0 Aggelis and Komaitis[138]

’’ Single-stage continuous Glycerol (raw) 8.1 43.0 Papanikolaou and Aggelis [47]

R. toruloides Fed-batch bioreactor Glucose 106.5 68.1 Li et al. [23]

’’ Shake flasks Glucose & sewage sludge 9.4 68.0 Angerbauer et al. [24]

Lipomyces starkeyi ’’ Glucose & xylose 20.5 61.5 Zhao et al. [25]

Trichosporon fermentans ’’ Glucose 24.1 56.6 Zhu et al. [27]

’’ ’’ Sucrose 19.5 62.6 ’’

’’ ’’ Xylose 17.1 57.8 ’’

’’ ’’ Lactose 16.9 49.6 ’’

’’ ’’ Fructose 21.5 40.7 ’’

’’ ’’ Molasses 36.4 35.3 ’’

Y. lipolytica ’’ Glycerol (raw) 6.5 30.7 Andre et al. [70]

R. toruloides ’’ Glucose 15.0 62.0 Hu et al. [136]

T. fermentans ’’ Rice straw hydrolysate 28.6 40.1 Huang et al. [48]

’’ ’’ Mannose 22.7 50.4 ’’

’’ ’’ Galactose 23.6 59 ’’

’’ ’’ Cellobiose 15.8 65.6 ’’

Rhodotorula mucilaginosa Shake-flasks Starch hydrolysate 21.8 53.0 Li et al. [76]

C. curvatus Fed-batch bioreactor Glycerol (raw) 32.9 52.9 Liang et al. [139]

Y. lipolyticac) Fed-batch bioreactor Glycerol (pure) 4.7 23.1 Makri et al. [72]

R. toruloides Shake-flasks Glucose 19.9 63.3 Wu et al. [29]

’’ Fed-batch bioreactor Jerusalem artichoke extract 39.6 56.5 Zhao et al. [79]

Rhodotorula mucilaginosa ’’ Inulin hydrolysate 14.4 49.0 Zhao et al. [77]

’’ Fed-batch bioreactor Jerusalem artichoke hydrolysate 19.5 52.1 ’’

Y. lipolyticad) Shake-flasks Inulin 13.3 48.3 Zhao et al. [78]

’’ ’’ Jerusalem artichoke extract 14.6 50.6 ’’

Trichosporon capitatum ’’ Molasses 17.0 35.2 Wu et al. [59]

’’ ’’ Glucose 15.4 43.0 ’’

a) X is the dry cell weight produced (in g/L).b) Culture at a constant dilution rate (¼ 0.04 h�1) and various pH values of the medium.c) During these cultures, nitrogen limitation led also to remarkable production of citric acid into the culture medium.d) Genetically engineered transformant Z31 capable of consuming inulin-based media.

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Numerous investigators have utilized oleaginous micro-

organisms, and principally yeasts, that can be considered as

‘‘perfect’’ candidates for this purpose [18, 22]. The fact that

thesemicroorganisms stock their lipids principally in the form

of TAGs esterified in the sn-2 position by unsaturated fatty

acids, has favored this approach [16, 17]. However, the main

drawback to alleviate was how to increase the C18:0 and,

generally, the TSAF content inside the yeast cells [20, 22,

140], whilst in most instances the oleaginous yeasts accumu-

late unsaturated fatty acids to more than 65% w/w, in their

total lipids. Many strategies concerning the increase of TSFA

content in the yeasts have been realized so far and they will be

presented in the next paragraphs.

2.3.1 Strategies dealing with the production of CBSsby oleaginous yeasts

The first of the approaches carried out in order to produce a

microbial analogous of cocoa butter, consisted of a conven-

tional culture of an oleaginous yeast (e.g., R. toruloides,

L. lipofer, or Rhodotorula graminis) on glucose in nitrogen-

limited media followed by a separation of the synthesized

cellular lipid. The unit operation proposed was either crys-

tallization fractional separation process in order to finally

recover the fractions S–U–S (S: saturated fatty acid, U:

unsaturated fatty acid) of the produced SCO [143].

Following this type of operation, TAGs of type P–O–P

and P–O–S have been purified and utilized in the fabrication

of chocolate, replacing cocoa butter. The result was revealed

satisfactory considering both fusion point and organoleptic

properties of the product [143]. The inconvenient of this

approach is the fact that only the 35% w/w, of the produced

fat is presented in the form S–U–S [18].

Another strategy used in order to increase the C18:0

amount of the yeast lipids produced, was based on the prin-

ciple that plants, animals, and microorganisms do not pro-

duce their unsaturated fatty acids directly; firstly, a formation

of a saturated precursor is performed, and then, by virtue of

sequential desaturation reactions, double bonds are intro-

duced firstly in the position D9 and subsequently in the

positions D12 and D15 (it is noted that the desaturation in

the position D15 is unusual in the oil-bearing yeasts, whereas

humans are incapable of de novo introducing double bonds

after the 9th carbon in the aliphatic chain) [15, 20, 50]. The

acylated groups desaturation pathway is illustrated in

Figure 2 [17]. Consequently the desaturation activity could

be restricted using various desaturase inhibitors. Sterculic

and malvalic acid are cyclopropene fatty acids found in the

seed-oil of different plants of the families Malvaceae and

Sterculiaceae like sterculia oil and to lesser extent to kapok

oil [144]. Being fatty acids of 17 or 18 carbons with a cyclo-

propenic group in the positionD9, sterculic and malvalic acid

have been reported to inhibit the desaturation activity in

various plants and animals. In fact, these cyclopropene fatty

acids are structural analogous of the natural substrates for the

D9 desaturase enzymes, which carry out the conversion:

C18:0 ! D9C18 : 1. These substances have been equally

utilized in order to modify the accumulated lipid produced

by the oleaginous yeasts [15, 50, 145]. Numerous strains

have been tested (Candida sp. 107, Trichosporon cutaneum,

L. starkeyi,R. toruloides etc). Although in some cases relatively

significant quantities of the cyclopropenic inhibitor have been

added into the culture medium (e.g., up to 4.0 mL/L of

sterculia oil, that indicates presence of more than 2.0 g/L

of cyclopropene fatty acids), cell growth and lipid accumu-

lation have not been altered (total biomass always ranging

between 6.4 and 12.3 g/L containing fat >25% w/w, inside

the yeast cells) [50]. Some results of the production of bio-

mass and lipid and the composition of cellular lipid produced

when cyclopropen fatty acids are into the medium are

depicted in Table 3.

The addition of sterculia oil (containing around 50%w/w,

of sterculic acid) into the culture medium resulted in the

synthesis of a yeast lipid in which the concentration of the

fatty acid C18:0 increased from 3–5% up to �40% w/w [15].

However, this inhibitor having no effect upon the D12 desa-

turation (D9C18 : 1 ! D9;12C18 : 2), the yeast oil presented

relatively high linoleic acid amounts (around 14–24%w/w, of

total lipid). This fatty acid, however, is found in minimal

amounts into the cocoa butter (ranging between 2 and 5%w/w,

of total lipid). In order to produce, hence, with this strategy a

cocoa butter-like yeast lipid, a second inhibition in the D12

position had to be performed. A synthetic D12 cyclopropene

inhibitor, namely ‘‘cis-methylen-octadecenoıc acid’’ was

used, and the obtained result is depicted in Table 4 [15, 145].

The utilization of desaturase inhibitors remarkably

increases the saturation of the SCO produced, having as a

result the synthesis of lipids presenting composition sim-

ilarities with the cocoa butter. However, this strategy presents

two fundamental drawbacks, namely the increased coast of

Triacylglycerols

Acetyl-CoA + Malonyl-CoA

Palmitoyl-CoA Elongase

Stearoyl-CoA ∆9-desaturase

Oleoyl-CoA Transferase

Oleoyl-phosphorolipid

Linoleoyl-phosphorolipid

∆12-desaturase

Figure 2. Pathways of desaturation of acylated groups in the olea-

ginous yeasts (from Ratledge [17], adapted).

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the inhibitors used and the fact that these products may

provoke mutagenesis and cancerogenesis [17]. As far as

the second disadvantage is concerned, this is of major import-

ance, taking into consideration the current trends of biotech-

nology, in which eco-friendly and ‘‘healthy’’ approaches

should be considered in order for the mass production of

biotechnological products [22]. By taking into consideration

that addition of other compounds could potentially interfere

with lipid desaturation [15], in order to alleviate this pre-

viously mentioned serious disadvantage, addition into the

culture medium of other natural compounds that are com-

pletely non-toxic could potentially be envisaged. One case

refers to the addition into the culture medium of the essential

oil of the plant Citrus sinensis cv New Hall –Citrus aurantium (a

natural and ‘‘healthy’’ compound), the addition of which,

even in very small concentrations (e.g., 0.3 mL/L) could

result in significant rise in the TSFA content of the yeast

Y. lipolytica [146]. Although in the culture conditions tested

no virtual accumulation of lipid inside the yeast cell occurred,

the fact that TSFAs significantly increased from 24% to 35–

43% w/w, indicates that addition of this compound into the

culture medium in conditions enhancing lipid accumulation,

could result in the synthesis of microbial CBSs [146].

Genetic manipulation strategies based on the destruction

of the gene encoding for D9 dehydrogenase, which is the

responsible enzyme for the biotransformation C18:0 !D9C18 : 1, have also been performed [35, 36, 38, 75, 147,

148]. In most of the cases D9-defective mutants of

Table 3. Effect of the utilization of sterculia oil upon biomass and lipid production and fatty acid composition of the cellular lipids produced by

oleaginous yeasts growing in shake-flask nitrogen-limited experiments with glucose utilized as the sole substrate

Fatty acid composition (% w/w)

Sterculia oil (mL/L) Xa) (g/L) Lb) (g/L) Lipid (% w/w) C16:0 C18:0 C18:1 C18:2

Candida sp. 107 0.0 9.9 3.1 31 28 5 33 27

0.2 6.4 1.5 25 28 33 10 23

2.0 7.1 2.7 37 25 44 9 15

Tricosporon cutaneum 0.0 9.9 2.8 28 29 7 50 13

0.2 8.4 2.5 30 32 24 30 14

2.0 7.6 2.4 32 35 23 25 13

R. toruloides 0.0 12.3 3.7 30 16 4 42 29

0.2 10.1 3.6 36 15 23 24 15

2.0 10.3 3.3 32 13 41 18 15

Cultivation was performed for 4 days (data from Moreton [50]).a) X is the dry cell weight produced (in g/L).b) L is the total cellular lipid produced (in g/L).

Table 4. Effect of the utilization ofD9 andD12 inhibitors upon biomass and lipid production and fatty acid the composition of the cellular lipids

by R. toruloides and A. curvatum

Fatty acid composition (% w/w)

Xa) (g/L) Lipid (% w/w) C16:0 C18:0 C18:1 C18:2

R. toruloides

Control 7.1 24 28 7 39 18

Sterculia oil (0.3 mL/L) 7.6 23 15 48 19 9

D12 inhibitor (0.4 ml/L) 7.8 20 36 8 45 2

Sterculia oil and D12 inhibitor (each 0.3 mL/L) 8.0 34 20 47 22 5

A. curvatum

Control 6.7 49 32 16 44 8

Sterculia oil (0.1 mL/L) 8.6 55 27 26 35 8

Sterculia oil (0.4 mL/L) and D12 inhibitor (4 mL/L) 8.0 52 41 25 28 T

Cocoa butter 23–30 32–37 30–37 2–4

Shake-flask nitrogen-limited experiments with glucose utilized as the sole substrate were done. Cultivation was performed for 4 days (data

from Moreton and Clode [145]).

T < 0.5% w/w.a) X is the dry cell weight produced (in g/L).

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A. curvatum have been constructed; cellular suspensions of

the wild strain were treated with various mutagenic factors

like N-methyl-N-nitro-N-nitrosoguanidine (MNNG), ethyl-

methanesulfonate or UV irradiation [35, 36, 38, 75, 141,

147, 148], and then have been grown in a medium supple-

mentary in oleic acid. The colonies were transferred in a

medium without supplementary oleic acid. In general, the

formed colonies in the two types of substrates have been

screened extensively, and small numbers of colonies of aux-

otroph mutants in the unsaturated fatty acids (Ufa mutants)

have been obtained [36, 141], since also fatty acid synthetase

(FAS) mutants have been created [35, 147]. In most of the

cases, the addition of a D9C18 : 1-donor (even in small con-

centrations) is obligatory, since the Ufa mutants may not be

capable at all of synthesizing the above-mentioned fatty acid.

The cellular lipids isolated from the Ufa mutants grown in a

medium supplementary in oleic acid, have been analyzed and

compared with these of the wild strain A. curvatum. The

result is illustrated in Table 5 [141]. A correct use of this

fatty acid with a sugar as co-substrate, results in the synthesis

of a cellular CBS by the various Ufa mutants [141]. For

instance, in the case of the Ufa-25, batch-bioreactor culti-

vations were performed in whey-permeate enriched with a

small quantity of D9C18 : 1 donor (namely rapeseed oil added

in a rate of 0.08 g/g of lactose into the medium), and after

120 h of cultivation a lipid quantity of around 6.0 g/L cor-

responding to lipid in dry weight 41.0% w/w, was achieved,

while SCO produced presented a composition in fatty acids

similar with that of cocoa butter (C18:0–40% w/w, C16:0–

25% w/w, D9C18:1–20% w/w, D9;12C18:2–10% w/w – Ykema

et al. [149]). In a similar manner, the Ufa mutant M3 was

cultivated in batch and single-stage continuous nitrogen-lim-

ited cultures with glucose utilized as substrate (initial or inlet

glucose concentration at 30 g/L, initial C/N � 40 g/g), and

oleic acid and Tween 80 added in small quantities into the

medium (0.2 and 0.5 g/L, respectively), and significant bio-

mass production (biomass yield on glucose consumed

�0.5 g/g) was observed, in both batch and continuous cul-

tures. For the chemostat experiments low D values (i.e.,

ranging between 0.040 and 0.068 h�1) were required for

high SCO production, and maximum lipid in dry weight

to �40–46% w/w, with dry cell mass of �15 g/L for several

steady-states was achieved [38]. The fatty acid composition

of cellular lipid produced was close to that of cocoa butter

[38]. In order to avoid oleic acid addition, revertants of the

Ufa mutants have been chosen, with partially restored D9

desaturase activity [36, 141]. The obtained result is presented

in Table 6.

In another genetic approach aiming at the production of

CBS-SCOs, the D9 fatty acid desaturase gene of C. curvatus

ATCC 20509 was cloned and characterized [148]. This gene

presented a high G þ C content (of 61%) and displayed a

codon usage different in comparison with the one of the non-

oleaginous yeast Saccharomyces cerevisiae, but similar to that

of the basidiomycete Schizophyllum commune [148].

Moreover, the expression of the above gene was studied in

the presence of several fatty acids found into the growth

medium. Repression of desaturase mRNA signals was found

if fatty acids with a double bond at the D9 position were

presented, while fatty acids with a double bond found at

another position (e.g., D10 or D6) or saturated fatty acids

Table 5. Composition of cellular lipids of A. curvatumwild type (WT

– ATCC 20509) strain and auxotroph mutant Ufa-33 (data from Smit

et al. [141]).

Cellular

fatty acids

(% w/w)

WT Ufa 33

Without

supplement

Supplement

C18:1

Supplement

C18:1

C16:0 28 17 22

C18:0 14 11 36

C18:1 44 55 24

C18:2 2 9 10

TSFAsa) 44 31 62

a) TSFAs is the percentage of saturated fatty acids (w/w) in the

microbial lipids produced.

Table 6. Production of lipid by mutant revertants cultivated on diluted whey-permeate in batch laboratory-scale bioreactor experiments and

comparisons with data obtained by A. curvatum wild-type (WT – ATCC 20509) strain and cocoa butter

Xa) (g/L) Lb) (g/L) Lipid (% w/w)

Fatty acid composition (% w/w)

C16:0 C18:0 C18:1 C18:2

WT 4.4 1.2 27.2 28 15 43 8

R22-72 3.0 1.4 46.6 26 36 20 9

R25-75 3.5 1.6 45.7 29 30 28 8

Cocoa butter 23–30 32–37 30–37 2–4

Initial lactose concentration at 10 g/L, initial molar C/N ratio 30 moles/moles, and fermentation time imposed until lactose exhaustion from

the culture medium (data from Ykema et al. [36]).a) X is the dry cell weight produced (in g/L).b) L is the total cellular lipid produced (in g/L).

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had no effect upon the transcription of the cloned gene [148].

It is evident that the genetic manipulation of the oleaginous

strains has presented very satisfactory results considering

both quantity of accumulated fats and cellular lipid compo-

sition, since the cellular stearic acid amount increases signifi-

cantly. However, not any process scale-up has been

performed, while, in addition, the wild cultures present

higher productivities compared with the mutants.

In order to obtain by a metabolic manipulation what the

genetic engineers have realized with the aid of mutations,

Davies et al. [37] have performed cultures of A. curvatum in

conditions of limited oxygen tension during the phase of lipid

accumulation. The idea was based on the fact that all of the

reactions of desaturation are oxygen dependant [18]. A

critical limitation of the oxygenation in the fermentation

medium could, hence, decrease the conversion percentage

of the reaction C18:0 ! D9C18:1. The obtained results (fer-

mentations performed in an air-lift bioreactor of 20 L

capacity) are presented in Table 7 [37]. It may be seen that

although the percentage of the fatty acid C18:0 into the

reserve lipids was not very high, the sum of saturated fatty

acids (C16:0, C18:0, C20:0, and C24:0) produced by

A. curvatum was in most of the cases higher than 50% w/

w, of total lipids. This fact represents, indeed, an interesting

result in a double coincidence; firstly because it has been

demonstrated with a completely eco-friendly method that the

oleaginous yeasts have in some circumstances the tendency to

accumulate lipids containing significant quantities of satu-

rated fatty acids. The second reason is that not any expensive

or dangerous method was applied [17]. Oxygen tension

decrease had almost not any negative effect upon cell growth

(�0.4 g of cells formed per 1 g of lactose consumed) and lipid

accumulation (around 37% w/w, of lipids in dry weight). In

addition, this approach is the only one having been extrapo-

lated in semi-industrial (500-L bubble column reactors –

Davies et al. [37]) and finally to industrial (13-m3 reactors

– Davies [42]) scale. In a similar manner, decrease in the

aeration rate during growth of oleaginousmolds of the species

M. circinelloides growing on small-chain organic acids,

resulted in increase of the TSFA content of the lipids pro-

duced, with concomitant increment of biosynthesis of sym-

metric TAGs of the type S–U–S, and therefore a CBS was

synthesized [51].

A simple method that has been applied in earlier or more

recent studies in relation with the production of SCOs pre-

senting somehow increased TSFA content is based on the

‘‘potentiality’’ itself of the oleaginous microorganism to store

relatively saturated fatty acids. For instance, in various cases

strains of A. curvatum (C. curvata – C. curvatus, i.e., strains D,

ATCC 20509, etc.) were capable to produce intra-cellular

lipids with somehow increased quantities of C16:0, resem-

bling, therefore, to palm oil [38, 49]. By performing simple

fermentation techniques like, i.e., the change in the incu-

bation temperature, it is possible to increase the TSFA con-

tent of lipids produced. In such a case, Wu et al. [30] have

performed growth of C. curvatus ATCC 20509 on N-acetyl-

glucosamine, and by shifting the incubation temperature

from T ¼ 308C to T ¼ 228C, the concentration of cellular

TSFAs remarkably increased (from�44 to�54%w/w) while

decrease of the temperature did not have remarkable effect

upon biomass and SCO produced. At T ¼ 228C, biomass of

�18 g/L containing fat at 50% w/w, was produced,

whereas the respective values at T ¼ 308C were 19.1 g/L

and 45%w/w, while interestingly enough, lipid was produced

as a primary anabolic activity despite the presence of nitrogen

into the medium [30]. Pan et al. [41] have performed an

extensive screening of soil-deriving yeasts, and have realized

cultures of the most promising strains on xylose and glycerol.

In some cases, SCO presenting composition close to cocoa

butter was synthesized, due to the spontaneous ability of

yeasts to produce lipids rich in the fatty acids C16:0 and

C18:0 [41]. Likewise, R. toruloides strain Y4 has been

revealed capable of producing significant lipid quantities

during growth on glucose-based media under phosphate-

[29] or sulphate- [150] limited conditions. The various

nutrient limitations applied induced differences (in some

Table 7. Effect of oxygen uptake rate (OUR) in the production and composition of cellular lipids byA. curvatum growing onwhey-permeate in

batch 20-L air-lift bioreactors

OUR (mmol/L/h) Xa) (g/L) Lipid (% w/w)

Fatty acid composition (% w/w)

C16:0 C18:0 C18:1 C18:2 C18:3 C20:0 C24:0

8–10 16.1 39.0 14 16 55 10 1 1 3

7.7 13.6 37.6 16 17 55 5 T 1 3

6.1 7.2 42.4 11 21 52 7 T 2 4

4.3 9.8 40.7 18 25 45 5 T 1 4

2.3 13.2 41.4 23 23 42 3 1 1 4

Initial lactose into the whey varying from 25 to 40 g/L (data from Davies et al. [37]).

T < 0.6% w/w.a) X is the dry cell weight produced (in g/L).

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cases remarkable) in the fatty acid composition of the SCO

produced. Therefore, with increasing phosphate limitation

imposed, lipids remarkably enriched in the fatty acid D9C18:1

and less rich in the fatty acid C18:0 were produced [29].

Thus, at initial C/P ratio 72 moles/moles, the cellular con-

centrations of D9C18:1 and C18:0 were 50 and 19% w/w,

while at initial C/P ratio 9552 moles/moles, the respective

values were 62 and 6% w/w. On the other hand, with increas-

ing sulphate limitation imposed, the concentration of TSFAs

remarkably increased, and thus a CBS was created; when the

initial C/S molar ratio imposed was 255 moles/moles, the

concentration of TSFAs was�43% w/w, while the respective

value of TSFAs was �63% w/w (a value very close to cocoa

butter) when the initial C/S molar ratio was 18 310 moles/

moles [150]. In all cases, the TSFAs of R. toruloides were

C16:0 and to lesser extent C18:0 [29, 150].

The last strategy related with the production of a CBS by

oleaginous microorganisms, was based on the yeasts capacity

to grow on fat substrates rich in C18:0, C16:0, and D9C18:1

or their fatty derivatives (e.g., FAMEs of the above-men-

tioned fatty acids) alone or on mixtures with sugars or polyols

[39, 40, 90, 105, 110, 113, 116]. The hydrophobic materials

used as substrates were pure fatty acids like stearic acid [110],

methyl-, ethyl-, butyl-, or vinyl-esters of stearic and palmitic

acid [113] or mixtures of stearin (a negligible-cost industrial

derivative of tallow composed of saturated free-fatty acids

and mainly of C16:0 and C18:0) and hydrolyzed oleic rape-

seed oil (composed mainly of D9C18:1) [39, 105, 107, 108].

The microorganisms Candida sp., C. tropicalis,

C. guillermondii, Torulopsis sp., T. versatilis, Trichosporon sp.

[113], R. toruloides [110, 116], and Y. lipolytica [39, 40, 90,

105, 107, 108] have been used for this purpose. Inmany cases

the microorganisms possessed active desaturase systems (i.e.,

D9 and D12 dehydrogenases) [110, 113]. Even if the initial

fatty substrates used was globally saturated (utilization of

pure stearic acid or mixtures of stearic and palmitic acid

esters as substrates), the cellular lipids contained D9C18:1

(22–44% w/w) and D9;12C18:2 (4–8% w/w) in significant

quantities (Table 8, entries A–F – Matsuo et al. [113];

Gierhart [110]). It is interesting to indicate that in this

strategy relatively large-scale stirred tank bioreactors (100-

L) were utilized [113].

The case of Y. lipolytica strain ACA-DC 50109 [former

registration LGAM S(7)1] was relatively more complicated.

The response of this microorganism was studied on stearin

and this fat permitted an efficient cell growth and significant

lipid accumulation inside the yeast cells (�55%w/w, of lipids

in dry matter, lipid up to�8.0 g/L – Papanikolaou et al. [105,

106]). The microorganism assimilated for growth and main-

tenance the lower aliphatic chain fatty acids (C12:0 and

C14:0) and accumulated as reserve lipid mainly the fatty

acid C18:0. TSFAs at almost at 100% w/w, principally

composed of C18:0 (i.e., average cellular fatty acid compo-

sition at C18:0 ¼ 80–83% w/w, C16:0 ¼ 14–16% w/w,

and traces of C12:0, C14:0, and D9C18:1) were stored, indi-

cating preferential accumulation of C18:0 as storage cellular

fatty acid and negligible D9 desaturation activity. Therefore,

the obtained result was rather unexpected considering

the predominance of unsaturated fatty acids into the

reserve lipids of the yeasts [17]. An oleic acid donor thus,

Table 8. Utilization of pure fatty acids, their derivatives (fatty esters) or low-cost industrial fats in order to produce microbial substitutes of

cocoa butter

A B C D E F G H I Cocoa butter

La) (g/L) 8.7 15.5 12.3 4.1 10.9 9.8 3.4 2.9 1.9

Lipid (% w/w) 35 61 51 35 29 34 42 36 46

C16:0 26 32 26 13 35 39 17 14 21 23–29

C18:0 26 29 37 49 17 19 63 50 68 32–37

C18:1 39 25 22 26 32 23 12 24 8 30–37

C18:2 5 8 8 4 6 6 4 4 2 2–4

TSFAsb) 52 61 63 62 52 58 80 64 89 55–67

A: Candida guillermondii on 14.0 g/L of methyl-stearate and 6.0 g/L of methyl-palmitate in 150-L bioreactor [113].

B: Trichosporon sp. on 14.0 g/L of methyl-stearate and 6.0 g/L of methyl-palmitate in 150-L bioreactor [113].

C: Torulopsis sp. on 13.0 g/L of vinyl-stearate and 7.0 g/L of vinyl-palmitate in 150-L [113].

D: R. toruloides on 40 g/L of pure stearic acid in shake flasks [110].

E: C. guilliermondii on 14.0 g/L of methyl-stearate and 6.0 g/L of methyl-palmitate in 150-L bioreactor [113].

F: Torulopsis versatilis on 14.0 g/L of methyl-stearate and 6.0 g/L of methyl-palmitate in 150-L bioreactor [113].

G: Y. lipolytica on 7.5 g/L of stearin (industrial tallow derivative composed of saturated free-fatty acids) and 2.5 g/L of hydrolyzed oleic

rapeseed oil (HORO) in shake flasks [39].

H: Y. lipolytica on 5 g/L of stearin and 5 g/L of HORO in shake flasks [39].

I: Y. lipolytica on 3.5 g/L of stearin and 1.5 g/L of HORO in 1.5-L bioreactor [108].a) L is the total cellular lipid produced (in g/L).b) TSFAs is the percentage of saturated fatty acids (w/w) in the lipid.

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was required, in order to produce a SCO-CBS byY. lipolytica,

and a widely available low-value fatty material (namely the

chemically hydrolyzed oleic rapeseed oil-HORO) was used as

substrate, and cell growth of Y. lipolytica cultivated on rich-

oleic acid media was significant but it was accompanied by a

very limited accumulation inside the yeast cells (SCO pro-

duced �5–7% w/w, in dry weight basis, with lipids produced

rich in D9C18:1) [39]. Therefore, the main problem to solve

in the case of Y. lipolytica was the adverse than that met in the

literature, namely how to decrease the amount of TSFAs,

increasing the unsaturated fatty acid content and simul-

taneously maintaining SCO accumulation in relatively high

amounts. Several shake-flask or batch-bioreactor exper-

iments were performed in various stearin-HORO mixtures

that in some cases resulted in relatively high lipid accumu-

lation whilst the accumulated lipid presented significant

C18:0 and some (non-negligible in various instances) unsa-

turated fatty acid percentage (Table 8, entries G–I –

Papanikolaou et al. [39]; Papanikolaou and Aggelis [108]),

since the microorganism used tended to rapidly incorporate

and oxidize for growth needs the unsaturated fatty acids

(principally D9C18:1 and D9;12C18:2) while it incorporated

with a lower rate but principally used as storage material the

fatty acid C18:0.

A similar strategy in order to produce CBSs, includes

growth of oleaginous yeasts on mixtures of sugars or polyols

with fatty materials [40, 90, 113, 116]. The presence of a fatty

material in some cases does not completely cease the process

of de novo fatty acid synthesis attributed from sugar or polyol

break-down, thus, a correct co-utilization of a fatty material

with a sugar (or a polyol) may result in the synthesis of lipids

with composition similarities with the cocoa butter. For

instance, Torupopsis sp. was cultivated on mixtures of

FAMEs of C16:0 C18:0 and glucose, and lipids with com-

position similar to that of cocoa butter were obtained

(Table 9, entry A – Matsuo et al. [113]). Moreover, two-

stage cultures of R. toruloides (first stage on glucose, second

stage on glucose or glycerol/pure stearic acid mixtures –

Gierhart [116]), or of single-stage cultures of Y. lipolytica

(growth biodiesel derived waste glycerol/stearin or glucose/

stearin mixtures – Papanikolaou et al. [40, 90, 105]) have

been assessed. For the case of R. toruloides, the sugars were

mainly used for growth needs while the fatty acids served

mainly to enhance the accumulation of storage lipid inside the

yeast cells, and for this reason, cultures presenting low con-

centrations of glucose or glycerol in the second stage showed

higher accumulation of storage lipid (Table 9, entries C and

E –Gierhart [116]). The bio-modification of pure stearic acid

Table 9. Utilization of mixtures of fatty materials (pure fatty acids, fatty esters, or low-cost fats) and hydrophilic substrates [glucose and/or

(biodiesel-derived waste) glycerol] in order to produce microbial substitutes of cocoa butter

A B C D E F G H I Cocoa butter

La) (g/L) n.r.b) 1.8 4.3 2.1 4.6 3.4 1.7 2.7 2.9

Lipid (%, w/w) n.r. 18 43 22 47 30 19 27 32

C16:0 21 15 10 13 14 14 17 16 16 23–29

C18:0 35 24 29 39 39 67 68 68 72 32–37

C18:1 36 42 44 32 37 10 11 7 5 30–37

C18:2 4 5 8 11 8 3 4 2 2 2–4

TSFAs 56 43 39 52 53 81 85 84 88 55–67

A: Torulopsis sp. on mixtures of methyl-palmitate, methyl-stearate and glucose [113].

B: Two-stage fermentation where R. toruloides was grown firstly on 50 g/L of glucose and then on 10 g/L of pure stearic acid and 10 g/L of

glucose in shake flasks [116].

C: Two-stage fermentation where R. toruloides was grown firstly on 50 g/L of glucose and then on 10 g/L of pure stearic acid and 2.0 g/L of

pure glycerol in shake flasks [116].

D: Two-stage fermentation where R. toruloides was grown firstly on 50 g/L of glucose and then on 10 g/L of pure stearic acid and 5.0 g/L of

glucose in shake flasks [116].

E: Two-stage fermentation where R. toruloides was grown firstly on 50 g/L of glucose and then on 10 g/L of pure stearic acid and 1.0 g/L of

glucose [116].

F: One-stage culture where Y. lipolytica was grown on 10 g/L of stearin and 34 g/L of glycerol in shake flasks (utilization of waste biodiesel

derived glycerol) [40].

G: One-stage culture where Y. lipolytica was grown on 11 g/L of stearin and 21 g/L of glucose in shake flasks (utilization of low-cost unpurified

glucose) [90].

H: One-stage culture where Y. lipolytica was grown on 10 g/L of stearin and 23 g/L of glycerol in shake flasks (utilization of waste biodiesel

derived glycerol) [40].

I: One-stage culture where Y. lipolytica was grown on 10 g/L of stearin and 10 g/L of glycerol in 1.5-L bioreactor (utilization of waste biodiesel

derived glycerol) [105].a) L is the total cellular lipid produced (in g/L).b) Not reported in the manuscript.

Eur. J. Lipid Sci. Technol. 2011, 113, 1052–1073 The biotechnology of oleaginous yeasts 1067

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found into the culture medium and the subsequent CBS

production was secured by the active D9 dehydrogenase

activity (Gierhart [110, 116]). Concerning Y. lipolytica,

growth on biodiesel derived glycerol or glucose (alone) in

nitrogen-limited flask cultures resulted in low lipid accumu-

lation, while cellular lipids were mainly composed of unsa-

turated fatty acids [68, 90]. Since growth on stearin alone

resulted in high SCOproduction with cellular lipid composed

mainly of the fatty acid C18:0 and given that some de novo

fatty acid accumulation occurs despite the presence of stearin

into the medium [40], waste glycerol (or glucose) were used

as unsaturated fatty acid precursors so as to produce SCO

with a remarkable content of TSFAs but also containing non-

negligible unsaturated fatty acids (Table 9, entries F–I –

Papanikolaou et al. [40, 90, 105]). Specifically the utilization

of biodiesel derived crude glycerol is of importance since even

though the fatty acid composition of the produced SCO

presented lower similarities with the cocoa butter compared

with the utilization of HORO as D9C18:1 fatty acid (compare

Tables 8 and 9), the current low cost of industrial glycerol

(ranging from 0 to 0.05US $ per kg) indicates the potentiality

of utilization of this co-substrate in the above-mentioned

strategy.

3 Concluding remarks – future perspectives

SCO fermentation attracts much interest the last years.

Amongst microbial lipids, yeast lipids present importance

in both academic and industrial point of view. Due to their

unicellular nature and their potentiality to grow on a plethora

of hydrophilic or hydrophobic substrates, the oleaginous

yeasts are considered as perfect ‘‘tools’’ for studying phenom-

ena of advanced lipid biochemistry and biotechnology [17,

22]. The increasing demand and utilization of 1st generation

biodiesel has notably increased the cost of various food-stuffs,

and this led to the necessity of discovery of non-conventional

sources of oils, that could be subsequently converted into

biodiesel. The oleaginous yeasts, which in most cases are

categorized as GRAS microorganisms and due to their fast

growth rates and their potentiality to grow on a plethora of

hydrophilic substrates, are considered as potential and inter-

esting candidates for the production of this 2nd generation

biodiesel.Moreover, the prospective remarkable increment in

the price of cocoa butter as well as the capability of various

oleaginous yeast strains to produce CBS-SCOs is also of

importance. Utilization of low- or even negative-cost

materials as substrates should be envisaged for both yeast

lipid applications previously mentioned. The removal of a

non-toxic and non-hazardous waste material from the food

industry, that will further be subjected to compost process

costs�0.1–0.3 US $ per kg of waste, therefore valorization of

these wastes with simultaneous production of cocoa butter-

like lipid or 2nd generation biodiesel with the aid of oleagi-

nous yeasts would have much to offer in both economical and

ecological points of view. As far as CBSs are concerned, waste

materials (like cheese–whey) can certainly be considered

again in the future as potential substrates for A. curvatum

strains. The same can be stressed in relation with the utiliz-

ation of stearin or other tallow derivatives (current cost of

tallow is around 0.3–0.4 US $ per kg) or even waste fats of

animal origin as substrates. However, in contrast with the use

of Torulopsis sp., R. toruloides, etc., employment of Y. lipolytica

as microorganism requires a donor of the fatty acid D9C18:1,

that should be cheaper than the rapeseed oil (crude glycerol

could be an option, but also other unsaturated fatty wastes

deriving from food production plants). Our research team is

willing to study in the future the biochemical and physiologi-

cal response of various Y. lipolytica strains (wild or genetically

engineered ones – e.g., strains lacking acyl-CoA oxidases) or

other oleaginous yeast strains (e.g., R. toruloides, L. starkeyi)

during their growth on various renewable substrates so as to

produce metabolites of added-value useful for the food and

chemical industry (SCO, citric acid, lipases, and exotic fats

substitutes).

Financial support concerning the results achieved by our research

team has been kindly provided by: Agricultural University of

Athens; University of Patras; Dracoil SA; Project BIOSIS

(INTERREG III GREECE – ITALY); State Scholarship

Foundation (Athens, Greece); General Secretary of Research and

Technology (Ministry of Development, Greek Government).

The authors have declared no conflict of interest.

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