**5. Cultivation condition of oleaginous yeasts**

Lipid accumulation by oleaginous yeasts depends mostly on nutrient limitation conditions when excess carbon is present in the medium. Nutrient limitation prevents cells from being generated, while the carbon excess is converted into storage TAG. Published studies reports that phosphorus, magnesium, zinc, or iron limitation lead to lipid accumulation in model oleaginous yeasts (Hall & Ratledge, 1977; Beopoulos et al., 2009; Wu et al., 2010). However, nitrogen limitation is the most efficient form of nutrient limitation for lipogenesis induction, leading to the highest values of substrate/lipid conversion yield and lipid content within biomass (Hall & Ratledge, 1977; Wynn et al., 2001). Thus, nitrogen limitation is commonly used to induce lipogenesis in oleaginous fungi and the utilization of cultural media with appropriate C/N ratio is crucial to maximize lipid production.

Several studies focused on determining the optimal composition of cultural media for oleaginous fungi with the aim to optimize the performance of lipid-producing bioprocesses. The effect of the C/N ratio on lipid metabolism has been investigated for a number of oleaginous yeasts and molds, such as *Y. lipolytica* and many oleaginous species of *Rhodotorula*, *Candida*, *Apiotrichum/Cryptococcus*, *Mortierella* (Hall & Ratledge, 1977; Papanikolau et al., 2003; Granger et al., 1992; Wu et al., 2010; Park et al., 1990; Jang et al., 2005; Amaretti et al., 2010), and has been mathematically modeled for some of these

has been used mostly as a model to investigate and understand the lipid metabolism, in *Y. lipolytica* several attempts have been done in order to address the carbon flux toward TAG production and accumulation. Similar approaches are precluded to other oleaginous fungi since they lack genome information and the necessary tools for gene manipulation and

In *Y. lipolytica*, the role of glycerol-3-phosphate (G3P) in triacylglycerol (TAG) biosynthesis and accumulation has been investigated (Beopoulos et al., 2008). In this yeast G3P is formed from glycerol by the glycerol kinase encoded by *GUT1*, or it is synthetized from dihydroxyacetone phosphate (DHAP) by the G3P dehydrogenase (*GPD1*). The antagonist reaction, which produces DHAP from G3P, is carried out in competition by a second isoform of the G3P dehydrogenase, encoded by *GUT2*. In order to force the conversion of DHAP into G3P, the gene *GPD1* was over-expressed and the gene *GUT2*

A diverse strategy to increase lipid accumulation was based on the disruption of the βoxidative metabolism, through the deletion of the 6 *POX* genes (*POX1 to POX6*) that encode the peroxisomal acyl-coenzyme oxidases (Mlickowa et al., 2004a; Mlickowa et al., 2004b; Beopoulos et al., 2008). As a whole, the best results in terms of percentage of lipids per dry biomass, were reached coupling the increased level of G3P with the disactivation of the β-

Metabolic engineering strategies have been recently exploited to expand the range of substrates used by oleaginous fungi, also through functional expression of heterologous genes. Recently, it has been found that inulin is a good material for bio-productions (Chi et al., 2009). In order to make the oleaginous yeast *Y. lipolytica* able to accumulate lipids on inulin containing materials, the *Kluyveromyces marxianus* exo-inulinase gene (*INU1*) was heterologously expressed on a high copy plasmid (Zhao et al., 2010). The inulinase was efficiently secreted by *Y. lipolytica*, and inulin was hydrolyzed, assimilated and converted

Lipid accumulation by oleaginous yeasts depends mostly on nutrient limitation conditions when excess carbon is present in the medium. Nutrient limitation prevents cells from being generated, while the carbon excess is converted into storage TAG. Published studies reports that phosphorus, magnesium, zinc, or iron limitation lead to lipid accumulation in model oleaginous yeasts (Hall & Ratledge, 1977; Beopoulos et al., 2009; Wu et al., 2010). However, nitrogen limitation is the most efficient form of nutrient limitation for lipogenesis induction, leading to the highest values of substrate/lipid conversion yield and lipid content within biomass (Hall & Ratledge, 1977; Wynn et al., 2001). Thus, nitrogen limitation is commonly used to induce lipogenesis in oleaginous fungi and the utilization of cultural media with

Several studies focused on determining the optimal composition of cultural media for oleaginous fungi with the aim to optimize the performance of lipid-producing bioprocesses. The effect of the C/N ratio on lipid metabolism has been investigated for a number of oleaginous yeasts and molds, such as *Y. lipolytica* and many oleaginous species of *Rhodotorula*, *Candida*, *Apiotrichum/Cryptococcus*, *Mortierella* (Hall & Ratledge, 1977; Papanikolau et al., 2003; Granger et al., 1992; Wu et al., 2010; Park et al., 1990; Jang et al., 2005; Amaretti et al., 2010), and has been mathematically modeled for some of these

strain improvement.

was deleted.

into TAG.

oxidation pathway (65%) (Dulermo et al., 2011).

**5. Cultivation condition of oleaginous yeasts** 

appropriate C/N ratio is crucial to maximize lipid production.

organisms (Ykema et al., 1986; Granger et al., 1993; Economou et al., 2011). *Y. lipolytica* is the oleaginous microorganism for which information about the metabolic response to different C/N ratios is most abundant (Beopoulos et al., 2009a), particularly due to the availability of molecular tools for genetic engineering of this organism. Therefore, *Y. lipolytica* is regarded as a model organism for microbial oil production and the main traits of its metabolism can be used to give a general description of the metabolic response to different C/N ratios in the majority of oleaginous yeasts. With the increase of the C/N ratio, different metabolic behaviors were observed in *Y. lipolytica*: i) growth with mobilization of storage lipids, ii) growth of fat-free biomass, iii) growth with accumulation of lipids, and iv) growth with lipid accumulation and production of organic acids (Fig. 6).

Fig. 6. Metabolic activity of oleaginous fungi (e.g. *Y. lipolitica*) as a function of carbon flow rate for a fixed nitrogen flow rate. Arrows indicate the consumption of nitrogen and carbon sources by the cells; squares indicate production rate. The dimension of arrows and squares is proportional to flow. (adapted from Beopoulos et al., 2009a)

If the medium is carbon limited or when the extracellular carbon supply gets exhausted, previously stored intracellular lipid can be mobilized and utilized by oleaginous microorganisms to sustain cells generation and production of lipid-free biomass (Park et al., 1990) (Fig. 6, i). If the medium is balanced and/or furnishes just the right amount of carbon flow to satisfy the growth need, balanced growth occurs without any accumulation of storage lipids (Fig. 6, ii). In conditions of carbon excess, a part of the carbon flow, which is proportional to nitrogen availability (Granger et al., 1993), is directed toward cells generation, whereas the carbon exceeding growth needs is channeled to the production of storage lipids (Fig. 6, iii). In some oleaginous fungi, the presence of a large carbon excess leads to the production of great amounts of organic acids, such as pyruvic acid and diverse TCA-cycle intermediates, at the expenses of lipid accumulation (Fig. 6, iv). In these latter conditions, *Y. lipolytica* produces citric acid (Levinson et al., 2007) but other oleaginous yeasts have never been reported to behave this way.

Getting Lipids for Biodiesel Production from Oleaginous Fungi 83

mol) and q is the nitrogen/carbon content of biomass (N-mol/C-mol) (Ykema et al., 1986). However, it should be considered that extremely high C/N ratios may cause the production of unwanted byproducts, such as organic acids (Fig. 6 iv), or may lead to severe nitrogen

Unlike batch processes, in fed-batch mode, nutrients are fed into the bioreactor in a controlled manner, with the purpose to monitor and control the specific growth rate and the flows of nitrogen and carbon utilization. Through the judicious management of the feeding rate and composition, it is possible to control the C/N ratio within the culture and maintain the oleaginous microorganism in the optimal metabolic status, as appropriate, first for the growth phase, and later for the lipogenic phase. The lipogenic phase is the most extensive, corresponding to lipid production under nitrogen limitation, with constant C/N ratio,

In continuous cultures, at the steady state, the assimilation of C and N sources and the microbial growth occur at constant rates, which ultimately depend by the dilution rate (D). The concentration of the substrates within the bioreactor is steady and depends by the dilution rate as well, the actual C/N ratio of the culture remaining constant unlike in batch cultures. Likewise in batch cultures, in continuous cultures the C/N ratio of the fresh medium needs to be higher than (YX/S· q)-1 to obtain some lipid accumulation (Ykema et al., 1986). However, at the steady-state with this medium, the C/N ratio within the culture is higher than in the fresh medium, due to nitrogen consumption. The extent of substrates utilization, and also the biomass and lipid concentrations are the highest at low D values and decrease with the increase of D (Fig. 7). While low D values promote lipid production and a more complete substrate utilization, on the contrary, the volumetric productivity of continuous processes is positively affected by the increase of the dilution rate (Ykema et al., 1986; Meeuwse et al., 2011b). Therefore, both the C/N content of the medium and the dilution rate need to be thoroughly tuned to maximize lipid productivity of continuous

The demand for the inexpensive production of biofuels has intensified due to increasing concerns of climate change, depletion of petroleum-based fuels, and environmental problems. In a market economy, corporations aim to maximize profit, seeking the most competitive feedstock. To produce single-cell oils for biodiesel production, the carbon source has necessarily to be cheap and available in large quantities. Therefore, while the first investigations on oleaginous fungi most commonly employed glucose as carbon source, nowadays the production of single-cell oils is predominantly addressed to transformation of

Glucose is the carbon source most commonly employed for growth of oleaginous fungi and lipid production (Boulton & Ratledge, 1984; Hansson & Dostalek, 1986; Hassan et al., 1993; Heredia & Ratledge, 1988; Jacob, 1991; Jacob, 1992; Johnson et al., 1992; Li et al., 2007; Pan et al., 1986; Ratledge, 2004; Rau et al., 2005; Saxena et al., 2008; Zhao et al., 2008). High glucose concentrations enhance the carbon flow that is directed toward TAG production, thus improving lipid production in several yeasts. However, growth of some yeasts (e.g. *R. toruloides)* is inhibited by high concentration of glucose, (Li et al., 2007). Furthermore, in batch cultures, initial glucose concentration also affects the fatty acids composition of the

preventing loss of viability and acids production (Beopoulos et al., 2009a).

deficiency, causing a rapid decrease in cells viability.

processes.

**7. Substrates and raw material** 

raw materials, by-products and surplus.

lipids (Amaretti et al., 2010).

#### **6. Batch, fed-batch and fermentation processes**

Batch, fed batch, and continuous modes of culture have been developed to culture oleaginous microorganisms. Lipid production in batch cultures is carried out in a cultural medium with a high initial C/N ratio, the carbon source being present in an adequate excess with respect to the nitrogen source. In fact, in this condition, the flow of carbon utilization is limited only by the substrate uptake system of the cell, while the changes in nitrogen concentration determine the passage from a phase of balanced growth to a phase of lipid accumulation, causing the process to proceed through two phases. As nitrogen is consumed from the culture the C/N ratio tends to increase, but growth remains exponential and balanced until nitrogen is not the limiting substrate. During the growth phase, the carbon flow is mostly channeled to satisfy the growth need, therefore growth is balanced and lipidfree biomass is mostly produced (Fig. 6 ii). As nitrogen concentration becomes limiting, the growth rate and the carbon flow toward biomass generation decrease, while lipid production is triggered, resulting in a shift of microbial metabolism into the lipogenic phase (Fig. 6 iii, Fig. 7).

Fig. 7. Modeling and prediction of the timecourse of a batch fermentation (left) and the steady-state values of a continuous process (right) for microbial production of lipids. Axis are in arbitrary scales.

In batch cultures the initial C/N ratio of the cultural medium has a pivotal role in determining the bioprocess performance. In fact, both the rate and the yield of lipid production depend by the C/N ratio, which affects the duration of the exponential phase and the amount of biomass produced during growth. With a fixed carbon concentration, higher amounts of lipid-free biomass produced during the growth phase correspond to higher lipid production rates during the lipogenic phase, but to lower amounts of lipid content within cells and lipid/substrate conversion yields. Therefore, the initial C/N ratio needs to be optimized to maximize lipid productivity in batch cultures. The optimal C/N value is always high (e.g. in the range between 80 and 350 mol/mol) and strongly depends on the microorganism, the medium composition, the carbon source (e.g. glucose, glycerol, etc.), and the nitrogen source (e.g. diverse organic or inorganic sources). The minimal C/N ratio suitable for lipid accumulation can be estimated as (YX/S· q)-1, where YX/S is the biomass/carbon source yield coefficient under conditions of carbon limitations (C-mol/C-

Batch, fed batch, and continuous modes of culture have been developed to culture oleaginous microorganisms. Lipid production in batch cultures is carried out in a cultural medium with a high initial C/N ratio, the carbon source being present in an adequate excess with respect to the nitrogen source. In fact, in this condition, the flow of carbon utilization is limited only by the substrate uptake system of the cell, while the changes in nitrogen concentration determine the passage from a phase of balanced growth to a phase of lipid accumulation, causing the process to proceed through two phases. As nitrogen is consumed from the culture the C/N ratio tends to increase, but growth remains exponential and balanced until nitrogen is not the limiting substrate. During the growth phase, the carbon flow is mostly channeled to satisfy the growth need, therefore growth is balanced and lipidfree biomass is mostly produced (Fig. 6 ii). As nitrogen concentration becomes limiting, the growth rate and the carbon flow toward biomass generation decrease, while lipid production is triggered, resulting in a shift of microbial metabolism into the lipogenic phase

Fig. 7. Modeling and prediction of the timecourse of a batch fermentation (left) and the steady-state values of a continuous process (right) for microbial production of lipids. Axis

In batch cultures the initial C/N ratio of the cultural medium has a pivotal role in determining the bioprocess performance. In fact, both the rate and the yield of lipid production depend by the C/N ratio, which affects the duration of the exponential phase and the amount of biomass produced during growth. With a fixed carbon concentration, higher amounts of lipid-free biomass produced during the growth phase correspond to higher lipid production rates during the lipogenic phase, but to lower amounts of lipid content within cells and lipid/substrate conversion yields. Therefore, the initial C/N ratio needs to be optimized to maximize lipid productivity in batch cultures. The optimal C/N value is always high (e.g. in the range between 80 and 350 mol/mol) and strongly depends on the microorganism, the medium composition, the carbon source (e.g. glucose, glycerol, etc.), and the nitrogen source (e.g. diverse organic or inorganic sources). The minimal C/N ratio suitable for lipid accumulation can be estimated as (YX/S· q)-1, where YX/S is the biomass/carbon source yield coefficient under conditions of carbon limitations (C-mol/C-

**6. Batch, fed-batch and fermentation processes** 

(Fig. 6 iii, Fig. 7).

are in arbitrary scales.

mol) and q is the nitrogen/carbon content of biomass (N-mol/C-mol) (Ykema et al., 1986). However, it should be considered that extremely high C/N ratios may cause the production of unwanted byproducts, such as organic acids (Fig. 6 iv), or may lead to severe nitrogen deficiency, causing a rapid decrease in cells viability.

Unlike batch processes, in fed-batch mode, nutrients are fed into the bioreactor in a controlled manner, with the purpose to monitor and control the specific growth rate and the flows of nitrogen and carbon utilization. Through the judicious management of the feeding rate and composition, it is possible to control the C/N ratio within the culture and maintain the oleaginous microorganism in the optimal metabolic status, as appropriate, first for the growth phase, and later for the lipogenic phase. The lipogenic phase is the most extensive, corresponding to lipid production under nitrogen limitation, with constant C/N ratio, preventing loss of viability and acids production (Beopoulos et al., 2009a).

In continuous cultures, at the steady state, the assimilation of C and N sources and the microbial growth occur at constant rates, which ultimately depend by the dilution rate (D). The concentration of the substrates within the bioreactor is steady and depends by the dilution rate as well, the actual C/N ratio of the culture remaining constant unlike in batch cultures. Likewise in batch cultures, in continuous cultures the C/N ratio of the fresh medium needs to be higher than (YX/S· q)-1 to obtain some lipid accumulation (Ykema et al., 1986). However, at the steady-state with this medium, the C/N ratio within the culture is higher than in the fresh medium, due to nitrogen consumption. The extent of substrates utilization, and also the biomass and lipid concentrations are the highest at low D values and decrease with the increase of D (Fig. 7). While low D values promote lipid production and a more complete substrate utilization, on the contrary, the volumetric productivity of continuous processes is positively affected by the increase of the dilution rate (Ykema et al., 1986; Meeuwse et al., 2011b). Therefore, both the C/N content of the medium and the dilution rate need to be thoroughly tuned to maximize lipid productivity of continuous processes.

#### **7. Substrates and raw material**

The demand for the inexpensive production of biofuels has intensified due to increasing concerns of climate change, depletion of petroleum-based fuels, and environmental problems. In a market economy, corporations aim to maximize profit, seeking the most competitive feedstock. To produce single-cell oils for biodiesel production, the carbon source has necessarily to be cheap and available in large quantities. Therefore, while the first investigations on oleaginous fungi most commonly employed glucose as carbon source, nowadays the production of single-cell oils is predominantly addressed to transformation of raw materials, by-products and surplus.

Glucose is the carbon source most commonly employed for growth of oleaginous fungi and lipid production (Boulton & Ratledge, 1984; Hansson & Dostalek, 1986; Hassan et al., 1993; Heredia & Ratledge, 1988; Jacob, 1991; Jacob, 1992; Johnson et al., 1992; Li et al., 2007; Pan et al., 1986; Ratledge, 2004; Rau et al., 2005; Saxena et al., 2008; Zhao et al., 2008). High glucose concentrations enhance the carbon flow that is directed toward TAG production, thus improving lipid production in several yeasts. However, growth of some yeasts (e.g. *R. toruloides)* is inhibited by high concentration of glucose, (Li et al., 2007). Furthermore, in batch cultures, initial glucose concentration also affects the fatty acids composition of the lipids (Amaretti et al., 2010).

Getting Lipids for Biodiesel Production from Oleaginous Fungi 85

by-products such as raw glycerol, producing large amounts of SCO and organic acids (Papanikolaou et al., 2003; Papanikolaou & Aggelis, 2002; Rymowicz et al., 2010; Rywinska et al., 2009). Biochemistry of lipid production on glycerol has been investigated in this organism: glycerol passes into the microbial cell by facilitated diffusion and the conversion is carried out via phosphorylation pathway, with direct phosphorylation to G3P and subsequent dehydrogenation. Recently, *Y. lipolytica* has been subjected to targeted and purposeful alteration of G3P shuttle pathway to better utilize glycerol for lipid production. In the genetically manipulated strains, lipid accumulation resulted from a complex interrelation between different processes in diverse cell compartments, such as lipid synthesis in the cytosol, location and storage in ER and LB, mobilization and degradation

Pure glycerol supported growth and lipid accumulation of *Rhodotorula glutinis* and *Candida freyschussii* (Easterling et al., 2009; Amaretti et al., 2011), being used as sole carbon and energy source or in addition to xylose or glucose. The diverse composition of the medium affected not only the lipid/biomass yield, but also the TAG composition, in terms of ration of saturated, monounsaturated, and polyunsaturated fatty acids (Easterling et al., 2009). Attempts to convert crude glycerol into lipids have been successfully performed exploiting the oleaginous yeast *Cryptococcus curvatus* (Liang et al., 2010)*.* Different processes have been developed with very efficient yields and productivities. In a 12 days two-stage fed-batch where raw glycerol was fed, the biomass density and the lipid content reached 32.9 g/l and 52%, respectively. Methanol of crude glycerol did not pose a significant inhibitory effect even though it was existent in the bioreactor. Lipid accumulated by *C. curvatus* on glycerol presented high amount of monounsaturated fatty acid, turning out as excellent substrate for

Oleaginous fungi, and particularly yeasts, are very efficient in the accumulation of intracellular TAG and it is expected that they will be exploited by the biofuel industry in the future. Nonetheless, the costs of microbial lipids are still too high in order to compete with plant oils for biodiesel manufacturing. Cheap carbon sources have necessarily to be used as carbon sources for the cultivation of these microorganisms and the performance of the bioprocess has to be further improved in terms of both the yield and the productivity. The exploration of the natural biodiversity is a promising strategy to identify novel oleaginous species that assimilate and get fat on agro-industrial residues, particularly the lingo-cellulosic biomass and crude glycerol from biodiesel industry. Further approaches combining genomic, transcriptomic, metabolomics, and lipidomic techniques will undoubtedly provide deeper information of lipid production by oleaginous fungi. A metabolic engineering approach is very promising, but it is still precluded for the most oleaginous species, for which genome disclosure has not been

Adachi, D., Hama, S., Numata, T., Nakashima, K., Ogino, C., Fukuda, H., & Kondo, A.,

*Technology*. Vol. 102, No. 12, pp. 6723-6729, ISSN 0960-8524.

(2011) Development of an *Aspergillus oryzae* whole-cell biocatalyst coexpressing triglyceride and partial glyceride lipases for biodiesel production. *Bioresource* 

processes (Dulermo & Nicaud, 2011).

transformation into biodiesel.

**9. References** 

**8. Conclusions and perspectives** 

accomplished and genetic tools are not available yet.

Carbon sources other than glucose, such as xylose (Chistopher et al., 1983; Heredia & Ratledge, 1988;), lactose (Christopher et al., 1983; Daniel et al., 1999;), arabinose, mannose (Hansson & Dostalek, 1986), mannitol (Hansson & Dostalek, 1986), ethanol (Chistopher et al., 1983; Eroshin & Krylova, 1983), have been also investigated in the 80s and 90s for the production of microbial lipids.

Albeit glucose is a very good carbon source for lipid production with oleaginous fungi, molasses, which carbohydrate fraction is mainly composed of sucrose, glucose, and fructose, do not represent a promising raw material for lipid production, since they are characterized by a high nitrogen content which delays the unbalanced growth, where number of cells can not augment anymore and lipids are accumulated (Johnson et al., 1995).

Carbons sources obtained from ligno-cellulosic biomasses represent one of the most important potential to produce biodiesel. In fact, several waste biomasses containing forest residues, agricultural residues, food wastes, municipal wastes, and animal wastes can be utilized for the production of lignocellulosic based microbial lipids. Microbial oil production from sulphuric acid treated rice straw hydrolysate (SARSH) by the yeast *Trichosporon fermentans* pointed out the difficulty to perform the process of lipid accumulation in presence of the inhibitory compounds released during hydrolysis, such as acetic acid, furfural, 5-hydroxymethylfurfural, and water soluble lignin (Huang et al., 2009). Selected strains were able to grow on xylose and glucose (Zhu et al., 2008), but the crude hydrolyzate did not result an optimal substrate for a high yield process of lipid production. Cellulose and hemicellulose are generally hardly hydrolyzed and assimilated by yeasts, while they can be degraded and used as carbon source by filamentous fungi. A screening of endophytic fungi from the oleaginous plants was the selection of strains belonging to the genera *Microsphaeropsis, Phomopsis, Cephalosporium, Sclerocystis* and *Nigrospora* that simultaneously accumulated lipids (21.3 to 35.0% of dry weight) and produced cellulase (Peng & Chen, 2007). Albeit these strains could be exploited as microbial oil producers by utilising straw as substrate, they have never been claimed again as a SCO producers on lingo-cellulosic biomass. Attempts to carry out lipid production in Solid State Fermentation (SSF) on wheat straw have been performed exploiting a cellulolytic strain of *Aspergillus oryzae* (Lin et al., 2010). This strain is able to use cellulose as substrate and accumulate lipids in a low cost fermentation system on this abundant cellulosic by-product.

Other complex matrices have been used, such as solids from wheat bran fermentation (Jacob 1991), sewage sludge (Angerbauer et al., 2008), wastewaters of animal fat treatment (Papanikolaou et al., 2002), whey derivatives (Ykema et al., 1989; Vamvakaki et al., 2010), olive oil mill wastewaters (Yousuf et al., 2010), and tomato waste hydrolysate (Fakas et al., 2008).

Nowadays, lipid production with oleaginous yeasts is focused on selection and development of yeasts as converters of glycerol into lipid for biodiesel production, since it is the major side-product of the biodiesel production process. The biotransformation of glycerol into TAG is therefore regarded as a promising way to decrease the cost of biodiesel process through simultaneous reutilization of its major byproduct. In general, for every 100 kg of biodiesel produced, approximately 10 kg of crude glycerol are created. Crude glycerol is a mixture of glycerol (65–85%, w/w), methanol, and soap, and contains macro elements such as calcium, potassium, magnesium, sulfur and sodium. In order to minimize unknown variables introduced through the use of crude glycerol, several studies to determine whether or not glycerol could be used as substrate or co-substrate for growth have been conducted using purified glycerol.

A deep characterization of lipid accumulation on glycerol has been carried out with *Yarrowia lipolytica*, that is able to metabolize several important industrial and agro-industrial

Carbon sources other than glucose, such as xylose (Chistopher et al., 1983; Heredia & Ratledge, 1988;), lactose (Christopher et al., 1983; Daniel et al., 1999;), arabinose, mannose (Hansson & Dostalek, 1986), mannitol (Hansson & Dostalek, 1986), ethanol (Chistopher et al., 1983; Eroshin & Krylova, 1983), have been also investigated in the 80s and 90s for the

Albeit glucose is a very good carbon source for lipid production with oleaginous fungi, molasses, which carbohydrate fraction is mainly composed of sucrose, glucose, and fructose, do not represent a promising raw material for lipid production, since they are characterized by a high nitrogen content which delays the unbalanced growth, where number of cells can

Carbons sources obtained from ligno-cellulosic biomasses represent one of the most important potential to produce biodiesel. In fact, several waste biomasses containing forest residues, agricultural residues, food wastes, municipal wastes, and animal wastes can be utilized for the production of lignocellulosic based microbial lipids. Microbial oil production from sulphuric acid treated rice straw hydrolysate (SARSH) by the yeast *Trichosporon fermentans* pointed out the difficulty to perform the process of lipid accumulation in presence of the inhibitory compounds released during hydrolysis, such as acetic acid, furfural, 5-hydroxymethylfurfural, and water soluble lignin (Huang et al., 2009). Selected strains were able to grow on xylose and glucose (Zhu et al., 2008), but the crude hydrolyzate did not result an optimal substrate for a high yield process of lipid production. Cellulose and hemicellulose are generally hardly hydrolyzed and assimilated by yeasts, while they can be degraded and used as carbon source by filamentous fungi. A screening of endophytic fungi from the oleaginous plants was the selection of strains belonging to the genera *Microsphaeropsis, Phomopsis, Cephalosporium, Sclerocystis* and *Nigrospora* that simultaneously accumulated lipids (21.3 to 35.0% of dry weight) and produced cellulase (Peng & Chen, 2007). Albeit these strains could be exploited as microbial oil producers by utilising straw as substrate, they have never been claimed again as a SCO producers on lingo-cellulosic biomass. Attempts to carry out lipid production in Solid State Fermentation (SSF) on wheat straw have been performed exploiting a cellulolytic strain of *Aspergillus oryzae* (Lin et al., 2010). This strain is able to use cellulose as substrate and accumulate lipids in a low cost

Other complex matrices have been used, such as solids from wheat bran fermentation (Jacob 1991), sewage sludge (Angerbauer et al., 2008), wastewaters of animal fat treatment (Papanikolaou et al., 2002), whey derivatives (Ykema et al., 1989; Vamvakaki et al., 2010), olive oil mill wastewaters (Yousuf et al., 2010), and tomato waste hydrolysate (Fakas et al., 2008). Nowadays, lipid production with oleaginous yeasts is focused on selection and development of yeasts as converters of glycerol into lipid for biodiesel production, since it is the major side-product of the biodiesel production process. The biotransformation of glycerol into TAG is therefore regarded as a promising way to decrease the cost of biodiesel process through simultaneous reutilization of its major byproduct. In general, for every 100 kg of biodiesel produced, approximately 10 kg of crude glycerol are created. Crude glycerol is a mixture of glycerol (65–85%, w/w), methanol, and soap, and contains macro elements such as calcium, potassium, magnesium, sulfur and sodium. In order to minimize unknown variables introduced through the use of crude glycerol, several studies to determine whether or not glycerol could be used as substrate or co-substrate for growth have been

A deep characterization of lipid accumulation on glycerol has been carried out with *Yarrowia lipolytica*, that is able to metabolize several important industrial and agro-industrial

not augment anymore and lipids are accumulated (Johnson et al., 1995).

fermentation system on this abundant cellulosic by-product.

conducted using purified glycerol.

production of microbial lipids.

by-products such as raw glycerol, producing large amounts of SCO and organic acids (Papanikolaou et al., 2003; Papanikolaou & Aggelis, 2002; Rymowicz et al., 2010; Rywinska et al., 2009). Biochemistry of lipid production on glycerol has been investigated in this organism: glycerol passes into the microbial cell by facilitated diffusion and the conversion is carried out via phosphorylation pathway, with direct phosphorylation to G3P and subsequent dehydrogenation. Recently, *Y. lipolytica* has been subjected to targeted and purposeful alteration of G3P shuttle pathway to better utilize glycerol for lipid production. In the genetically manipulated strains, lipid accumulation resulted from a complex interrelation between different processes in diverse cell compartments, such as lipid synthesis in the cytosol, location and storage in ER and LB, mobilization and degradation processes (Dulermo & Nicaud, 2011).

Pure glycerol supported growth and lipid accumulation of *Rhodotorula glutinis* and *Candida freyschussii* (Easterling et al., 2009; Amaretti et al., 2011), being used as sole carbon and energy source or in addition to xylose or glucose. The diverse composition of the medium affected not only the lipid/biomass yield, but also the TAG composition, in terms of ration of saturated, monounsaturated, and polyunsaturated fatty acids (Easterling et al., 2009).

Attempts to convert crude glycerol into lipids have been successfully performed exploiting the oleaginous yeast *Cryptococcus curvatus* (Liang et al., 2010)*.* Different processes have been developed with very efficient yields and productivities. In a 12 days two-stage fed-batch where raw glycerol was fed, the biomass density and the lipid content reached 32.9 g/l and 52%, respectively. Methanol of crude glycerol did not pose a significant inhibitory effect even though it was existent in the bioreactor. Lipid accumulated by *C. curvatus* on glycerol presented high amount of monounsaturated fatty acid, turning out as excellent substrate for transformation into biodiesel.
