There are many microalgae, yeasts (e. g. Candida, Cryptococcus, Lipomyces, Rhodotorula, Rhodosporidium, Trichosporon), fungi (e. g. Mortierella, Cunninghamella) and bacteria that can accumulate intra-cellularly high amounts of SCO that has fatty acid composition similar to vegetable oils (Meng et al., 2009). Microorganisms can be characterized as oleaginous in the case that they can accumulate SCO to more than 20% of their total cellular dry weight (Ratledge, 1991). SCO could be used either for value-added applications (e. g. food additives) or commodity uses (e. g. biodiesel production). The industrial application of SCO for biodiesel production is dependent on the development of a fermentation process that provides high carbon source to SCO conversion yields, high productivities, high lipid content in cellular biomass and high S CO concentrations. The previous criteria constitute a useful tool so as to select the appropriate microorganisms that will facilitate the industrial implementation of biodiesel production from SCO. For instance, microalgae may accumulate high amounts of microbial lipids but they cannot compete with oleaginous yeast and fungi because their cultivation requires a big area and long fermentation duration. Furthermore, bacteria may achieve high growth rates but the majority of bacterial strains accumulate relatively low amounts of SCO (up to 40% of total cellular dry weight) (Meng et al., 2009). Some yeast strains (e. g. Rhodosporidium sp., Rhodotorula sp., Lipomyces sp.) may accumulate intra-cellularly around 70% (w/w) of SCO (Guerzoni et al, 1985; Li et al, 2007; Angerbauer et al., 2008; Meng et al., 2009).

Table 8.1 shows that mainly yeasts and some fungi may offer appropriate cell factories for the production of SCO. Table 8.1 clearly demonstrates that cell densities up to 185 g/L with a lipid content up to 67.5% (w/w) have been achieved mainly in fed-batch cultures or continuous fermentations with recycling (Yamauchi et al., 1983; Pan et al., 1986; Ykema et al., 1988; Meesters et al., 1996; Li et al., 2007). In many cases, SCO has similar fatty acid composition as in the case of vegetable oils used for biodiesel production. SCO is mainly composed of triacylglycerols — TAGs — with a fatty acid composition rich in C16 and C18, namely palmitic (16:0), palmitoleic (16:1), stearic (18:0), oleic (18:1) and linoleic (18:2) acids (Meesters et al., 1996; Ratledge and Wynn, 2002; Li et al., 2007; Meng et al., 2009). The SCO produced by C. curvatus has similar composition to palm oil (Davies, 1988). The SCO produced by Yarrowia lipolytica contains stearic, oleic, linoleic and palmitic acid (Papanikolaou et al., 2002a).

There is a remarkable plethora of (pure or raw agro-industrial) substrates that can be used by oleaginous microorganisms for microbial growth and accumulation of microbial lipids (Table 8.1). Production of SCO implicates utilization of pure sugars as substrates (e. g. analytical glucose, lactose, etc.) (Moreton, 1985; Moreton and Clode, 1985; Aggelis et al., 1996; Papanikolaou et al., 2004a, 2004b; Li et al., 2007; Zhao et al., 2008; Fakas et al., 2009a), sugar-based renewable materials or sugar-enriched wastes (Ykema et al., 1989, 1990; Davies et al., 1990; Papanikolaou et al., 2007a; Fakas et al., 2006, 2007, 2008a, 2008b, 2009a), vegetable oils (Bati et al., 1984; Koritala et al., 1987; Aggelis and Sourdis, 1997), crude-waste industrial hydrophobic materials (e. g. industrial free-fatty acids, waste fats, crude fish oils, soap-stocks etc) (Guo et al., 1999; Guo and Ota, 2000; Papanikolaou et al., 2001, 2002a, 2007b; Papanikolaou and Aggelis, 2003a, 2003b), pure fatty acids (Mlickova et al. 2004a, 2004b) or glycerol (Meesters et al., 1996; Papanikolaou and Aggelis 2002; Mantzouridou et al., 2008; Andre et al., 2009; Makri et al., 2010). This indicates that it is feasible to utilize various natural resources for the production of SCO providing the opportunity to develop processes producing SCO-derived biodiesel either integrated in existing food industries or as individual production plants (e. g. in agricultural areas so as to utilize various lignocellulosic feedstocks).

Starch-based waste or by-product streams (e. g. wheat flour milling by-products, waste bread, flour-based waste or by-product streams from the confectionary industry) generated by the food industry or collected as disposed food by dedicated companies could be used for the production of glucose-based fermentation media. Wheat flour milling by-products has been considered for the production of biofuels and platform chemicals (Neves et al., 2007; Dorado et al., 2009) and therefore could be regarded as a potential feedstock for the production of SCO-derived biodiesel. In the case of SCO production, certain oleaginous microorganisms have the ability to consume both glucose and xylose. This

Table 8.1 SCO production from various microorganisms, carbon sources and cultivation modes Microorganism Cultivation Carbon Total dry MO content Productivity Reference mode source weight (g/L) (%, w/w) (g L-1 h-1) Yeast species Yarrowia lipolytica single-stage Glucose 9.2 25 0.08 Aggelis and continuous Komaitis, 1999 Yarrowia single-stage Crude glycerol 8.1 43 0.11 Papanikolaou and lipolytica continuous Aggelis, 2002 Yarrowia shake flask Stearin 15.2 52 N. A. Papanikolaou lipolytica etai, 2007 b Candida sp. 107 single-stage continuous Glucose 18.1 37.1 0.4 Gill etai, 1977 single-stage continuous Glucose 13.5 29 0.16 single-stage continuous Sucrose 16 28 0.18 Candida curvata single-stage Lactose 18 31 0.22 Evans and continuous single-stage continuous Xylose 15 37 0.27 Ratledge, 1983 single-stage continuous Ethanol 11.5 35 0.2 Apiotrichum curvatum batch Glucose 14.5 45.6 N. A. Hassan etai., 1993 Apiotrichum curvatum batch Whey 21.6 36 0.119 Ykema et ai, 1988 recycling 85 35 0.372 continuous 20 36 0.382 partial recycling 91.4 33 0.995 Cryptococcus curvatus fed-batch Glycerol 118 25 0.59 Meesters etai., 1996 Lipomyces starkeyi shake flask Glucose & Xylose 20.5 61.5 N. A. Zhao et ai, 2008 Lipomyces starkeyi shake flask Glucose & 9.4 68 N. A. Angerbauer

153

24.1

19.5

17.1 16.9

21.5 36.4

28.6

22.7 23.6

15.8 106.5

9.60

25 185

15

13.5

4.1

27

10.4

8.4

27

62

indicates that it will be feasible to consume the major carbon sources in wheat flour milling by-products (i. e. glucose from starch and xylose from hemicelluloses). Waste bread and other starch-based food could be collected prior to disposal by dedicated companies and could be used for the production of SCO derived biodiesel. Waste bread has been evaluated for the production of bioethanol (Ebrahimi et al., 2008). Furthermore, waste or by-product streams from the confectionary industry that contain mainly starch and sucrose as carbon sources could be considered as potential feedstocks for SCO production.

Other waste streams from the food industry that could be used for the production of SCO-derived biodiesel are whey and molasses. Whey constitutes a significant waste stream from the dairy industry and its valorization is an important environmental target. The yeast strain Cryptococcus curvatus can accumulate intra-cellularly a SCO content of around 60% (w/w) of the total cell dry weight during fermentation on whey or other agricultural and food processing wastes (Ratledge 1991; Meesters et al., 1996). In addition, molasses (a by-product from sugar refining) has been used as fermentation medium in shake flask cultures for the production of SCO by the yeast Trichosporon fermentans to produce 36.4 g/L total dry weight with an SCO content of 35.3% (w/w) (Zhu et al., 2008).

As indicated in Table 8.1, certain oleaginous microorganisms can utilize glycerol for the production of SCO (Meesters et al., 1996; Papanikolaou and Aggelis, 2002). Therefore, crude glycerol generated from biodiesel production plants could be recycled for the production of SCO-derived biodiesel. More importantly, the ability of some oleaginous microorganisms to consume various sugars derived from lignocellulosic biomass (e. g. xylose, mannose, galactose, cellobiose) could lead to the utilisation of lignocellulosic biomass for the production of SCO-derived biodiesel (Zhu et al., 2008; Huang et al., 2009).

Biorefineries should depend entirely on crude biological entities for the formulation of fermentation media that will contain all the necessary nutrients for microbial growth and SCO accumulation. In order to implement this principle, protein-rich industrial waste streams should be used for the production of fermentation media enriched in organic sources of nitrogen (e. g. amino acids, peptides), phosphorus, minerals, vitamins and trace elements. Such nutrient supplements for fermentation processes could be produced from oilseed residues generated after oil extraction in the first-generation biodiesel production plants (e. g. protein-rich rapeseed or sunflower cakes), meat-and-bone meal, sewage sludge, protamylase (residual stream enriched in amino acids and peptides that is generated during the industrial production of starch from potatoes), corn steep liquor and residual yeast from potable or fuel ethanol production plants. Protein and other nutrients are also contained together with carbon sources in various food waste streams (e. g. waste bread, whey). Therefore, in many cases, a single waste stream from the food industry could be sufficient for the production of nutrient-complete fermentation media for SCO production. It should be stressed that organic N-sources may enhance lipid accumulation (even two or three times higher than the amount of lipids accumulated with inorganic N-sources) in certain oleaginous microorganisms (e. g. Rhodosporidium toruloides, Trichosporon cutaneum and T. fermentans) (Evans and Ratledge, 1984a, 1984b; Zhu et al., 2008).

The conversion of waste streams into fermentation media would require the development of advanced upstream processing strategies that exploit the full potential of complex biological entities. Similar upstream processing schemes have been developed in the case of cereal conversion into bioethanol, biodegradable plastics and platform chemicals (Arifeen et al., 2007; Koutinas et al., 2007b; Du et al., 2008; Xu et al., 2010). In addition, pre-treatment technologies that have been developed for the generation of fermentation feedstocks for bioethanol production could be adapted in the case of SCO-derived biodiesel production (Lloyd and Wyman, 2005; Zhu et al., 2009).

Based on the maximum theoretical conversion yields of glucose to SCO (0.33 g/g) and bioethanol (0.51 g/g) and the lower heating values (LHVs) for SCO — derived biodiesel (37.5 MJ/kg) and bioethanol (26.7 MJ/kg), then the LHV per kg glucose that could be generated via fermentative production of SCO and bioethanol is 9% higher in the case of ethanol. However, the overall energy balance (output/ input) could be favourable in the case of SCO-derived biodiesel because it is expected that the energy required to produce biodiesel after SCO fermentation would be lower than the energy required to purify bioethanol from fermentation broths. This will also result in surplus lignin that will be used for chemical production when lignocellulosic biomass is used as raw material. In the case of bioethanol production, all lignin is required for energy generation for the plant. In addition, biodiesel production from SCO would create a sustainable supply of glycerol that is regarded as an important building block for the chemical industry. For instance, we could combine biodiesel production from SCO with biodegradable polymer (e. g. polyhydroxyalkanoates) and platform chemical (e. g. 1,3-propanediol, succinic acid, itaconic acid) production from crude glycerol generated during biodiesel production (Jarry and Seraudie, 1997; Papanikolaou et al., 2000; Lee et al., 2001). We should also highlight the well-understood efficiency of diesel engines which lead to a lower level of CO2 emitted per kilometre travelled.