Biodiesel production from microbial oil

8 Biodiesel production from microbial oil A.A. KOUTINAS and S. PAPANIKOLAOU, Agricultural University of Athens, Greece Abstract: Biodiesel and bioethanol constitute the main biofuels produced currently at industrial scale from renewable resources (mainly oilseeds, waste oils, starchy crops and sucrose-rich biomass). However, the limited availability of conventional raw materials and/or the direct competition with food production restricts the growth of first-generation biodiesel and bioethanol production. In the last few years, there is a growing interest in biodiesel production from microbial oil accumulated by oleaginous microorganisms cultivated on waste streams from the food industry and agricultural residues. This chapter focuses on the description of the potential of microbial oil production by yeast and fungi, the biochemistry of oil accumulation and the prospect of biodiesel production from microbial oil. Key words: biodiesel, microbial oil, biorefinery, oleaginous microorganisms, biomass.

8.1

Introduction

Bioethanol (mainly from sucrose and starchy crops) and biodiesel production (via transesterification of triglycerides) are the main first-generation biofuels that are currently produced on industrial scale. Biodiesel is produced by transesterification of triacylglycerols with short-chain alcohols (mainly methanol or ethanol) to produce monoalkyl esters, namely fatty acid methyl esters (FAMEs) and fatty acid ethyl esters (FAEEs). The worldwide production of biodiesel is mainly dependent on the utilization of waste oils, animal fats and oilseeds such as rapeseed, sunflower and soybeans. The recent food crisis has shown that research should focus on the development of second-generation biofuels generated from lignocellulosic raw materials and industrial waste streams (e.g. food industry wastes). In the past few years, research has focused on the development of biodiesel production from single cell oil (SCO) that can be produced via fermentation using various oleaginous microorganisms (i.e. microorganisms that are able to accumulate lipids intra-cellularly at more than 20% of the total cellular dry weight). The proposed strategy may provide a more eco-efficient and sustainable option as compared to first-generation biofuels and second-generation bioethanol production routes utilising lignocellulosic biomass. Potential advantages include: – The raw materials that will be used for the production of SCO-derived biodiesel do not compete with food production. In this way, cultivation of land 177 © Woodhead Publishing Limited, 2011

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for food production as well as industrial food processes could coincide with biodiesel production by utilizing residues and agro-industrial wastes. Microbial oil could be produced from various carbon sources (e.g. glucose, lactose, xylose, sucrose, glycerol) using natural microorganisms contrary to bioethanol production where natural microorganisms that are traditionally used in industrial processes utilize mainly glucose and sucrose. Bioethanol separation is an energy intensive technology with significant capital investment requirements, while separation of intra-cellularly accumulated SCO is likely to be achieved at significantly lower capital cost and energy requirements. Biodiesel production from oilseeds and waste oils will never provide adequate quantities of biodiesel to sustain the worldwide demand. In addition, the production cost of oilseeds is approximately 70–80% of the total biodiesel production cost. Biodiesel production from SCO will depend on the utilization of low-value waste streams or residues and therefore will offer a sustainable option for biofuel production. Transesterification of SCO results in the production of crude glycerine that could be used as a platform intermediate for the production of biofuels, chemicals and biodegradable plastics (Koutinas et al., 2007a; Aggelis, 2009).

8.2

Microorganisms and raw materials used for microbial oil production

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 SCO 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%

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(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 (Mlicˇková et al. 2004a, 2004b) or glycerol (Meesters et al., 1996; Papanikolaou and Aggelis 2002; Mantzouridou et al., 2008; André 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

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Cultivation mode

Carbon source

Total dry weight (g/L)

MO content (%, w/w)

Yeast species Yarrowia lipolytica single-stage Glucose 9.2 25 0.08 continuous Yarrowia single-stage Crude glycerol 8.1 43 0.11 lipolytica continuous Yarrowia shake flask Stearin 15.2 52 N.A. lipolytica Candida sp. 107 single-stage Glucose 18.1 37.1 0.4 continuous single-stage Glucose 13.5 29 0.16 continuous single-stage Sucrose 16 28 0.18 continuous Candida curvata single-stage Lactose 18 31 0.22 continuous single-stage Xylose 15 37 0.27 continuous single-stage Ethanol 11.5 35 0.2 continuous Apiotrichum curvatum batch Glucose 14.5 45.6 N.A. Apiotrichum curvatum batch Whey 21.6 36 0.119 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 Lipomyces starkeyi shake flask Glucose & Xylose 20.5 61.5 N.A. Lipomyces starkeyi shake flask Glucose & 9.4 68 N.A. sewage sludge

Microorganism

Productivity (g L–1 h–1)

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Meesters et al., 1996 Zhao et al., 2008 Angerbauer et al., 2008

Hassan et al., 1993 Ykema et al., 1988

Evans and Ratledge, 1983

Aggelis and Komaitis, 1999 Papanikolaou and Aggelis, 2002 Papanikolaou et al., 2007b Gill et al., 1977

Reference

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Table 8.1  SCO production from various microorganisms, carbon sources and cultivation modes

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Lipomyces starkeyi fed-batch Glucose 153 54 0.59 Glucose 24.1 56.6 N.A. Sucrose 19.5 62.6 N.A. Xylose 17.1 57.8 N.A. Trichosporon shake flask Lactose 16.9 49.6 N.A. fermentans Fructose 21.5 40.7 N.A. Molasses 36.4 35.3 N.A. Trichosporon shake flask Rice straw 28.6 40.1 N.A. fermentans hydrolysate Mannose 22.7 50.4 N.A. Galactose 23.6 59 N.A. Cellobiose 15.8 65.6 N.A. Rhodosporidium fed-batch Glucose 106.5 67.5 0.54 toruloides Rhodotorula gracilis continuous Glucose 9.60 49.8 0.096 Rhodotorula shake flask Monosodium 25 20 N.A. glutinis glutamate wastewater Rhodotorula glutinis fed-batch Glucose 185 40 0.88     Fungal species Cunninghamella shake flask Glucose 15 46 N.A. echinulata Cunninghamella shake flask Starch 13.5 28 N.A. echinulata Pectin 4.1 10 N.A. Mortierella isabellina shake flask Glucose 27 44.6 N.A. Mortierella shake flask Starch 10.4 36 N.A. isabellina Pectin 8.4 24 N.A. Mucor sp. RRL001 shake flask Tapioca starch 28 17.8 N.A. Mortierella commercial-scale Glucose 62 46.1 N.A. ramanniana batch bioreactor

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Ahmed et al., 2006 Hiruta et al., 1997

Fakas et al., 2009a Papanikolaou et al., 2007a

Papanikolaou et al., 2007a

Fakas et al., 2009a

Pan et al., 1986

Choi et al., 1982 Xue et al., 2008

Li et al., 2007

Huang et al., 2009

Zhu et al., 2008

Yamauchi et al., 1983

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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

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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 SCOderived 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 CO 2 emitted per kilometre travelled.

8.3

The biochemistry of lipid accumulation in the oleaginous microorganisms

8.3.1 General remarks When various sugars or similarly metabolized compounds (e.g. glycerol, polysaccharides, etc.) are utilized for the production of SCO, accumulation of lipid in the microbial cells or mycelia (the so-called ‘de novo’ lipid accumulation process) is triggered by exhaustion of nitrogen from the growth medium, which allows the conversion of sugar to storage lipid (Ratledge, 1988, 1994; Ratledge and Wynn, 2002; Wynn and Ratledge, 2006; Papanikolaou and Aggelis, 2009;

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Fakas et al., 2009b). In contrast, when growth is conducted on hydrophobic carbon sources (e.g. fats, oils), accumulation of storage lipids (the so-called ‘ex novo’ lipid accumulation process) is a primary anabolic process occurring simultaneously with the production of lipid-free material, being independent from the nitrogen exhaustion in the medium (Fickers et al., 2005; Papanikolaou and Aggelis, 2010). In the case of SCO utilization for biodiesel production, research interest is focused only upon the process of de novo lipid accumulation. In this case, there is continuously increasing interest upon the potentiality of transforming abundant renewable materials (like waste glycerol, flour-rich waste streams, cellulose and hemicellulose hydrolysates, etc.) into SCO that will be further transformed into biodiesel. The process of ex novo lipid accumulation aims at adding value to low-cost fatty materials so that speciality high-value lipids (e.g. cocoa-butter or other exotic fats substitutes) will be produced (Papanikolaou et al., 2001; 2003; Papanikolaou and Aggelis 2003a, 2003b, 2010). The lipids produced by oleaginous microorganisms are mainly composed of neutral fractions [principally triacylglycerols (TAGs) and to lesser extent sterylesters (SEs)] (Ratledge, 1994; Ratledge and Wynn, 2002). As a general remark it must be stressed that when growth is carried out on various hydrophobic substances, the microbial lipid produced contains lower quantities of accumulated TAGs compared with growth elaborated on sugar-based substrates (Koritala et al., 1987; Guo et al., 1999; Kinoshita and Ota, 2001; Papanikolaou et al., 2001, 2002a; Fakas et al. 2006, 2007, 2008a). In any case, accumulation of storage lipids is accompanied by morphological changes in the oleaginous microorganisms, since ‘obese’ cells with large lipid globules can generally appear during the lipid-accumulating phase (Figure 8.1). Storage lipids, unable to integrate into

8.1  ‘Obese’ cells of the yeast Yarrowia lipolytica with large lipid globules appeared during lipid-accumulating growth phase. Magnification ×100 (Makri et al., 2010).

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8.2  Lipid bodies in the yeast Yarrowia lipolytica as shown by electron microscopy (Mlicˇ ková et al., 2004a).

phospholipid bi-layers, cluster to form the hydrophobic core of the so-called ‘lipid bodies’ or ‘oil bodies’ (Mlicˇková et al., 2004a, 2004b). Lipid bodies of the oleaginous Y. lipolytica yeast are illustrated in Figure 8.2. As previously stressed, the biochemical pathways of de novo and ex novo lipid accumulation process present fundamental differences. These differences will be presented, explained, clarified and comprehensively discussed in the following sections.

8.3.2 Lipid accumulation from fermentation of sugars and related substrates used as the sole carbon source De novo accumulation of cellular lipids is an anabolic biochemical process in which, by virtue of quasi-inverted β-oxidation reaction series, acetyl-CoA issued by the intermediate cellular metabolism, generates the synthesis of intra-cellular fatty acids. Fatty acids will be then esterified in order to synthesize structural (phospholipids, sphingolipids, etc.) and reserve lipids (TAGs and SEs) (Moreton, 1988; Ratledge, 1988, 1994; Davies and Holdsworth, 1992; Ratledge and Wynn, 2002; Papanikolaou and Aggelis, 2009). In oleaginous microorganisms in which de novo lipid accumulation is conducted, acetyl-CoA that constitutes the precursor of intra-cellular fatty acids, derives from breakdown of citric acid that under some circumstances cannot be catabolized through the reactions performed in the Krebs cycle, but it is accumulated inside the mitochondria. This occurs when its concentration becomes higher than a critical value resulting in citric acid transportation into the cytosol (Ratledge, 1988, 1994; Ratledge and Wynn, 2002; Wynn and Ratledge, 2006; Fakas et al., 2009b). The key-step for citric acid accumulation inside the mitochondrion matrix is the change of intra-cellular concentration of various metabolites, conducted after exhaustion of some nutrients (mainly nitrogen) in the culture medium (Ratledge, 1988, 1994; Ratledge and Wynn, 2002; Wynn and Ratledge, 2006). This exhaustion provokes a rapid decrease of the concentration of intra-cellular AMP, since, by virtue of AMPdesaminase, the microorganism cleaves AMP into IMP and NH 4+ ions in order to utilize nitrogen, in the form of NH 4+ ions, as a complementary nitrogen source, necessary for synthesis of cell material (Evans and Ratledge, 1985).

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The excessive decrease of intra-cellular AMP concentration alters the Krebs cycle function; the activity of both NAD + and NADP +-isocitrate dehydrogenases, enzymes responsible for the transformation of iso-citric to α-ketoglutaric acid, lose their activity, since they are allosterically activated by intra-cellular AMP, and this event results in the accumulation of citric acid inside the mitochondrion (studies performed in the oleaginous microorganisms Candida sp. 107, Rhodosporidium toruloides, Y. lipolytica, Mortierella isabellina, Mortierella alpina, Mucor circinelloides and Cunningamella echinulata) (Botham and Ratledge, 1979; Evans and Ratledge, 1985; Wynn et al., 2001; Finogenova et al., 2002; Papanikolaou et al., 2004b). When the concentration of citric acid becomes higher than a critical value, it is secreted into the cytosol. Finally, in the case of lipogenous (lipidaccumulating) microorganisms, cytosolic citric acid is cleaved by ATP-citrate lyase (ACL), the key-enzyme of lipid accumulation process in the oil-bearing microorganisms, in acetyl-CoA and oxaloacetate, with acetyl-CoA being converted, by an inversion of β -oxydation process, to cellular fatty acids. In contrast, nonoleaginous microorganisms (e.g. various Y. lipolytica and Aspergillus niger strains) secrete the accumulated citric acid into the culture medium (Ratledge, 1994; Anastassiadis et al., 2002; Papanikolaou et al., 2002b) instead of accumulating significant quantities of reserve lipid. In general, production of citric acid by citrate-producing strains is a process carried out when extra- and hence intracellular nitrogen is depleted [overflow metabolism phenomenon (see Anastassiadis et al., 2002)], while studies of the intra-cellular enzyme activities and co-enzyme concentrations have somehow identified and clarified the biochemical events leading to citric acid biosynthesis (Finogenova et al., 2002; Morgunov et al., 2004; Makri et al., 2010) and indeed it has been demonstrated that citric acid secretion and SCO accumulation are processes indeed identical into their first steps. In a third category of microorganisms, the accumulated (inside the cytosol) citric acid provokes inhibition of the enzyme 6-phospho-fructokinase, and the above fact results in the intra-cellular accumulation of polysaccharides based on the 6-phospho-glucose (Evans and Ratledge, 1985). Schematically, the intermediate cellular metabolism resulting in the synthesis of either citric acid or storage lipid is presented in Figure 8.3 (Ratledge, 1994; Ratledge and Wynn, 2002; Papanikolaou and Aggelis, 2009). After the biosynthesis of intra-cellular fatty-CoA esters, an esterification with glycerol takes place in order for the reserve lipids to be stocked in the form of TAGs (Ratledge, 1988, 1994). This synthesis in the oleaginous microorganisms is conducted by virtue of the so-called pathway of α-glycerol phosphate acylation (Ratledge, 1988; Davies and Holdsworth, 1992; Athenstaedt and Daum, 1999; Müllner and Daum, 2004; Fakas et al., 2009b). In this metabolic pathway, free fatty acids are activated by coenzyme A and are subsequently used for the acylation of the glycerol backbone to synthesize TAGs. In the first step of TAGs assembly, glycerol-3-phosphate (G-3-P) is acylated by G-3-P acyltranferase (GAT) at the sn-1 position to yield 1-acyl-G-3-P (lysophospatidic acid-LPA), which is then

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8.3  Pathways involved in the breakdown of glucose by microbial strains capable of producing SCO and/or citric acid in nitrogen-limited conditions. FFA: free-fatty acids; TRSP: citric acid transporting system; a, b, c: systems transporting pyruvic acid from cytosol to mitochondrion and inversely; d: system transporting citric and malic acid from cytosol to mitochondrion and inversely; ACL: ATP-citrate lyase; FAS: fatty acid synthetase; ICDH: iso-citrate dehydrogenase; MD c: malate dehydrogenase (cytoplasmic); MD m: malate dehydrogenase (mitochondrial); ME: NADPH +-malic enzyme; PD: pyruvate dehydrogenase; CS: citrate synthase; ICL: iso-citrate lyase; EMP: Embden-Mayerhoff-Parnas pathway. Pathways described by Ratledge (1994), Ratledge and Wynn (2002), Papanikolaou and Aggelis (2009).

further acylated by lysophosphatidic acid acyltransferase (also named 1-acyl-G3-P acyltransferase-AGAT) in the sn-2 position to yield phosphatidic acid (PA). This is followed by dephosphorylation of PA by phosphatidic acid phosphohydrolase (PAP) to release diacylglycerol (DAG). In the final step DAG is acylated either by diacylglycerol acyltransferase or phospholipid diacylglycerol acyltransferase to produce TAGs (Ratledge, 1988; Davies and Holdsworth, 1992; Athenstaedt and Daum, 1999; Müllner and Daum, 2004; Fakas et al., 2009b). As far as the structure of the microbial TAGs produced is concerned, although their final composition could theoretically be a random substitution of acyl-CoA

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groups into glycerol, in the case of the oleaginous microorganisms that have been examined, the glycerol sn-2 position is almost always occupied by unsaturated fatty acids [production of vegetable-type TAGs (see Ratledge, 1988; 1994; Guo and Ota, 2000)]. Therefore, various oleaginous microorganisms (principally yeasts belonging to the species Rhodosporidium toruloides, Apiotrichum curvatum and Y. lipolytica) have long been considered as promising candidates for the production of equivalents of exotic fats (fats that are principally saturated but containing unsaturated fatty acids esterified in the sn-2 glycerol position) (Moreton 1985, 1988; Moreton and Clode 1985; Ykema et al., 1989, 1990; Davies et al., 1990; Lipp and Anklam, 1998; Papanikolaou et al., 2001, 2003; Papanikolaou and Aggelis 2003b; Papanikolaou and Aggelis, 2010).

8.3.3 Lipid production from fermentation of hydrophobic materials used as the sole carbon source It is known that when microorganisms are cultivated on fat-type substrates (e.g. long-chain free-fatty acids, TAGs, fatty-esters, etc.) production of (intracellular, cell-bounded or extra-cellular) lipases is performed as a physiological response to the presence of fatty materials into the growth medium (Fickers et al., 2005). This secretion is obligatory in the case that TAGs or fatty-esters are used as substrates (Fickers et al., 2005; Papanikolaou and Aggelis, 2010). In contrast, a large variety of microorganisms are capable of utilizing soaps as well as freefatty acids as sole carbon and energy source, regardless of the lipolytic capacities of the microorganisms used in order to break down fatty materials (Ratledge and Boulton, 1985; Papanikolaou and Aggelis, 2010). Specifically, for the case of the yeast Y. lipolytica, its culture on TAG-type substrates is accompanied by secretion of an extra-cellular lipase called Lip2p, encoded by the LIP2 gene (Pignède et al., 2000). This gene encoded for the biosynthesis of a precursor premature protein with Lys-Arg cleavage site. The secreted lipase was reported to be a 301-aminoacid glycosylated polypeptide which belongs to the TAGs hydrolase family (EC 3.1.1.3) (Pignède et al., 2000; Fickers et al., 2005). The Lip2p precursor protein was processed by the KEX2-like endoprotease encoded by the gene XPR6, whereas deletion of the above gene resulted in the secretion of an active but fewer stable pro-enzyme (Pignède et al., 2000). Simultaneously, other intra-cellular lipases (Lip7p, Lip8p) may also be produced and secreted into the culture medium, that present different fatty acid specificities, with maximum activity being displayed against ∆918:1 (oleic acid), 6:0 (capronic) and 10:0 (caprinic) fatty acids (Fickers et al., 2005). The free-fatty acids (existed as initial substrate or produced after lipase hydrolysis of the TAGs/fatty-esters) will be incorporated, with the aid of active transport, inside the microbial cell. It is interesting to state that for the case of Y. lipolytica yeast, the various individual substrate fatty acids would be removed from the medium (and hence incorporated inside the microbial cell) with different

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rates (Papanikolaou et al. 2001, 2002a; Papanikolaou and Aggelis, 2003b). Specifically, regardless of the initial concentrations of the extra-cellular fatty acids, the incorporation rate of the lower aliphatic chain (lauric acid-12:0 and myristic acid-14:0) or unsaturated (∆918:1 and linoleic acid-∆9,1218:2) fatty acids is significantly higher than that of principally stearic (18:0) and to lesser extent palmitic (16:0) acid (Papanikolaou et al., 2001; Papanikolaou and Aggelis, 2003b). Moreover, the incorporated fatty acids will be either dissimilated for growth needs or become a substrate for endo-cellular bio-transformations (synthesis of ‘new’ fatty acid profiles which did not exist previously in the substrate) (Ratledge and Boulton, 1985; Koritala et al., 1987; Aggelis and Sourdis, 1997; Guo et al., 1999; Kinoshita and Ota, 2001; Papanikolaou et al., 2001, 2002a, 2007b; Papanikolaou and Aggelis, 2003a, 2003b, 2010). The intra-cellular dissimilation of the various catabolized fatty acids is performed by reactions catalyzed by the various intra-cellular acyl-CoA oxidases (Aox). A significant amount of experimental work has been performed in relation with the elucidation of the above-mentioned reactions by using strains of the nonconventional yeast Y. lipolytica (Fickers et al., 2005). In fact, it has been revealed that the aforementioned biochemical process is a multi-step reaction requiring different enzymatic activities of five acyl-CoA oxidase isozymes (Aox1p through Aox5p), encoded by the POX1 through POX5 genes (Luo et al. 2002; Mlicˇková et al. 2004a, 2004b; Fickers et al. 2005). Aox3p is specific for short chain acyl-CoAs, Aox2p preferentially oxidizes long-chain acyl-CoAs while Aox1p, Aox4p and Aox5p do not appear to be sensitive in the chain length of the aliphatic acyl-CoA chain (Mauersberger et al. 2001; Luo et al. 2002; Fickers et al. 2005). It should also be noticed that genetically modified strains of Y. lipolytica namely JMY 798 (MTLY 36-2P) and JMY 794 (MTLY 40-2P) have been created from the wild-type W29 strain (Mlicˇková et al. 2004a, 2004b). These strains were subjected to disruptions of the genes implicated in the encoding of various intracellular Aox. The genetically engineered strains, hence, either under-expressed or did not at all express several of the enzymes implicated in the catabolism (β-oxidation) of aliphatic chains. When cultures were performed on oleic acid utilized as the sole substrate, although the genetically engineered strains showed almost equivalent microbial growth compared with the wild strain (W29) from which they derived, in contrast with W29 strain they presented significantly higher formation of lipid bodies and, hence, increased lipid accumulation (Mlicˇková et al. 2004a, 2004b). Therefore, the above-mentioned studies as well as various others reported in the literature (Aggelis and Sourdis, 1997; Papanikolaou et al., 2003; Szcze˛sna-Antczak et al., 2006; Mantzouridou and Tsimidou, 2007) indicate that external addition of fat (ex novo lipid accumulation) can significantly enhance the bio-process of SCO production in various oleaginous microorganisms, but external utilization of fat mainly serves for the ‘improvement’ and ‘upgrade’ of a fatty material utilized as substrate [e.g. valorization of low-cost or waste fats so as to produce specialty lipids like cocoa-butter substitutes or substitutes of other

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high-added value lipids like illipé butter, shea butter, sal fat (Papanikolaou and Aggelis, 2010)] and not for the use of the SCO produced in the manufacture of bio-diesel.

8.4

Biodiesel production from single cell oil

The conversion rate of TAGs to FAMEs, changes in the composition of biodiesel during transesterification and analysis of biodiesel characteristics are the main aspects that are investigated in most studies about biodiesel production from vegetable oils (Darnoko and Cheryan, 2000; Dorado et al., 2004; Vicente et al., 2005; Arzamendi et al., 2006). The above parameters are related to the FAME concentration resulting during transesterification and characterize biodiesel yield or purity (Vicente et al., 2007). Contrary to biodiesel production from vegetable oils, there are limited publications investigating the optimum conditions (e.g. reaction duration, reaction temperature, agitation, type and amount of catalyst, ratio of alcohol to SCO) for biodiesel production from SCO. SCO derived from various yeast and fungi should be thoroughly compared with vegetable oils in order to justify the possibility to substitute for the current raw materials used for biodiesel production. SCO-derived biodiesel should be characterized according to biodiesel standards ASTMD 6751 (USA), DIN 51606 (Germany) and EN 14214 (European Organization). Preliminary results indicate that SCO could be regarded as a potential raw material for biodiesel production. Li et al. (2007) claimed that the fatty acid distribution of the SCO produced during fed-batch fermentations by Rhodosporidium toruloides could be converted into biodiesel with a cetane number (CN) higher than 51, which meet the minimal CN standards (47, 49 and 51) set by ASTMD 6751, DIN 51606 and EN 14214. Zhu et al. (2008) reported that the SCO produced by T. fermentans contained an unsaturated fatty acid content of 64% which is similar to that of vegetable oils but a relatively high acid value of 5.6 mg KOH/g. After pretreatment of SCO, transesterification via methanolysis resulted in a methyl ester yield of 92% (Zhu et al., 2008). Transesterification of SCO could be carried out either directly without extraction of SCO from the microbial biomass or indirectly after extraction of SCO from microbial cells. Extraction of SCO from cellular biomass components by solvent extraction or other means will increase production cost and capital investment. It is therefore evident that future research should investigate more thoroughly the direct transesterification of SCO without extraction from microbial cells. The processing steps of direct transesterification involves separation of cellular biomass by centrifugation after the end of fermentation, washing the cells with water, drying the cells to constant weight and finally mix the dry cells with a mineral acid solution (HCL or H2SO 4) and methanol (Liu and Zhao, 2007). Liu and Zhao (2007) reported that direct acid-catalyzed transesterification of SCOrich microbial biomass from two yeast (Lipomyces. starkeyi and R. toruloides) and one fungal strain (M. isabellina) resulted in FAMEs with CN of 59.9, 63.5

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and 56.4 respectively and lipid to FAME yields higher than 90% (w/w). The optimum reaction conditions applied by Liu and Zhao (2007) were 0.2 mol/L H2SO 4 at 70 °C for 20 h with a biomass-to-methanol ratio of 1:20 (w/v). Vicente et al. (2009) compared the efficiency of direct transesterification with indirect transesterification (lipid extraction was carried out by 3 solvent systems including chloroform:methanol, chloroform:methanol:water and n-hexane) for biodiesel production from SCO produced by the fungal strain Mucor circinelloides. The direct transesterification method produced FAMEs with higher purities (>99%) than those from the indirect process (91.4–98.0%) and a significantly higher yield due to a more efficient lipid extraction when the acid catalyst was present (Vicente et al., 2009). The reaction conditions applied by Vicente et al. (2009) were 8% (w/w relatively to the microbial oil) BF 3, H2SO 4 or HCl for 8 h at 65 °C with a methanol to oil molar ratio of 60:1.

8.5

Future trends

Future research incentives on the development of biorefineries should focus on all aspects of the process regarding upstream processing (i.e. evaluation of various renewable raw materials and conversion strategies), bioconversion for SCO production, downstream conversion of SCO into biodiesel and generation of co-products through valorization of crude glycerol or other side/waste streams. Furthermore, it is essential to evaluate the economic viability and sustainability of industrial-scale SCO-based biodiesel production. In the 1980s, Davies (1992) reported the most thorough economic analysis for SCO production ($0.8 – 1 per kg MO) from waste lactose (200 000 m3 whey per year) utilizing the yeast strain Candida curvata. Based on this cost and using an order-of-magnitude approximation, the SCO production cost in 2008 would have been $1.4 and 1.8/kg (this value does not include the biodiesel production cost from SCO) in the case that whey is used as carbon source. If we consider that biodiesel production from SCO is still at an early research stage, then the above economic considerations demonstrate that SCO production deserves more thorough research and development. For instance, maximization of SCO production would only be achieved by optimizing fed-batch fermentations due to the nature of SCO biosynthesis. In fed-batch bioconversions, a nutrientcomplete feedstock could be provided in the first stage to achieve high microbial growth, while in the second stage we could provide a nitrogen limited medium that contains a high amount of a carbon source in order to promote SCO accumulation. Li et al. (2007) achieved 106.5 g/L total dry weight, 67.5% (w/w) SCO content and 0.54 g/(L.h) SCO productivity during fed-batch fermentations in a 15 L bioreactor. Meesters et al. (1996) employed a fed-batch fermentation process using glycerol as carbon source to produce high cell densities of 118 g/L with a lipid production rate of 0.59 g/(L.h) and a cellular lipid content of 25% (w/w). Higher total dry weights (185 g/L and 153 g/L) with reasonably high

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lipid contents (40% and 54%, w/w) have been achieved in other studies employing fed-batch fermentations with the yeast strains Rhodotorula glutinis and L. starkeyi, respectively (Yamauchi et al., 1983; Pan et al., 1986). It should be stressed that a relatively low number of publications have been published in the literature regarding SCO production via fed-batch fermentations. Therefore, research should focus on the optimization of fed-batch bioconversions for SCO production. Furthermore, the application of genetic engineering and metabolic engineering to oleaginous microorganisms will lead to enhanced SCO production. Previous studies on SCO production focus on the utilization of commercial nutrient supplements (e.g. yeast extract, inorganic chemicals) to formulate the fermentation medium. Li et al. (2007) reported that the utilization of commercial sources of glucose, inorganic salts and protein supplements result in a higher biodiesel production cost as compared to the cost of biodiesel production from vegetable oils. Future research should focus on the utilization of agro-industrial residues and wastes for the formulation of the required media for SCO fermentation. It was previously stressed that the addition of protein hydrolysates could enhance SCO accumulation by certain microorganisms. Future research should also focus on the complete characterization of SCO as raw material for biodiesel production, including analysis of free-fatty acid composition, water content, acidity, peroxide value, density and kinematic viscosity. The efficiency of transesterification and the performance of biodiesel in diesel engines are strongly dependent on these properties. For instance, in the case that SCO or any other renewable source of oil has a high FFA content, the use of homogeneous acid catalysts rather than alkaline catalysts is recommended, because the utilization of alkaline catalysts will hinder separation and purification of the final product due to excessive soap formation. In addition, the selection of the most appropriate microorganism will be dependent on the quality of the SCO required for biodiesel production.

8.6

References

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