Traditionally, yeasts have been used in the food and beverage industry, so the major­ity of yeasts have been adapted to meet these procedures. The ability to accumu­late lipids above 20 % of its weight is achieved by only 5 % of the known yeasts (Beopoulos et al. 2011). Lipid accumulation in oleaginous yeast occurs under excess of carbon sources, being scarce the nitrogen source, so the carbon excess is channeled into triglycerides (Ageitos et al. 2011). Similar to other microorganisms, yeast is able to consume different sources of carbon and nitrogen, from waste to laboratory-pure sources. However, to take advantage of this technology, the use of widely available waste is a key parameter. According to this, the main by-products of the rapeseed oil-based biodiesel industry, glycerol (carbon source) and rape — seed meal (nitrogen source), were used as culture medium for the oleaginous yeast Rhodosporidium toruloides Y4 and the accumulation of oil was analyzed. Results showed that the accumulation of oil reached up to 19.7 g/L, higher than 16.2 g/L achieved when a medium composed of glycerol and yeast extract as nitrogen source was used. Besides, the oil fatty acid composition comprised a high content of mon­ounsaturated fatty acids, which makes it suitable for biodiesel production (Uckun Kiran et al. 2013). Many authors have proposed the use of glycerol as carbon source to grow different oleaginous yeasts, i. e., Cryptococcus curvatus (Liang et al. 2010), Rhodotorula glutinis (Saenge et al. 2011), Rhodotorula graminis (Galafassi et al. 2012), and R. toruloides (Xu et al. 2012). In all cases, it was considered a suitable carbon source for lipogenesis. Also, the hydrolyzate from lignocellulosic materials has been considered an interesting substrate due to the availability and economic feasibility (Yu et al. 2011; Gong et al. 2012; Uckun Kiran et al. 2012).

The culture conditions, such as C/N ratio (close to 100), substrate, culture mode, microelements, and inorganic salts, are crucial in lipid accumulation (Ageitos et al. 2011). While the ratio C/N plays the most important role in lipid accumulation, the culture mode is also of special interest. For this reason, Zhao et al. (2011) used dif­ferent feeding strategies with yeast R toruloides Y4 and concluded that the fed-batch strategy exhibited the largest oil accumulation potential under large-scale production plant, while keeping the residual glucose concentration to 5 g/L of carbon source and the fed-batch cycles were multiple times repeated. Authors removed the majority of the mature culture at the end of each cycle, keeping 900 ml of the culture in the bioreactor. Then, fresh media were added and a new cultivation cycle was initiated. As a result, the highest amount of lipids reported in the literature, 78.7 g/L, was achieved (Table 7).

The main disadvantage of oleaginous yeast is the extraction of the oil, due to the resistance of the cell walls to different solvents. In most cases, a chloroform methanol stream has been used, although this solution is not environmentally friendly because of the toxicity of reagents. An interesting alternative is provided by an enzyme-assisted method, consisting in a microwave-aided heating pretreat­ment, further enzymatic treatment with the recombinant P-1,3-glucomannanase and plMAN5C, and later oil extraction with ethyl acetate. The percentage of extraction with this method is close to 96.6 % of the total oil (Zeng et al. 2013).

Table 7 shows the fatty acid composition of yeast oil. Although it varies depending on the species and substrate, it is mostly composed of palmitic and oleic acid, the lat­ter being preferred for the biodiesel industry due to its high unsaturation degree (Pinzi et al. 2011). Wahlen et al. (2012) compared biodiesel properties, performance, and emissions in a diesel engine, biodiesel being produced from soybean, algae, bacteria, and yeast oil. Only small differences in terms of exhaust emissions were detected, as biodiesel from yeast oil emitted lower hydrocarbon but higher NOx emissions.

4 Conclusion

Many studies have demonstrated that the use of oleaginous macro — and microor­ganisms has a great interest to the biodiesel industry, as an alternative to first — and second-generation biodiesel. Although each species has its own characteristics that make it suitable to the production of biodiesel, insects posses the ability to recycle organic waste like manure and produce high amount of good-quality oil, while micro­organisms may be fermented on conventional bioreactors, which is a very attractive feature. In the improvement of these technologies, genetic engineering provides a key tool, besides the increase of knowledge about organisms, i. e., culture media and growing conditions. Moreover, the oil composition of oleaginous organisms may be genetically modified to meet the ideal biodiesel requirements, but also it can be modi­fied in pursuit of the best combination of substrate, species, or culture mode. It may be concluded that yeast is the preferred oleaginous microorganism among those ana­lyzed in this chapter, due to its rapid growth, ability to be scaled up, production of lipids, and suitable fatty acid composition to be transesterified into biodiesel.

Acknowledgments This research was supported by the Spanish Ministry of Education and Science (ENE2010-15159) and the Andalusian Economy, Innovation and Enterprise Council, Spain (TEP-4994).