Among the oleaginous microorganisms reported in the literature, filamentous fungi show the highest lipid accumulation after yeast, besides the capacity to produce a wide range of products, i. e., enzymes, antibiotic, and chemicals (Karimi and Zamani 2013). Some of the main differences between filamentous fungi and other oleaginous species (yeast, microalgae, and bacteria) on the production of oils are based on the capability of filamentous fungi to build pellets in submerged cultures, due to filamen­tous growth during fermentation. Moreover, the viscosity of the broth is reduced, thus improving the mixing and mass transfer performance. Finally, due to the formation of pellets, they are easy to harvest from broth by simple cell filtration, which reduces the cost compared with traditional methods like centrifugation (Xia et al. 2011).

To decrease the cost of the process, methanolysis from fungal biomass has been proposed as an alternative to the oil extraction process. Through the use of metha­nol and a catalyst, usually H2SO4 or HCl, some authors reported a yield of FAME conversion of 91 %, being the cetane number 56.4, thus making this technique an attractive alternative for the biodiesel industry (Liu and Zhao 2007).

The stored lipids in filamentous fungi contain a high percentage of saturated (Venkata Subhash and Venkata Mohan 2011) and polyunsaturated fatty acids (Mitra et al. 2012), accumulated during the stationary phase in special organelles, named lipid granules. Like bacteria, filamentous fungi may also consume a wide range of carbon sources, including lignocellulosic biomass (Table 6), thus provid­ing inexpensive raw material for biodiesel.

Although lignocellulose comprises hemicellulose, cellulose, and lignin, only hemicellulose and cellulose may be consumed as feedstock for biological conver­sion. For this purpose, to make carbohydrates accessible to microorganisms, lig — nocellulose needs a pretreatment before hydrolysis (Zeng et al. 2013). Zikou et al. (2013) used a mixture of xylose and glucose, which are abundant sugars from ligno — cellulosic biomass, to produce y-linolenic acids (GLA) by Zygomycetes T. elegans. Results showed that the best combination of xylose to glucose is 1:1, achieving 12.6 g/L lipids and 936 mg/L GLA. Instead, when glucose was used as the sole medium, the values were 15 g/L and 1,014 mg/L, respectively. M. isabellina was also tested, and a positive influence of the increment of these sugars separately in the medium over the accumulation of lipids was found (Ruan et al. 2012). The same filamentous fungus was used for the production of oil when rice hulls hydrolyz — ate, which is a lignocellulosic material, was used as a substrate. Authors proposed a mathematical model to simulate the consumption of sugar and nitrogen, the fat-free biomass formation, and the accumulation of lipids (Economou et al. 2011). Khot et al. (2012) isolated fungi of tropical mangrove wetlands, but only five out of 14 showed lipid accumulation above 20 % dry cell biomass. Fungi from this ecosystem were also used for the production of lignocellulosic enzymes. The oil of three out of the previous five was transesterified, the biodiesel properties predicted, and it was found that the most appropriate fungus was IBB M1, known as A. terreus strain. Another important issue to be fixed when lignocellulosic biomass is used consists in the inhibitory effects of the lignocellulose-derived compounds over oil accumulation

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D. E. Leiva-Candia and M. P. Dorado

(lignin aldehydes, furan aldehydes, and weak acid). When M. isabellina was used to determine the inhibitory effect of these compounds, the lignin derivative was found to be the main inhibitor considering lipid accumulation, while acetic and formic acid doubled the lipid accumulation with respect to the control test (Zeng et al. 2013). It was concluded that the most suitable combination of fungus and lignocellulosic material substrate for fungal oil production was provided by the strain M. isabellina when it consumed non-detoxified lignocellulosic hydrolyzate, due to both the high oil content and the simplified process of fermentation (Zheng et al. 2012b).

In terms of environmental preservation, the bioremediation of soils contami­nated by hydrocarbons is an important issue. For this purpose, the use of A. terreus has been investigated to transform petroleum hydrocarbons in oils to be used in the biodiesel industry. Results showed that the use of hydrocarbons as carbon source provides sevenfold higher lipid accumulation compared to the use of glucose as sub­strate (Kumar et al. 2010). Crude glycerol is a by-product of the biodiesel industry, which has recently been released in high quantities due to the increasing biodiesel demand. It usually comprises residues of alcohol (methanol or ethanol) and a basic catalyst. This by-product has been tested as a carbon source for Mucor sp., C. echi — nulata, M. ramanniana, T. elegans, Z. moelleri (Chatzifragkou et al. 2011; Bellou et al. 2012), and M. isabellina (Chatzifragkou et al. 2011). Chatzifragkou et al. (2011) used the fungi mentioned above and compared lipid accumulation with that of yeasts. Results showed that all fungi were able to accumulate higher amount of oil than yeasts under nitrogen-limited conditions. Bellou et al. (2012) focused their research on the production of PUFA produced by filamentous fungi. In the majority of the tested fungi, authors observed that PUFA was mainly accumulated in myce­lial membranes during mycelial growth. However, one of the studied filamentous fungi (Mortierella ramannniana) depicted the opposite trend. In this sense, PUFA continued decreasing after the end of the growth phase, thus suggesting PUFA is involved in primary metabolism of this microorganism (Bellou et al. 2012).

Filamentous fungi have been genetically engineered focusing on lipid produc­tion, giving relevance to metabolic routes governing fatty acid synthesis and lipid storage. Unique metabolic features have been identified in Mortierella alpina and Mortierella circinelloides, particularly with respect to NADPH metabolism and sterol biosynthesis, which might be related to differences in fungal lipid phenotype (Vongsangnak et al. 2013). The gene coding for acetyl-CoA carboxylase (ACC) was isolated from Mucor rouxii. This gene is able to increase by 40 % the total fatty acid content of non-oleaginous microorganism (Ruenwai et al. 2009). Wynn et al. (1999) studied the significant role of malic enzyme on lipid accumulation. Authors used a fungus with low lipid accumulation (M. circinelloides) and found out that the enzyme disappeared 15 h after the depletion of the nitrogen source, which was coincident with the end of lipid accumulation. Instead, when a high-lipid accumula­tion fungus like Mortierella alpine was used, the enzyme was held 60 h after the completion of the nitrogen source, which lasted longer than the lipid accumulation.

The accumulation of lipids from filamentous fungi is increasingly attractive because of the high oil yields, versatility of the microorganisms to use different car­bon sources (including wastes like lignocellulosic material), and the possibility to

be grown in submerged cultures, which give the opportunity to easily collect the biomass. In this context, genetic engineering may be a magnificent tool to help in the inclusion of these microorganisms to provide an alternative oil to the biodiesel industry. Although most research in this area is focused on the production of high — value-added products such as enzymes and polyunsaturated fatty acids, among many others, the production of microbial oil could provide an extra value to the process.