# Lignocellulosic Biomass as a Substrate for Oleaginous Microorganisms: A Review
## 2 Oleaginous microorganisms
### Growth Conditions That Promote Lipid Accumulation  
The concentration of nutrients, such as nitrogen and carbon, in culture medium is decisive for lipid production [69; 6; 70; 71; 39]. In heterotrophs, usually a high C:N molar ratio is needed to achieve an important lipid accumulation [4; 5; 72; 73; 74], while the nature of the nitrogen source plays a critical role in the process [5; 75]. Detailed mathematical models have been developed in order to predict growth and lipid accumulation, as well as nutrient (i.e., sugar and nitrogen) consumption by oleaginous microorganisms (see for instance [74]). In some cases, ammonium nitrogen favors lipid accumulation [76], while in others, organic nitrogen is preferred for lipogenesis [4; 5; 76; 77]. In addition, the limitations of minerals such as magnesium, iron, zinc, phosphorous or the double limitations of minerals including nitrogen and magnesium or nitrogen and phosphorus, induce the production of lipids [5; 78; 79]. Temperature, pH, dissolved oxygen and culture agitation are other important variables that affect lipid accumulation [72; 79]. Studies have shown low biosynthesis of unsaturated fatty acids (FAs) at temperatures above 30 \({}^{\circ}\)C [47] and increased lipid accumulation under neutral and basic pH conditions [26]. It has been reported that biomass and lipid production are high between 200 and 300 rpm, speeds that allow for high oxygenation. However, lipid production decreases as agitation increases, while at lower agitation speeds the carbon metabolism is diverted to extracellular products such as alcohols (e.g., ethanol, mannitol, arabitol and 2,3-butanediol) [47; 80]. The concentration and chemical forms of nitrogen influence the growth and lipid production in autotrophic microorganisms such as microalgae [81]. Studies have shown that many species of microalgae accumulate a greater amount of lipids, especially TAGs, under nitrogen limited conditions, compared to nitrogen excess conditions [82]. On the contrary, other studies suggest that nitrogen deficiency adversely affects photosynthesis, thus biomass production and lipid productivity, due to decreased biosynthesis of NADPH, which is the main source of reducing power [83; 84]. Furthermore, it is possible that nitrogen limitation also affects the lipid profile and several biochemical pathways of the cells [85; 86; 87]. Therefore, new approaches, such as metabolic engineering, may be needed to increase the understanding of the effect of nitrogen on microalgae physiology and on the synthesis and accumulation of TGAs. In many microalgae species, lipid accumulation is favored in phosphate limited conditions [3; 88], as well as, at pH \(<\) 8. Specifically, the dissociation of NH4\({}^{+}\) to NH3\({}^{-}\) occurs at pH \(>\) 8; thus, at lower pH values, nitrogen exists as NH4\({}^{+}\) ions, which is preferred by microalgae over NH3 [89; 90]. Furthermore, studies have also demonstrated the lipid content increases with light intensity, CO2 concentration and oxidative stress caused by salinity [91; 92; 93]. Growth rate as well as the productivity and lipid composition can be affected by temperature changes [94; 95]. The different factors that are involved in the growth and lipid biosynthesis in microalgae can affect the different species in different ways, so their optimum values are species- or even strain-specific and should be experimentally determined. Recently, the effect of reactive oxygen compounds (ROS) or free radicals on the synthesis and accumulation of lipids of heterotrophic and autotrophic oleaginous microorganisms has been evaluated. ROS are generated during microbial metabolism as signs of stress and include superoxide (O2\({}^{-}\)), hydroxyl (OH\({}^{-}\)), perhydroxyl (HO2\({}^{-}\)) and the alkoxy radical (RO\({}^{-}\)), and non-radical forms such as hydrogen peroxide (H2O2) and oxygen (O2). Their accumulation usually results in lipid peroxidation and protein and DNA denaturation.