Different extraction methods were carried out as described below. Response factors (RF) were determined for all components (Cn) in the mixture based on corresponding calibration curves and were used to quantify the FAME content of the samples by the following equation: For FTIR measurements, a High Throughput Screening eX-Tension (HTS-XT) unit coupled to a Vertex 70 spectrometer (both Bruker Optik GmbH, Germany) equipped with a globular mid-IR source and a DTGS detector were used. The growing global demand of lipids as a source of food, feed and fuel has caused an increasing interest in microbial production of lipids. The IR spectrum of M.alpina biomass after Folch extraction (Fig 1B) did not show absorbance peaks related to unsaturated hydrocarbons, C = O stretch and CH2 rocking at wavenumbers 3010, 1745 and 720 cm-1, respectively. The hexane peak was identified by comparison with the retention time of the authentic standard. in these sites and their terms of usage. The FTIR spectra of the biomass before (BE) and after extraction (AE) using the different solvent/sample ratios in the Bligh method are given in the supporting information (Fig B in S1 Fig). Infrared spectra of fungal biomass before and after lipid extraction when a modified Bligh method was applied (Fig C). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Residual lipids could not be detected in the infrared spectra of M.alpina biomass after extraction using the Folch and Lewis methods, indicating their complete lipid extraction in this species. While a wide variety of lipid extraction methods are in use, it is known that variations in experimental conditions and solvent polarities between the different methods result in a high variability in the reported fatty acid yields and profiles from similar SCO producers [5–7]. We hypothesize that the shifted bands are not associated with lipids but with CH2 bending signals in either proteins or chitin, a major component (present as chitin-glucan complexes) in the fungal cell wall [53, 54]. This may also explain the more efficient lipid extraction from M.alpina biomass compared to M.circinelloides using the Folch method, since this method does not include acid hydrolysis. (Table C in S1 Table). The hexane spectrum had the major peaks of alkanes: a C–H stretch at 3000–2875 cm−1, a CH 2 bending absorption at In accordance with previous studies, the gravimetric lipid yield was shown to overestimate the potential of the SCO producers and FAME quantification in GC-FID was found to be the best-suited method for lipid quantification. Because hexane has only C-H and C-C bonds (and no functional groups), this spectrum can help orient you to the important regions in an IR spectrum. FTIR spectra of glucuronic acid and glycerol standards (Fig A). The internal standards, tridecanoic acid (C13:0, Sigma Aldrich, USA) and tricosanoic acid (C23:0, Sigma Aldrich, USA) were dissolved in hexane and added to a final amount of 1.0 and 0.7 mg per sample, respectively. The presence of lipids was determined by recording infrared spectra of all components in the lipid extraction procedure, such as the biomass before and after extraction, the water and extract phases. The spots corresponding to FFAs, PLs, MDGs and TAGs were identified by comparison with known standards by a Bioscan AR-2000 Radio-TLC & Imaging Scanner (Bioscan Inc., Washington, DC, USA). (1) Gravimetric approach: lipids were quantified gravimetrically by evaporating the solvent under a steam of nitrogen and determining the weight of the lipid extract.