Date Published: January 24, 2017
Publisher: Public Library of Science
Author(s): Kristin Forfang, Boris Zimmermann, Gergely Kosa, Achim Kohler, Volha Shapaval, Thomas L. Dawson.
To assess whether Fourier Transform Infrared (FTIR) spectroscopy could be used to evaluate and monitor lipid extraction processes, the extraction methods of Folch, Bligh and Lewis were used. Biomass of the oleaginous fungi Mucor circinelloides and Mortierella alpina were employed as lipid-rich material for the lipid extraction. 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. Infrared spectra revealed the incomplete extraction after all three extraction methods applied to M.circinelloides and it was shown that mechanical disruption using bead beating and HCl treatment were necessary to complete the extraction in this species. FTIR spectroscopy was used to identify components, such as polyphosphates, that may have negatively affected the extraction process and resulted in differences in extraction efficiency between M.circinelloides and M.alpina. 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. Bligh extraction underestimated the fatty acid content of both M.circinelloides and M.alpina biomass and an increase in the initial solvent-to-sample ratio (from 3:1 to 20:1) was needed to achieve complete extraction and a lipid-free IR spectrum. 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. We conclude that FTIR spectroscopy can serve as a tool for evaluating the lipid extraction efficiency, in addition to identifying components that may affect lipid extraction processes.
The growing global demand of lipids as a source of food, feed and fuel has caused an increasing interest in microbial production of lipids. Numerous microalgae, yeasts, filamentous fungi and some bacteria are referred to as oleaginous microorganisms due to their ability to accumulate over 20% (w/w) lipids [1–4]. Microbial lipids, often referred to as single cell oils (SCOs), mainly consist of fatty acids in the form of triglycerides. Depending on the fatty acid composition, the SCO can be exploited in the production of nutraceuticals, food, feed or biodiesel. Knowledge of the fatty acid yield and profile of the SCO are crucial for determining the potential application. The first step in determining the yield and fatty acid profile is lipid extraction. 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].
Lipid extraction is a crucial step in the screening of oleaginous microorganisms and the following optimization of SCO production. Thus, the selection of a suitable extraction method is important in order to perform an adequate evaluation of the lipid yield and profile. Lipid losses can occur for any extraction method applied for microbial biomass. This study has shown that, depending on the extraction method and microbial biomass employed, significant variations in lipid yield can be obtained. Thus, it is difficult to appoint a single extraction method as a golden standard, especially when extraction methods are applied to microorganisms that have not been extensively studied previously. FTIR spectroscopy is commonly used in microbial research for the identification and characterization of microorganisms, but this study has shown that FTIR spectroscopy applied to intact biomass and biomass residual after lipid extraction can serve as a tool for evaluating the lipid extraction efficiency. The presence or absence of lipid bands in the IR spectrum of the biomass after extraction can function as a measure for the efficiency of the extraction method. Since an FTIR spectrum represents an overall biochemical fingerprint, it can be used in parallel to identify components that may affect lipid extraction processes, for example chitin, glucuronans and polyphosphates in fungal biomass.