Comparison of commonly used extraction methods for ergosterol in soil samples

Abstract

A b s t r a c t. Concerning the contribution of fungi to soil carbon sequestration, various methods have been used to extract ergosterol from soil samples. This study aims to explore the extraction ability and applicability of commonly used methods to extract ergosterol from two contrasting soils. An agricultural soil (chernozem) and a forest soil (podzol) were extracted with different types of cell lysis such as alkaline, glass bead, and ultrasonication methods in association with simple shaking. The ergosterol concentration was measured by high pressure liquid chromatography. Regardless of the method applied, ergosterol yield was higher in podzol than in chernozem. Alkaline extraction resulted in the highest ergosterol concentrations for both soils and miniaturized glass bead extraction produced comparable results in the case of chernozem. In terms of applicability, the non-alkaline methods were simpler to conduct and less demanding of labour, chemical use and glassware and more flexible in terms of the equipment used for mechanical disruption. Despite the limit of the two soil types in the present study, only the simple shaking method was revealed to be dependent on soil type. Based on our results, we recommend the miniaturized glass bead method for agricultural soils, low in organic matter for high throughput. However, not all of the methods described allow for the proper separation of coextracted organic substances from organic-rich soil.

Limosilactobacillus fermentum Limits Candida glabrata Growth by Ergosterol Depletion

“…Coculture samples were separated by centrifugation (2,000 rpm for 5 min). The quantification method was described previously ( 67 ). In short, pellets were resuspended with 250 μL 10% (wt/wt) KOH in MeOH, followed by sonication (15 min) and 70°C for 50 min.…”

Revisiting soil fungal biomarkers and conversion factors: Interspecific variability in phospholipid fatty acids, ergosterol and rDNA copy numbers

“…The large deviation among fungal ITS copy contents in different studies was likely driven by methodological differences: especially differences in DNA extraction methods and primers applied (Table S2) can result in varying DNA extraction efficiencies (Delmont et al, 2012) and qPCR results, even when different primer pairs target the same gene (Thijs et al, 2017). To allow for comparability among datasets, methods of qPCR quantification of soil fungi need to be standardized, as is the case for PLFA or ergosterol analyses (Frostegård et al, 1991;Sae-Tun et al, 2020). In our study, fungal ITS1 copy contents led to a conversion factor of 0.264 pg fungal C copy −1 ITS1.…”

Ergosterol extraction: a comparison of methodologies

“…Current ergosterol extraction procedures typically employ the use of cyclohexane as an organic solvent along with alkaline methanol [9–11, 15, 16]. The use of cyclohexane requires additional safety measures to be implemented and exposes the user to long-term health hazards [17].…”

“…The use of cyclohexane requires additional safety measures to be implemented and exposes the user to long-term health hazards [17]. Other extraction methods, with the absence of cyclohexane, utilize methanol and alkaline methanol -potassium hydroxide and methanol [methanol hydroxide (MeOH)] -for varying lengths of extraction time [10,15,16].…”