Adapting an Ergosterol Extraction Method with Marine Yeasts for the Quantification of Oceanic Fungal Biomass

Abstract

Ergosterol has traditionally been used as a proxy to estimate fungal biomass as it is almost exclusively found in fungal lipid membranes. Ergosterol determination has been mostly used for fungal samples from terrestrial, freshwater, salt marsh- and mangrove-dominated environments or to describe fungal degradation of plant matter. In the open ocean, however, the expected concentrations of ergosterol are orders of magnitude lower than in terrestrial or macrophyte-dominated coastal systems. Consequently, the fungal biomass in the open ocean remains largely unknown. Recent evidence based on microscopy and -omics techniques suggests, however, that fungi contribute substantially to the microbial biomass in the oceanic water column, highlighting the need to accurately determine fungal biomass in the open ocean. We performed ergosterol extractions of an oceanic fungal isolate (Rhodotorula sphaerocarpa) with biomass concentrations varying over nine orders of magnitude. While after the initial chloroform-methanol extraction ~87% of the ergosterol was recovered, a second extraction recovered an additional ~10%. Testing this extraction method on samples collected from the open Atlantic Ocean, we successfully determined ergosterol concentrations as low as 0.12 pM. Thus, this highly sensitive method is well suited for measuring fungal biomass from open ocean waters, including deep-sea environments.

Extracellular Enzymatic Activities of Oceanic Pelagic Fungal Strains and the Influence of Temperature

“…In order to have fresh cultures, the pure isolates were cultured on yeast malt extract agar [ 23 , 24 ] for one week. Afterwards, an initial amount of each fungus was diluted in artificial seawater (30 g/L sea salts S9883 Sigma-Aldrich, Vienna, Austria) to obtain an OD 660 ≈ 1 [ 25 ]. The optical density (OD) was measured with a UV-1800 Shimadzu spectrophotometer.…”

Ergosterol extraction: a comparison of methodologies

“…A biphasic layer is formed upon extraction, with lipids contained in the chloroform layer and non-lipid compounds within the Milli-Q water aqueous layer. Alekseyeva et al [18] experimentally showed the presence of 98 % of the total extracted ergosterol was present within the chloroform layer. Both the present study and Alekseyeva et al [18] dried the extracts upon completion before reconstituting in methanol.…”

“…The use of methanol alone for the extraction of ergosterol has been utilized by several investigations with a range of success [18,23,24]. The main difference in methanol alone to extract ergosterol is the duration of a heat treatment in a water bath or heating block dependent on the overall volume of the sample.…”

“…Furthermore, the chemical nature of the solvents allows flexibility to extract ergosterol from a range of environmental samples, and should be tested further in continued study. Bligh and Dyer [26] and Alekseyeva et al [18] provide details regarding the chemical processes around the functionality of the chloroform/methanol extraction of ergosterol. A biphasic layer is formed upon extraction, with lipids contained in the chloroform layer and non-lipid compounds within the Milli-Q water aqueous layer.…”

“…A modified method of Alekseyeva et al . [ 18 ] was used for ergosterol extraction via methanol and chloroform. Of both growth substrate and root samples, 300 mg was weighed into 50 ml centrifuge tubes, as summarized in Fig.…”