Incomplete recovery of intact polar glycerol dialkyl glycerol tetraethers from lacustrine suspended biomass

Abstract

Branched and isoprenoid glycerol dialkyl glycerol tetraethers (GDGTs) are membrane lipids of bacteria and archaea, respectively, and their core lipid distributions are used as proxy indicators in paleolimnological studies. In addition, the amount and composition of intact polar lipid (IPL) GDGTs yield information on the presence and abundance of GDGT‐producing microbes within the water column. GDGTs are, however, not always easily recovered from cultured microbial cells by commonly applied extraction methods, and this may also apply to suspended particulate matter (SPM) in aquatic systems. In order to investigate potential biases induced by incomplete GDGT recovery, we analyzed both core‐ and IPL‐GDGTs in SPM from Lake Lugano (Switzerland) using (1) ultrasonic solvent extraction with mixtures of methanol (MeOH) and dichloromethane (DCM), and (2) two modifications of the Bligh–Dyer (BD) protocol. Acid hydrolysis of the post‐extraction residues revealed that particularly branched GDGTs were poorly recovered from SPM by the MeOH/DCM mixtures (25–62%). Much better extraction yields (> 85%) were achieved with the BD method, however, during subsequent phase separation, up to 75% of the extracted branched IPL‐GDGTs partitioned into the aqueous phosphate buffer that is usually discarded. In contrast, when this buffer was substituted with 5% trichloroacetic acid, only 13% were lost into the aqueous phase. Depending on the protocol used, the distribution of the IPL‐derived GDGTs varied substantially. Our results indicate that both bacterial‐ and archaeal IPL‐GDGTs can be difficult to extract from lacustrine microbial communities, and caution is advised when targeting the “viable” GDGT pool.

Redox-dependent niche differentiation provides evidence for multiple bacterial sources of glycerol tetraether lipids in lakes

“…Sediments and soils were extracted three times by accelerated solvent extraction (ASE300; Thermo Fisher Scientific) ( 5 ). For SPM samples, we employed a sequential solvent extraction and acid hydrolysis procedure to assure complete recovery of brGDGTs from living cells (intact polar brGDGTs; see also SI Appendix ), and to avoid losses and biases associated with modified Bligh−Dyer extraction ( 39 ). Briefly, the freeze-dried SPM was extracted by ultrasonication with methanol and dichloromethane as solvents, and the residual material was subsequently subjected to acid hydrolysis.…”

“…The brGDGT-containing lipid fractions were prepared as previously described ( 39 ), spiked with an internal standard ( 40 ), and analyzed by ultra-high-performance liquid chromatography (UHPLC) positive ion atmospheric pressure chemical ionization mass spectrometry in selected ion monitoring mode. For all samples from Lake Lugano, analytical separation of GDGTs was achieved using two UHPLC columns in series (ACQUITY UPLC BEH HILIC, 130 Å, 1.7 µm, 2.1 mm × 150 mm; Waters) ( 41 ), whereas, for all remaining sites, we used an array of four HPLC columns (Alltima Silica, 100 Å, 3 µm, 2.1 mm × 150 mm; W. R. Grace & Co.) ( 4 ).…”

Energy flux controls tetraether lipid cyclization in Sulfolobus acidocaldarius

“…Core GDGTs were isolated from freeze-dried biomass by acid hydrolysis followed by 350 ultrasonic solvent extraction (e.g. Weber et al, 2017). To this end, freeze-dried cell pellets 351 representing 50 mL aliquots of cell culture were submerged in 3 N methanolic HCl (33% H2O) for 352 3 hours at 70 °C.…”

Determination of the δ2H values of high molecular weight lipids by high‐temperature gas chromatography coupled to isotope ratio mass spectrometry

“…Cells were collected by centrifugation at 4°C, frozen in liquid nitrogen, and freeze‐dried. Then 0.5 g of the freeze‐dried cell pellet was subjected to acid hydrolysis in 5 mL of 1.5 N methanolic HCl (10 % H 2 O made from 37 % HCl) for 3 h at 70°C, and lipids were extracted by ultrasonication in dichloromethane/methanol (1:1, v/v) as previously described 56 . The total lipid extract (TLE) was dried under a stream of N 2 , dissolved in 1 mL of n‐hexane/isopropanol (97:3, v/v), and filtered through a 0.45‐μm PTFE filter.…”