Determination of the sedimentary microbial biomass by extractible lipid phosphate

Abstract

The measurement of lipid phosphate is proposed as an indicator of microbial biomass in marine and estuarine sediments. This relatively simple assay can be performed on fresh, frozen or frozen-lyophilized sediment samples with chloroform methanol extraction and subsequent phosphate determination. The sedimentary lipid phosphate recovery correlates with the extractible ATP and the rate of DNA synthesis. Pulse-chase experiments show active metabolism of the sedimentary phospholipids. The recovery of added C-labeled bacterial lipids from sediments is quantitative. Replicate analyses from a single sediment sample gave a standard deviation of 11%. The lipid extract can be fractionated by relatively simple procedures and the plasmalogen, diacyl phospholipid, phosphonolipid and non-hydrolyzable phospholipid content determined. The relative fatty acid composition can be readily determined by gas-liquid chromatography.The lipid composition can be used to define the microbial community structure. For example, the absence of polyenoic fatty acids indicates minimal contamination with benthic micro-eukaryotes. Therefore the high content of plasmalogen phospholipids in these sediments suggests that the anaerobic prokaryotic Clostridia are found in the aerobic sedimentary horizon. This would require anaerobic microhabitats in the aerated zones.

Post-Sampling Changes in Microbial Community Composition and Activity in a Subsurface Paleosol

“…Storage time and sediment condition contributed to the response ( Table 2). The rapid dephosphorylation of cellular phospholipids upon cell death makes total PLFA a useful measure of the biomass of living cells [40]. Total PLFA was lower and relatively constant after t = 0, except for elevated values in intact samples at 112 and 224 d (Fig.…”

“…Subsamples (100 g) were frozen at −70°C and shipped on dry ice, by overnight express mail, to the laboratory of D.C. White. Samples (75 g) were placed into 250-ml glass centrifuge bottles, and 142.5 ml of a Bligh and Dyer extraction solvent system [2], modified to include a phosphate buffer [40], was added. Following 2 min of sonication at room temperature, the single-phase extraction was allowed to progress for 3 h. Samples were centrifuged at 650× g for 30 min, after which the liquid phase was decanted into a separatory funnel.…”

“…The total lipid extract was then further separated into neutral, glyco-, and polar lipids on a silicic acid column (100-200 mesh Unisil, Clarkson Chemical Co., Williamsport, PA) [17]. The polar lipid fraction was subjected to a mild alkaline methanolysis, from which the transesterified fatty acid methyl esters were recovered [40]. Fatty acid methyl esters were further separated and quantified by capillary column gas chromatography (GC)/mass spectrometry, as described by Ringelberg et al [28].…”

Gas chromatography coupled to mass spectrometry for analyses of organic compounds and biomarkers as tracers for geological, environmental, and forensic research

“…However, it is in the characterization of soil microbial communities using microbial biomarkers that the greatest application of biomarkers in soil studies resides. The use of phospholipid fatty acids (PLFA) patterns as a way of acquiring a view of the microbial community in soil samples has become increasingly popular since the work of White et al [253]. PLFA and, more recently, neutral lipid fatty acids (NLFA) have been applied as a measure of biomass and nutritional status, respectively, of soil microorganisms [e. g., 157, 161, 254 -258].…”

Detection of Sphingomonas spp in soil by PCR and sphingolipid biomarker analysis

“…S. aromaticivorans B0695R (50 mg fresh weight) or 10 g soil sample was used for sphingolipid analysis. Total lipid of the sample was extracted by the modified Bligh and Dyer procedure [25]. The lipid extract was dried under a stream of nitrogen gas at room temperature, re-dissolved in 1 ml of 3 M hydrochloric acid and heated in a capped 15-ml glass tube at 100°C for 3 h. The acid digest was cooled to room temperature and adjusted to pH 12 by KOH.…”