Spore dipicolinic acid contents used for estimating the number of endospores in sediments

Abstract

Endospores are heat-resistant bacterial resting stages that can remain viable for long periods of time and may thus accumulate in sediments as a function of sediment age. The number of spores in sediments has only rarely been quantified, because of methodological problems, and consequently little is known about the quantitative contribution of endospores to the total number of prokaryotic cells. We here report on a protocol to determine the number of endospores in sediments and cultures. The method is based on the fluorimetric determination of dipicolinic acid (DPA), a spore core-specific compound, after reaction with terbium chloride. The concentration of DPA in natural samples is converted into endospore numbers using endospore-forming pure cultures as standards. Quenching of the fluorescence by sediment constituents and background fluorescence due to humic substances hampered direct determination of DPA in sediments. To overcome those interferences, DPA was extracted using ethyl acetate prior to fluorimetric measurements of DPA concentrations. The first results indicated that endospore numbers obtained with this method are orders of magnitude higher than numbers obtained by cultivation after pasteurization. In one of the explored sediment cores, endospores accounted for 3% of all stainable prokaryotic cells.

Pre‐column liquid chromatographic determination of dipicolinic acid from bacterial endospores

“…In short, subsamples of hydrolyzate are dried under vacuum, redissolved in Milli-Q water, and dried again before they are dissolved in Buffer A. This procedure is simple and faster than the repeated ethyl acetate extraction procedure described by Fichtel et al (2007a), and we do not experience problems with high background fluorescence as they described.…”

“…Bacterial cell counts were obtained from Jørgensen et al (1990). In comparison, Fichtel et al (2007a) estimated bacterial endospore numbers of ~3 ¥ 10 6 endospores gdw -1 and ~5 ¥ 10 5 endospores gdw -1 in surface sediment at two sites from the North Sea tidal flat. In this study, endospores represented < 1% of the total cell counts in the upper 50 cm of the sediment, whereas the contribution of endospores reached 10% in deeper layers of the sediment.…”

“…Recent studies have shown that bacterial endospores are abundant in subsurface marine sediment and that the relative contribution of endospores to the microbial community increased with depth in up to 5.5 m long sediment cores from tidal flats in the southern North Sea (Fichtel et al 2007a(Fichtel et al , 2008. Isolation of endospore-forming microorganisms from the deep marine sediment of the Equatorial Pacific and Peru Margin (Batzke et al 2007) indicates that endospore formation might be a survival strategy.…”

“…DPA is an excellent biomarker for intact endospores, because it is uniquely produced by bacterial endospores (Powel 1953), and it is rapidly degraded even under strictly anoxic conditions (Seyfried and Schink 1990). Hence, DPA quantification serves as a specific and culture-independent method of endospore abundance (e.g., Warth 1979;Hindle and Hall 1999;Fichtel et al 2007aFichtel et al , 2007b.…”

Amyloid Histology Stain for Rapid Bacterial Endospore Imaging

“…For the last decade, dipicolinic acid (DPA) has gained considerable popularity as a marker for the detection of bacterial endospores (16,22,25,59,67,83,92,97). Emission enhance-ment assays for DPA and bacterial endospores, utilizing lanthanide ions, were developed at the U.S. Army Research Laboratory at the end of the 20th century (59,67) and were recently improved by a research team at the Jet Propulsion Laboratory (9,10).…”