Evaluation of Sample Preservation and Storage Methods for Metaproteomics Analysis of Intestinal Microbiomes

Metaproteomics is a powerful tool to study the intestinal microbiome. By identifying and quantifying a large number of microbial, dietary, and host proteins in microbiome samples, metaproteomics provides direct evidence of the activities and functions of microbial community members.

Stable Isotope Mixing Models Are Biased by the Choice of Sample Preservation and Pre-treatment: Implications for Studies of Aquatic Food Webs

Stable isotope analysis has become one of the most widely used techniques in ecological studies. However, there are still uncertainties about the effects of sample preservation and pre-treatment on the ecological interpretation of stable isotope data and especially on Bayesian stable isotope mixing models. Here, Bayesian mixing models were used to study how three different preservation methods (drying, freezing, formalin) and two pre-treatments (acidification, lipid removal) affect the estimation of the utilized organic matter sources for two benthic invertebrate species (Limecola balthica, Crangon crangon) collected in the Baltic Sea. Furthermore, commonly used mathematical lipid normalization and formalin correction were applied to check if they were able to adjust the model results correctly. Preservation effects were strong on model outcomes for frozen as well as formalin preserved L. balthica samples, but not for C. crangon. Pre-treatment effects varied with species and preservation method and neither lipid normalization nor mathematical formalin correction consistently resulted in the desired model outcomes. Our analysis highlights that particularly small, not significant changes in stable isotope ratios introduced by different preservation and pre-treatments display a so far unrecognized source of error in stable isotope mixing models. We conclude that mathematical correction of benthic invertebrate stable isotopes data should be avoided for Bayesian mixing models and that previously unaddressed effects of sample preservation (especially those arising from preservation by freezing) have potentially biased our understanding of the utilization of organic matter in aquatic food webs.

A Novel Method for Determination of the Natural Toxin Ptaquiloside in Ground and Drinking Water

Ptaquiloside (PTA) is a carcinogenic compound naturally occurring in bracken ferns (Pteridium aquilinum). It is highly water soluble and prone to leaching from topsoil to surface and groundwaters. Due to possible human exposure via drinking water, PTA is considered as an emerging contaminant. We present a sensitive and robust method for analysis of PTA and its degradation product pterosin B (PtB) in groundwater. The method comprises two steps: sample preservation at the field site followed by sample pre-concentration in the laboratory. The preservation step was developed by applying a Plackett–Burman experimental design testing the following variables: water type, pH, filtering, bottle type, storage temperature, transportation conditions and test time. The best sample preservation was obtained by using amber glass bottles, unfiltered solutions buffered at pH 6, transported without ice, stored at 4 °C and analysed within 48 h. The recovery was 94% to 100%. The sample purification step had a pre-concentration factor of 250, and the recovery percentages of the entire method were 85 ± 2 (PTA) and 91 ± 3 (PtB). The limits of detection (LOD) of the full method were 0.001 µg L−1 and 0.0001 µg L−1 for PTA and PtB, respectively. The method enables sensitive monitoring of PTA and PtB in groundwater. Carcinogenic PTA was detected in one groundwater well (0.35 µg L−1).

Sample preservation and pre-treatment in stable isotope analysis: Implications for the study of aquatic food webs

Stable isotope analysis has become one of the most widely used techniques in ecology. However, uncertainties about the effects of sample preservation and pre-treatment on the ecological interpretation of stable isotope data and especially on Bayesian stable isotope mixing models remain. Here, Bayesian mixing models were used to study how three different preservation methods (drying, freezing, formalin) and two pre-treatments (acidification, lipid removal) affect the estimation of diet composition for two benthic invertebrate species (Limecola balthica, Crangon crangon). Furthermore, commonly used mathematical lipid normalization and formalin correction were applied to check if they improve the model results. Preservation effects were strong on model outcomes for frozen as well as formalin preserved L. balthica samples, but not for C. crangon. Pre-treatment effects varied with species and preservation method and neither lipid normalization nor mathematical formalin correction consistently resulted in improved model outcomes. Our analysis highlights that particularly small changes in δ15N introduced by different preservation and pre-treatments display a so far unrecognized source of error in stable isotope studies. We conclude that mathematical correction of stable isotopes data should be avoided for Bayesian mixing models and that previously unaddressed effects of sample preservation (especially those arising from preservation by freezing) have potentially biased our understanding of the utilization of organic matter in aquatic food webs.