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Community-Level Physiological Profiling of Microbial Communities in Constructed Wetlands: Effects of Sample Preparation

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Abstract

Community-level physiological profiling (CLPP) using BIOLOG® EcoPlates™ has become a popular method for characterizing and comparing the functional diversity, functional potential, and metabolic activity of heterotrophic microbial communities. The method was originally developed for profiling soil communities; however, its usage has expanded into the fields of ecotoxicology, agronomy, and the monitoring and profiling of microbial communities in various wastewater treatment systems, including constructed wetlands for water pollution control. When performing CLPP on aqueous samples from constructed wetlands, a wide variety of sample characteristics can be encountered and challenges may arise due to excessive solids, color, or turbidity. The aim of this study was to investigate the impacts of different sample preparation methods on CLPP performed on a variety of aqueous samples covering a broad range of physical and chemical characteristics. The results show that using filter paper, centrifugation, or settling helped clarify samples for subsequent CLPP analysis, however did not do so as effectively as dilution for the darkest samples. Dilution was able to provide suitable clarity for the darkest samples; however, 100-fold dilution significantly affected the carbon source utilization patterns (CSUPs), particularly with samples that were already partially or fully clear. Ten-fold dilution also had some effect on the CSUPs of samples which were originally clear; however, the effect was minimal. Based on these findings, for this specific set of samples, a 10-fold dilution provided a good balance between ease of use, sufficient clarity (for dark samples), and limited effect on CSUPs. The process and findings outlined here can hopefully serve future studies looking to utilize CLPP for functional analysis of microbial communities and also assist in comparing data from studies where different sample preparation methods were utilized.

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Acknowledgments

T. Aubron and J. Nivala were supported in part by funding from the German Ministry of Education and Research (BMBF) within the context of the SMART-MOVE Project (BMBF FKZ 02WM1355B). J. Nivala also kindly acknowledges the Helmholtz-Zentrum für Umweltforschung (UFZ) integrated projects “Water Scarcity” (T34) and “Urban Transformations” (T13). K. Weber gratefully acknowledges NSERC funding for this work. All authors are particularly grateful to Katy Bernhard for assistance with sample collection and operation of the research site at Langenreichenbach and to Grit Weichert and Johannes Boog for their support and coordination of laboratory analyses. Dr. Regina Stoltenburg, Dr. Christine Reinemann, and Kerstin Ethner are also kindly acknowledged for their logistical support in the laboratory.

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Authors and Affiliations

  1. Environmental Sciences Group, Department of Chemistry and Chemical Engineering, Royal Military College of Canada, Kingston, Ontario, K7K 7B4, Canada

    Mark Button & Kela Weber

  2. Helmholtz Center for Environmental Research (UFZ), Environmental and Biotechnology Center (UBZ), Permoserstrasse 15, Leipzig, 04318, Germany

    Jaime Nivala, Thomas Aubron & Roland Arno Müller

Authors
  1. Mark Button
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  2. Kela Weber
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  3. Jaime Nivala
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  4. Thomas Aubron
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Correspondence to Mark Button.

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SI Figure 1

Characteristics of water samples included in the study. 1 H50p refers to a planted horizontal subsurface flow system with a 50 cm water depth. 2 HAp refers to the effluent sample of an aerated horizontal subsurface flow system with a 100 cm water depth. Internal samples were taken from the middle of the water column at a fractional distance of 25% along the flow path. n/a = not available. Further details of these systems are provided elsewhere (25). (DOCX 45.6 kb)

SI Figure 2

The standard deviation of carbon source responses (wells) on the BIOLOG EcoPlate as a function of incubation time for the five water samples prepared using different methods. Readings were taken at 0, 12, 20, 37, 41, 49, 63, 72, 84, 96, 108, 133, and 158 hours. 84hr was used as the time point for further data analysis. (DOCX 1.41 mb)

SI Figure 3

The number of wells on the BIOLOG EcoPlate with an absorbance value above 2.0 AU as a function of incubation time for the five water samples prepared using different methods. (DOCX 2.38 mb)

SI Figure 4

Average Well Color Development (AWCD) as a function of time for each of the five water samples after preparation by each of the investigated methods. (DOCX 2.47 mb)

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Button, M., Weber, K., Nivala, J. et al. Community-Level Physiological Profiling of Microbial Communities in Constructed Wetlands: Effects of Sample Preparation. Appl Biochem Biotechnol 178, 960–973 (2016). https://doi.org/10.1007/s12010-015-1921-7

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