Microbiota Assembly and Dissolved Organic Matter Diversity in Natural Mineral Waters Celine C
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Lesaulnier et al. Microbiome (2017) 5:126 DOI 10.1186/s40168-017-0344-9 RESEARCH Open Access Bottled aqua incognita: microbiota assembly and dissolved organic matter diversity in natural mineral waters Celine C. Lesaulnier1†, Craig W. Herbold1†, Claus Pelikan1, David Berry1, Cédric Gérard4, Xavier Le Coz4, Sophie Gagnot4, Jutta Niggemann3, Thorsten Dittmar3, Gabriel A. Singer2† and Alexander Loy1* Abstract Background: Non-carbonated natural mineral waters contain microorganisms that regularly grow after bottling despite low concentrations of dissolved organic matter (DOM). Yet, the compositions of bottled water microbiota and organic substrates that fuel microbial activity, and how both change after bottling, are still largely unknown. Results: We performed a multifaceted analysis of microbiota and DOM diversity in 12 natural mineral waters from six European countries. 16S rRNA gene-based analyses showed that less than 10 species-level operational taxonomic units (OTUs) dominated the bacterial communities in the water phase and associated with the bottle wall after a short phase of post-bottling growth. Members of the betaproteobacterial genera Curvibacter, Aquabacterium, and Polaromonas (Comamonadaceae) grew in most waters and represent ubiquitous, mesophilic, heterotrophic aerobes in bottled waters. Ultrahigh-resolution mass spectrometry of DOM in bottled waters and their corresponding source waters identified thousands of molecular formulae characteristic of mostly refractory, soil-derived DOM. Conclusions: The bottle environment, including source water physicochemistry, selected for growth of a similar low-diversity microbiota across various bottled waters. Relative abundance changes of hundreds of multi-carbon molecules were related to growth of less than ten abundant OTUs. We thus speculate that individual bacteria cope with oligotrophic conditions by simultaneously consuming diverse DOM molecules. Keywords: Bottled water, Microbial diversity, Dissolved organic matter, Fourier transform ion cyclotron resonance mass spectrometry, Aquabacterium, Curvibacter, Polaromonas Background distinguished from other types of drinking water, e.g., by Bottled water, including natural mineral water, is an its characteristic content of minerals and trace elements. increasingly popular source of drinking water around Furthermore, disinfection or chemical treatment of nat- the world and represents a multi-billion-dollar industry. ural mineral water is not permitted, yet it is routinely The European Union regulates the exploitation and mar- tested for its number of cultivable bacteria including keting of natural mineral waters to protect their unique several marker organisms (Escherichia coli and other characteristics and original purity. The latest EU Directive coliforms, fecal streptococci, Pseudomonas aeruginosa, 2009/54/EC defines natural mineral water as microbio- and sporulated sulfite-reducing anaerobes). The largely logically wholesome water of underground origin that is untreated nature of natural mineral waters allows that mi- protected from all risk of pollution and can be clearly croorganisms from the source water aquifer and possibly the bottling plant (i.e., pipelines and storage tanks) act as * Correspondence: [email protected] inoculum for the bottle environment. Within a few days †Equal contributors after bottling members of this “seed microbiota” begin to 1 Department of Microbiology and Ecosystem Science, Division of Microbial grow during storage of natural mineral waters at ambient Ecology, Research Network Chemistry meets Microbiology, University of Vienna, Althanstrasse 14, A-1090 Vienna, Austria Full list of author information is available at the end of the article © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Lesaulnier et al. Microbiome (2017) 5:126 Page 2 of 17 temperature, with absolute cell counts reaching 105–106 investigation of two representative bottled waters over cells/mL [1–3]. 2 months of incubation and detailed comparative Such microbial growth in non-carbonated bottled analysis of all 12 bottled waters 1 day after filling in waters is a well-known fact, but the composition of the polyethylene terephthalate (PET) bottles. We revealed bottled water microbiota and its post-bottling dynamics corresponding patterns in DOM turnover and microbial have thus far been investigated by molecular techniques community development after bottling and thereby in only two natural mineral waters [2, 4]. These and provide novel insights into the molecular microbial and numerous isolation-dependent studies [5–8] have chemical ecology of this important drinking water source. established that Alpha-, Beta-,andGammaproteobac- teria are the prevalent microorganisms in bottled water. Methods Beyond this, many fundamental questions regarding the Sampling and storage of natural mineral waters microbial ecology of bottled waters still remain poorly We analyzed non-carbonated natural mineral waters re- answered. How different is the microbiota in bottled trieved from 12 European bottling plants of well-derived waters from different sources? How does the bottled mineral water in the years 2011 and 2012 (Additional file 1: water microbiota assemble? Are there differences be- Tables S1–S4). Samples from each plant included bottled tween the free-living community in the water phase water and corresponding well water; the latter was taken (plankton microbiota) and the inner-bottle-surface-as- before its entry into the bottling plant from a sampling sociated community (biofilm microbiota)? port located at the head of each source. Bottled water 6 re- A persistent question that has puzzled researchers for ceives water from two wells, 6a and 6b, while bottled wa- decades is what substrates in bottled water may fuel the ters 9a and 9b receive water from the same well 9. Bottled observed sudden microbial growth [8]? Although auto- and well waters were filled in brand-specific 0.5 L and trophic growth has been suggested [4], it is commonly standard 1 L PET bottles, respectively. Water bottles were assumed that bottled water microorganisms mainly transported to the laboratory within 24 h and were subse- generate energy and multiply through heterotrophic quently stored in a dark, climate-controlled room (20– utilization of dissolved organic matter (DOM) available 24 °C) for sampling at regular time points after bottling. in the bottle environment [8]. Ground water, treated All bottled waters used in this study complied with the drinking water, and oligotrophic surface freshwaters legal microbiological criteria (EU Directive 2009/54/EC). generally have low DOM concentrations in the range of 0.5–5 mg carbon/L. Microorganisms metabolize only a Recovery of plankton and biofilm biomass minor fraction (0.01–0.1 mg carbon/L) of this DOM All waters were sampled in triplicate at each time point. pool while a larger refractory fraction remains un- Each replicate sample consisted of biomass from one or touched [9]. The susceptibility of DOM to microbial more separate bottles of water. Planktonic microorgan- turnover depends on its origin and diagenetic history, isms were recovered on polycarbonate filters with a pore which define chemical composition [10, 11], and its size of 0.22 μm (GTTP04700; Millipore, Eschborn, concentration, which defines the probability of micro- Germany) using a stainless steel vacuum filtration unit bial encounter for a specific chemical structure [12]. equipped with three 500-mL filter funnels (Sartorius, DOM analytics have been dramatically advanced by the Göttingen, Germany). Per replicate sample, 3 L (e.g., advent of Fourier transform ion cyclotron resonance 6 × 0.5 L bottles or 3 × 1 L bottles) of water were mass spectrometry (FT-ICR-MS) with ultra-high mass filtered for nucleic acid extraction and 0.5 to 3 L for resolution allowing to accurately identify thousands of microscopy. Filters with cellular biomass were air dried, individual molecular formulae in a single environmen- cut in half, and stored at − 80 °C for DNA extraction or tal sample [13]. While FT-ICR-MS has provided funda- fixed with para-formaldehyde and stored at − 20 °C for mental insights into the molecular diversity of DOM microscopy [2]. Biofilm samples were recovered from and its turnover in various aquatic environments such the inner linings of the same bottles that were used for as oceans [12–14], lakes [15], glaciers [11], and ground- harvesting planktonic biomass. Bottles were cut in half water [16, 17], analogous information is not available with a sterile scalpel blade and the entire inner surface for bottled drinking water. of each bottle was swabbed with a sterile viscose collec- Here, we simultaneously analyzed microbiota and tion swab (Deltalab, Carcassonne, France). The swab tips DOM composition in 12 European non-carbonated were then cut off and stored at − 80 °C prior to DNA natural mineral waters and their corresponding source extraction. waters by multiplexed sequencing of