Canadian Journal of Microbiology Microbial diversity associated to the anaerobic sediments of a soda lake (Mono Lake, CA) Journal: Canadian Journal of Microbiology Manuscript ID cjm-2017-0657.R2 Manuscript Type: Article Date Submitted by the Author: 20-Feb-2018 Complete List of Authors: Rojas, Patricia; Universidad Autonoma de Madrid, Molecular Biology Rodriguez, Nuria; Centro de Astrobiologia, (INTA-CSIC) de la Fuente, Vicenta; Universidad Autonoma de Madrid, Biology Sánchez-Mata,Draft Daniel; Universidad Complutense de Madrid, Pharmacology, Pharmacognosy and Botany Amils, Ricardo; Centro de Biologia Molecular S.O., UAM-CSIC; Centro de Astrobiologia, (UAM-CSIC) Sanz Martin, Jose Luis; Universidad Autonoma de Madrid, Molecular Biology Is the invited manuscript for consideration in a Special N/A Issue? : Mono Lake, anaerobic sediments, microbial communities, soda lakes, Keyword: pyrosequencing https://mc06.manuscriptcentral.com/cjm-pubs Page 1 of 28 Canadian Journal of Microbiology Microbial diversity associated to the anaerobic sediments of a soda lake (Mono Lake, CA) Patricia Rojas1, Nuria Rodríguez2, Vicenta de la Fuente3, Daniel Sánchez-Mata4, 5 Ricardo Amils 2, 5, José L. Sanz1, * 1: Department of Molecular Biology, Universidad Autónoma de Madrid, Spain. E- mail: [email protected] 2: Centro de Astrobiología (INTA-CSIC), Spain. E-mail: [email protected] 10 3: Department of Biology, UniversidadDraft Autónoma de Madrid, Spain. E-mail: [email protected] 4: Department of Pharmacology, Pharmacognosy and Botany. Universidad Complutense de Madrid, Spain. E-mail: [email protected] 5: Centro de Biología Molecular Severo Ochoa (UAM-CSIC), Universidad Autónoma 15 de Madrid, Spain. E-mail: [email protected] *: corresponding author. E-mail: [email protected] Phone: +34 914 974 303 1 https://mc06.manuscriptcentral.com/cjm-pubs Canadian Journal of Microbiology Page 2 of 28 20 Abstract Soda lakes are inhabited by important haloalkaliphilic microbial communities that are well adapted to these extreme characteristics. The surface waters of the haloalkaline Mono Lake (CA, USA) are alkaline but, in contrast to its bottom waters, do not present high salinity. We have studied the microbiota present in the shoreline 25 sediments of Mono Lake using next-generation sequencing techniques. The statistical indexes showed that Bacteria had a higher richness, diversity and evenness compared to Archaea. 17 phyla and 8 "candidate divisions", were identified among the Bacteria, with a predominance of the phyla Firmicutes, Proteobacteria and Bacteroidetes. Among the Proteobacteria, there was a notable 30 presence of Rhodoplanes and Drafta high diversity of sulfate-reducing (SRB) Deltaproteobacteria, in accordance with the high sulfate-reducing activity detected in soda lakes. Numerous families of bacterial fermenters were identified among the Firmicutes. The Bacteroides were represented by several environmental groups that have not yet been isolated. Since final organic matter in anaerobic 35 environments with high sulfate contents is mineralized mainly by SRB, very little methanogenic archaeal biodiversity was detected. Only two genera, Methanocalculus and Methanosarcina, were retrieved. The species similarities described indicate that a significant number of the OTUs identified may represent new species. 40 Key words: Mono Lake, anaerobic sediments, microbial communities, soda lakes, pyrosequencing. 2 https://mc06.manuscriptcentral.com/cjm-pubs Page 3 of 28 Canadian Journal of Microbiology INTRODUCTION Mono Lake is an alkaline lake located in the rain shadow of the Sierra Nevada in 45 eastern California. The lake is elongated, 18–19 km by 13.5 km, with a surface area covering about 200 km2 (Jayko et al. 2013). The water table environment is covered by a typical highly-specialized phreatohalophytic salt-desert vegetation type: a plant community structured by Sarcobatus vermiculatus (black greasewood), an intricate shrub in the Chenopodiaceae family. This community grows around salt 50 lakes and on brackish valley bottoms with at least a seasonal water table. Soda lakes are widespread throughout the world: for instance the Siberian and Kulunda Steppes in Russia, the Mono and Big Soda Lakes in North America, the Magadi, Natron and Bogoria LakesDraft in Kenya, or the Wadi Natrun in Egypt (Zhao et al. 2014). Due to its high salinity and as consequence of variations in freshwater 55 input and climate conditions, Mono Lake, in common with other soda lakes, is subject to chemical stratification periods, from meromixis to monomixis. In general, the Mono Lake monimolimnion is anoxic and hypersaline, and salinities have been reported from 67 (Blinn 1993) to 94 g L-1 (Melack and Jellison 1998). Microbiologists have paid special attention to the microbiota present in hypersaline 60 lakes, as they are good models for studying mechanisms for coping with stress and survival in extreme conditions, and particularly the biotechnological potential of the microorganisms inhabiting these extreme environments. Exoenzymes, secondary metabolites and organic compatible solutes offer potential uses for numerous industries (Zhao et al. 2014). An overview of the microbial communities inhabiting 65 soda lake environments, with special emphasis on the Lonar Lake (India), was published by Antony and co-workers (Antony et al., 2013). These authors compiled 3 https://mc06.manuscriptcentral.com/cjm-pubs Canadian Journal of Microbiology Page 4 of 28 the prominent bacteria and archaea commonly detected in African, North American and Eurasian soda lakes in a single table. Water column inhabitants with metabolic abilities like phototrophic bacteria, sulfur-oxidizing bacteria and aerobic 70 methylotrophs are abundant in the aerobic layers of soda lakes and have been studied in depth (review by Antony et al., 2013). Organotrophs in the Clostridiales and Halanaerobiales orders were the predominant anaerobes in the anaerobic sediments of East African soda lakes (Jones et al., 1998). In a study by Wani et al. (2006), sequences retrieved from Lonar Lake 75 sediments were mostly related to Firmicutes, Proteobacteria and Actinobacteria. Sulfate-reduction is a frequent activity in soda lake sediments. A study by Foti et al. (2007) showed the dominance ofDraft phylotypes related to Desulfovibrionales and Desulfobacterales. Several litho- and heterotrophic sulfate-reducing bacteria (SRB) have been isolated from soda lakes in the Kulunda Steppe (Sorokin et al., 2011). 80 Despite the obvious domination of sulfidogenesis as the therminal anaerobic process in the hypersaline soda lakes of the Kulunda Steppe (Altai, southwestern Siberia), high concentrations of methane were detected in their anaerobic sediments (Sorokin et al., 2015a). In fact, this study identified the genera Methanolobus, Methanosalsum, Methanocalculus and Methanosaeta. 85 Methanocalculus spp. have been isolated from sediments of Russian soda lakes (Zhilina et al. 2013; Sorokin et al. 2015b). Other methanogenic archaea related to Methanosarcina, Methanocalculus and Methanoculleus were previously described in Lonar Lake sediments (rev. Antony et al., 2013). Sulfate-reducing activity (Scholten et al. 2005; Stam et al. 2010) and the microbial 90 arsenic cycle (Kulp et al. 2006, 2008; Oremland et al. 2004) have been the main 4 https://mc06.manuscriptcentral.com/cjm-pubs Page 5 of 28 Canadian Journal of Microbiology focus of microbiologists at Mono Lake. As gas seeps –mainly methane– are fairly common within and around Mono Lake (Oremland et al., 1987), the aim of the present study was to extend the knowledge of the prokaryotic communities (bacteria and archaea) inhabiting shoreline anaerobic lake sediments, an alkaline low-salt 95 content environment not yet explored from the microbiological point of view. To date only two reports have considered the Mono Lake microbial communities as a whole, both of which used classical molecular approaches (Hollibaugh el al. 2001; Humayoun el al. 2003). Since next-generation sequencing (NGS) techniques can overcome the constraints imposed by classical molecular techniques, our results 100 provide a new insight into the composition of the phylogenetic group at different taxonomic levels, indicating the anaerobic metabolic capabilities of the Mono Lake taxa identified by pyrosequencing. Draft MATERIALS AND METHODS 105 Sampling and physicochemical analysis Three sediment samples were collected along the Mono Lake shoreline (California: Mono Co. Mono Lake, flooded soils close to Test Station Road; 119 ̊ 13’ W, 37 ̊ 56’ N) and pooled together in a composite sample for metagenomic DNA extraction and pyrosequencing. 110 Redox potential, pH and dissolved oxygen were measured on-site with a YSI- 650MSD multiprobe-meter (YSI Inc., Ohio, USA). Conductivity and salinity were measured with a WTW LF320 conductivity meter equipped with a TetraCon 325 probe. Total dissolved solids were measured by drying a volume of water filtered by a 0.25 µm filter overnight at 105°C, and weighing the residue. Ions were measured 5 https://mc06.manuscriptcentral.com/cjm-pubs Canadian Journal of Microbiology Page 6 of 28 115 by ion chromatography using a Dionex DX600 equipped with an ED50 electrochemical conductivity detector. 1 ml/min 9mM Na2CO3, and 1 ml/min 25mN H2SO4, were used as eluents for anions and cations respectively. Analysis of bacterial communities: DNA extraction, PCR amplification and 454 pyrosequencing 120 Total DNA extractions were performed using the FastDNA Spin kit for soil BIO101 (MPBio). Invitrogen