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      Sulfur Biogeochemistry of an Oil Sands Composite Tailings Deposit

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          Abstract

          Composite tailings (CT), an engineered, alkaline, saline mixture of oil sands tailings (FFT), processed sand and gypsum (CaSO 4; 1 kg CaSO 4 per m 3 FFT) are used as a dry reclamation strategy in the Alberta Oil Sands Region (AOSR). It is estimated that 9.6 × 10 8 m 3 of CT are either in, or awaiting emplacement in surface pits within the AOSR, highlighting their potential global importance in sulfur cycling. Here, in the first CT sulfur biogeochemistry investigation, integrated geochemical, pyrosequencing and lipid analyses identified high aqueous concentrations of ∑H 2S (>300 μM) and highly altered sulfur compounds composition; low cell biomass (3.3 × 10 6– 6.0 × 10 6 cells g −1) and modest bacterial diversity (H' range between 1.4 and 1.9) across 5 depths spanning 34 m of an in situ CT deposit. Pyrosequence results identified a total of 29,719 bacterial 16S rRNA gene sequences, representing 131 OTUs spanning19 phyla including 7 candidate divisions, not reported in oil sands tailings pond studies to date. Legacy FFT common phyla, notably, gamma and beta Proteobacteria, Firmicutes, Actinobacteria, and Chloroflexi were represented. However, overall CT microbial diversity and PLFA values were low relative to other contexts. The identified known sulfate/sulfur reducing bacteria constituted at most 2% of the abundance; however, over 90% of the 131 OTUs identified are capable of sulfur metabolism. While PCR biases caution against overinterpretation of pyrosequence surveys, bacterial sequence results identified here, align with phospholipid fatty acid (PLFA) and geochemical results. The highest bacterial diversities were associated with the depth of highest porewater [∑H 2S] (22–24 m) and joint porewater co-occurrence of Fe 2+ and ∑H 2S (6–8 m). Three distinct bacterial community structure depths corresponded to CT porewater regions of (1) shallow evident Fe (II) (<6 m), (2) co-occurring Fe (II) and ∑H 2S (6–8 m) and (3) extensive ∑H 2S (6–34 m) (UniFrac). Candidate divisions GNO2, NKB19 and Spam were present only at 6–8 m associated with co-occurring [Fe (II)] and [∑H 2S]. Collectively, results indicate that CT materials are differentiated from other sulfur rich environments by modestly diverse, low abundance, but highly sulfur active and more enigmatic communities (7 candidate divisions present within the 19 phyla identified).

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          A rapid method of total lipid extraction and purification.

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            Dehalococcoides mccartyi gen. nov., sp. nov., obligately organohalide-respiring anaerobic bacteria relevant to halogen cycling and bioremediation, belong to a novel bacterial class, Dehalococcoidia classis nov., order Dehalococcoidales ord. nov. and family Dehalococcoidaceae fam. nov., within the phylum Chloroflexi.

            Six obligately anaerobic bacterial isolates (195(T), CBDB1, BAV1, VS, FL2 and GT) with strictly organohalide-respiring metabolisms were obtained from chlorinated solvent-contaminated aquifers, contaminated and uncontaminated river sediments or anoxic digester sludge. Cells were non-motile with a disc-shaped morphology, 0.3-1 µm in diameter and 0.1-0.2 µm thick, and characteristic indentations on opposite flat sides of the cell. Growth occurred in completely synthetic, reduced medium amended with a haloorganic electron acceptor (mostly chlorinated but also some brominated compounds), hydrogen as electron donor, acetate as carbon source, and vitamins. No other growth-supporting redox couples were identified. Aqueous hydrogen consumption threshold concentrations were <1 nM. Growth ceased when vitamin B(12) was omitted from the medium. Addition of sterile cell-free supernatant of Dehalococcoides-containing enrichment cultures enhanced dechlorination and growth of strains 195 and FL2, suggesting the existence of so-far unidentified stimulants. Dechlorination occurred between pH 6.5 and 8.0 and over a temperature range of 15-35 °C, with an optimum growth temperature between 25 and 30 °C. The major phospholipid fatty acids were 14 : 0 (15.7 mol%), br15 : 0 (6.2 mol%), 16 : 0 (22.7 mol%), 10-methyl 16 : 0 (25.8 mol%) and 18 : 0 (16.6 mol%). Unusual furan fatty acids including 9-(5-pentyl-2-furyl)-nonanoate and 8-(5-hexyl-2-furyl)-octanoate were detected in strains FL2, BAV1 and GT, but not in strains 195(T) and CBDB1. The 16S rRNA gene sequences of the six isolates shared more than 98 % identity, and phylogenetic analysis revealed an affiliation with the phylum Chloroflexi and more than 10 % sequence divergence from other described isolates. The genome sizes and G+C contents ranged from 1.34 to 1.47 Mbp and 47 to 48.9 mol% G+C, respectively. Based on 16S rRNA gene sequence comparisons, genome-wide average nucleotide identity and phenotypic characteristics, the organohalide-respiring isolates represent a new genus and species, for which the name Dehalococcoides mccartyi gen. nov., sp. nov. is proposed. Isolates BAV1 ( = ATCC BAA-2100  = JCM 16839  = KCTC 5957), FL2 ( = ATCC BAA-2098  = DSM 23585  = JCM 16840  = KCTC 5959), GT ( = ATCC BAA-2099  = JCM 16841  = KCTC 5958), CBDB1, 195(T) ( = ATCC BAA-2266(T)  = KCTC 15142(T)) and VS are considered strains of Dehalococcoides mccartyi, with strain 195(T) as the type strain. The new class Dehalococcoidia classis nov., order Dehalococcoidales ord. nov. and family Dehalococcoidaceae fam. nov. are described to accommodate the new taxon.
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              Unexpected diversity and complexity of the Guerrero Negro hypersaline microbial mat.

              We applied nucleic acid-based molecular methods, combined with estimates of biomass (ATP), pigments, and microelectrode measurements of chemical gradients, to map microbial diversity vertically on a millimeter scale in a hypersaline microbial mat from Guerrero Negro, Baja California Sur, Mexico. To identify the constituents of the mat, small-subunit rRNA genes were amplified by PCR from community genomic DNA extracted from layers, cloned, and sequenced. Bacteria dominated the mat and displayed unexpected and unprecedented diversity. The majority (1,336) of the 1,586 bacterial 16S rRNA sequences generated were unique, representing 752 species (> or =97% rRNA sequence identity) in 42 of the main bacterial phyla, including 15 novel candidate phyla. The diversity of the mat samples differentiated according to the chemical milieu defined by concentrations of O(2) and H(2)S. Bacteria of the phylum Chloroflexi formed the majority of the biomass by percentage of bulk rRNA and of clones in rRNA gene libraries. This result contradicts the general belief that cyanobacteria dominate these communities. Although cyanobacteria constituted a large fraction of the biomass in the upper few millimeters (>80% of the total rRNA and photosynthetic pigments), Chloroflexi sequences were conspicuous throughout the mat. Filamentous Chloroflexi bacteria were identified by fluorescence in situ hybridization within the polysaccharide sheaths of the prominent cyanobacterium Microcoleus chthonoplastes, in addition to free living in the mat. The biological complexity of the mat far exceeds that observed in other polysaccharide-rich microbial ecosystems, such as the human and mouse distal guts, and suggests that positive feedbacks exist between chemical complexity and biological diversity. The sequences determined in this study have been submitted to the GenBank database and assigned accession numbers DQ 329539 to DQ 331020, and DQ 397339 to DQ 397511.
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                Author and article information

                Contributors
                Journal
                Front Microbiol
                Front Microbiol
                Front. Microbiol.
                Frontiers in Microbiology
                Frontiers Media S.A.
                1664-302X
                03 February 2016
                2015
                : 6
                : 1533
                Affiliations
                School of Geography and Earth Sciences, McMaster University Hamilton ON, Canada
                Author notes

                Edited by: Maria Dittrich, University of Toronto, Canada

                Reviewed by: Dennis A. Bazylinski, University of Nevada at Las Vegas, USA; Seung Gu Shin, Pohang University of Science and Technology, South Korea; Chris Weisener, University of Windsor, Canada

                *Correspondence: Lesley A. Warren warrenl@ 123456mcmaster.ca

                This article was submitted to Microbiotechnology, Ecotoxicology and Bioremediation, a section of the journal Frontiers in Microbiology

                Article
                10.3389/fmicb.2015.01533
                4737920
                26869997
                48e18e61-81ed-44d9-a2fd-710aa686d67e
                Copyright © 2016 Warren, Kendra, Brady and Slater.

                This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

                History
                : 01 October 2015
                : 21 December 2015
                Page count
                Figures: 7, Tables: 2, Equations: 1, References: 87, Pages: 14, Words: 10145
                Funding
                Funded by: Natural Sciences and Engineering Research Council of Canada 10.13039/501100000038
                Award ID: CRDPJ 403361-10
                Categories
                Microbiology
                Original Research

                Microbiology & Virology
                oil sands,composite tailings (ct) deposit,sulfur biogeochemistry,pyrosequencing,plfa,active bacterial sulfur cycling

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