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      Evidence of the Generation of Isosaccharinic Acids and Their Subsequent Degradation by Local Microbial Consortia within Hyper-Alkaline Contaminated Soils, with Relevance to Intermediate Level Radioactive Waste Disposal

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          Abstract

          The contamination of surface environments with hydroxide rich wastes leads to the formation of high pH (>11.0) soil profiles. One such site is a legacy lime works at Harpur Hill, Derbyshire where soil profile indicated in-situ pH values up to pH 12. Soil and porewater profiles around the site indicated clear evidence of the presence of the α and β stereoisomers of isosaccharinic acid (ISA) resulting from the anoxic, alkaline degradation of cellulosic material. ISAs are of particular interest with regards to the disposal of cellulosic materials contained within the intermediate level waste (ILW) inventory of the United Kingdom, where they may influence radionuclide mobility via complexation events occurring within a geological disposal facility (GDF) concept. The mixing of uncontaminated soils with the alkaline leachate of the site resulted in ISA generation, where the rate of generation in-situ is likely to be dependent upon the prevailing temperature of the soil. Microbial consortia present in the uncontaminated soil were capable of surviving conditions imposed by the alkaline leachate and demonstrated the ability to utilise ISAs as a carbon source. Leachate-contaminated soil was sub-cultured in a cellulose degradation product driven microcosm operating at pH 11, the consortia present were capable of the degradation of ISAs and the generation of methane from the resultant H 2/CO 2 produced from fermentation processes. Following microbial community analysis, fermentation processes appear to be predominated by Clostridia from the genus Alkaliphilus sp, with methanogenesis being attributed to Methanobacterium and Methanomassiliicoccus sp. The study is the first to identify the generation of ISA within an anthropogenic environment and advocates the notion that microbial activity within an ILW-GDF is likely to influence the impact of ISAs upon radionuclide migration.

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          Isolation and direct complete nucleotide determination of entire genes. Characterization of a gene coding for 16S ribosomal RNA.

          Using a set of synthetic oligonucleotides homologous to broadly conserved sequences in-vitro amplification via the polymerase chain reaction followed by direct sequencing results in almost complete nucleotide determination of a gene coding for 16S ribosomal RNA. As a model system the nucleotide sequence of the 16S rRNA gene of M.kansasii was determined and found to be 98.7% homologous to that of M.bovis BCG. This is the first report on a contiguous sequence information of an entire amplified gene spanning 1.5 kb without any subcloning procedures.
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            Methanomassiliicoccus luminyensis gen. nov., sp. nov., a methanogenic archaeon isolated from human faeces.

            During attempts to obtain novel, human-associated species of the domain Archaea, a coccoid micro-organism, designated strain B10(T), was isolated in pure culture from a sample of human faeces collected in Marseille, France. On the basis of its phenotypic characteristics and 16S rRNA and mcrA gene sequences, the novel strain was classified as a methanogenic archaeon. Cells of the strain were non-motile, Gram-staining-positive cocci that were approximately 850 nm in diameter and showed autofluorescence at 420 nm. Cells were lysed by 0.1% (w/v) SDS. With hydrogen as the electron donor, strain B10(T) produced methane by reducing methanol. The novel strain was unable to produce methane when hydrogen or methanol was the sole energy source. In an atmosphere containing CO(2), strain B10(T) could not produce methane from formate, acetate, trimethylamine, 2-butanol, 2-propanol, cyclopentanol, 2-pentanol, ethanol, 1-propanol or 2,3-butanediol. Strain B10(T) grew optimally with 0.5-1.0% (w/v) NaCl, at pH 7.6 and at 37 °C. It required tungstate-selenite for growth. The complete genome of the novel strain was sequenced; the size of the genome was estimated to be 2.05 Mb and the genomic DNA G+C content was 59.93 mol%. In phylogenetic analyses based on 16S rRNA gene sequences, the highest sequence similarities (98.0-98.7%) were seen between strain B10(T) and several uncultured, methanogenic Archaea that had been collected from the digestive tracts of a cockroach, a chicken and mammals. In the same analysis, the non-methanogenic 'Candidatus Aciduliprofundum boonei' DSM 19572 was identified as the cultured micro-organism that was most closely related to strain B10(T) (83.0% 16S rRNA gene sequence similarity). Each of the three treeing algorithms used in the analysis of 16S rRNA gene sequences indicated that strain B10(T) belongs to a novel order that is distinct from the Thermoplasmatales. The novel strain also appeared to be distinct from Methanosphaera stadtmanae DSM 3091(T) (72.9% 16S rRNA gene sequence similarity), another methanogenic archaeon that was isolated from human faeces and can use methanol in the presence of hydrogen. Based on the genetic and phenotypic evidence, strain B10(T) represents a novel species of a new genus for which the name Methanomassiliicoccus luminyensis gen. nov., sp. nov. is proposed. The type strain of the type species is B10(T) ( = DSM 24529(T) = CSUR P135(T)).
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              Novel primers for 16S rRNA-based archaeal community analyses in environmental samples.

              Next generation sequencing technologies for in depth analyses of complex microbial communities rely on rational primer design based on up-to-date reference databases. Most of the 16S rRNA-gene based analyses of environmental Archaea community composition use PCR primers developed from small data sets several years ago, making an update long overdue. Here we present a new set of archaeal primers targeting the 16S rRNA gene designed from 8500 aligned archaeal sequences in the SILVA database. The primers 340F-1000R showed a high archaeal specificity (<1% bacteria amplification) covering 93 and 97% of available sequences for Crenarchaeota and Euryarchaeota respectively. In silico tests of the primers revealed at least 38% higher coverage for Archaea compared to other commonly used primers. Empirical tests with clone libraries confirmed the high specificity of the primer pair to Archaea in three biomes: surface waters in the Arctic Ocean, the pelagic zone of a temperate lake and a methanogenic bioreactor. The clone libraries featured both Euryarchaeota and Crenarchaeota in variable proportions and revealed dramatic differences in the archaeal community composition and minimal phylogenetic overlap between samples. Copyright © 2010 Elsevier B.V. All rights reserved.
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                Author and article information

                Contributors
                Role: Academic Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                6 March 2015
                2015
                : 10
                : 3
                : e0119164
                Affiliations
                [1 ]Department of Biological Sciences, School of Applied Sciences, University of Huddersfield, Queensgate, Huddersfield, HD1 3DH, United Kingdom
                [2 ]School of Geography, Earth and Environmental Sciences, University of Birmingham, Edgbaston, Birmingham, B15 2TT, United Kingdom
                Chengdu Institute of Biology, CHINA
                Author notes

                Competing Interests: Radioactive Waste Management Limited is a wholly owned subsidiary of the Nuclear Decommissioning Authority, which is an Executive Non-Departmental Public Body of the Department of the Energy and Climate Change of the UK government. This does not alter the authors' adherence to PLOS ONE policies on sharing data and materials

                Conceived and designed the experiments: PNH SR CC JG. Performed the experiments: SR CC EG. Analyzed the data: PNH SR CC AL. Wrote the paper: SR PNH AL.

                Article
                PONE-D-14-54960
                10.1371/journal.pone.0119164
                4351885
                25748643
                8865c9df-2aad-41f8-b4ec-063c970fc9ed
                Copyright @ 2015

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

                History
                : 12 December 2014
                : 28 January 2015
                Page count
                Figures: 6, Tables: 1, Pages: 13
                Funding
                The work was partially supported by both the Engineering and Physical Sciences Research council under grant EP/I036354/1 and by Radioactive Waste Management Ltd. The remaining funds being provided by the University of Huddersfield. The funders had no role in study design, data collection and analysis associated with the paper. RWM ltd did review the paper prior to submission.
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