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      Active microbial biofilms in deep poor porous continental subsurface rocks

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          Abstract

          Deep continental subsurface is defined as oligotrophic environments where microorganisms present a very low metabolic rate. To date, due to the energetic cost of production and maintenance of biofilms, their existence has not been considered in poor porous subsurface rocks. We applied fluorescence in situ hybridization techniques and confocal laser scanning microscopy in samples from a continental deep drilling project to analyze the prokaryotic diversity and distribution and the possible existence of biofilms. Our results show the existence of natural microbial biofilms at all checked depths of the Iberian Pyrite Belt (IPB) subsurface and the co-occurrence of bacteria and archaea in this environment. This observation suggests that multi-species biofilms may be a common and widespread lifestyle in subsurface environments.

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          Metabolic dependencies drive species co-occurrence in diverse microbial communities.

          Microbial communities populate most environments on earth and play a critical role in ecology and human health. Their composition is thought to be largely shaped by interspecies competition for the available resources, but cooperative interactions, such as metabolite exchanges, have also been implicated in community assembly. The prevalence of metabolic interactions in microbial communities, however, has remained largely unknown. Here, we systematically survey, by using a genome-scale metabolic modeling approach, the extent of resource competition and metabolic exchanges in over 800 communities. We find that, despite marked resource competition at the level of whole assemblies, microbial communities harbor metabolically interdependent groups that recur across diverse habitats. By enumerating flux-balanced metabolic exchanges in these co-occurring subcommunities we also predict the likely exchanged metabolites, such as amino acids and sugars, that can promote group survival under nutritionally challenging conditions. Our results highlight metabolic dependencies as a major driver of species co-occurrence and hint at cooperative groups as recurring modules of microbial community architecture.
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            Microbial life under extreme energy limitation.

            A great number of the bacteria and archaea on Earth are found in subsurface environments in a physiological state that is poorly represented or explained by laboratory cultures. Microbial cells in these very stable and oligotrophic settings catabolize 10⁴- to 10⁶-fold more slowly than model organisms in nutrient-rich cultures, turn over biomass on timescales of centuries to millennia rather than hours to days, and subsist with energy fluxes that are 1,000-fold lower than the typical culture-based estimates of maintenance requirements. To reconcile this disparate state of being with our knowledge of microbial physiology will require a revised understanding of microbial energy requirements, including identifying the factors that comprise true basal maintenance and the adaptations that might serve to minimize these factors.
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              Optimizing fluorescent in situ hybridization with rRNA-targeted oligonucleotide probes for flow cytometric identification of microorganisms.

              A combination of fluorescent rRNA-targeted oligonucleotide probes ("phylogenetic stains") and flow cytometry was used for a high resolution automated analysis of mixed microbial populations. Fixed cells of bacteria and yeasts were hybridized in suspension with fluorescein- or tetramethylrhodamine-labeled oligonucleotide probes complementary to group-specific regions of the 16S ribosomal RNA (rRNA) molecules. Quantifying probe-conferred cell fluorescence by flow cytometry, we could discriminate between target and nontarget cell populations. We critically examined changes of the hybridization conditions, kinetics of the hybridization, and posthybridization treatments. Intermediate probe concentrations, addition of detergent to the hybridization buffer, and a posthybridization washing step were found to increase the signal to noise ratio. We could demonstrate a linear correlation between growth rate and probe-conferred fluorescence of Escherichia coli and Pseudomonas cepacia cells. Oligonucleotides labeled with multiple fluorochromes showed elevated levels of nonspecific binding and therefore could not be used to lower the detection limits, which still restrict studies with fluorescing rRNA-targeted oligonucleotide probes to well-growing microbial cells. Two probes of different specificities--one labeled with fluorescein, the other with tetramethylrhodamine--could be applied simultaneously for dual color analysis.
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                Author and article information

                Contributors
                ramils@cbm.csic.es
                Journal
                Sci Rep
                Sci Rep
                Scientific Reports
                Nature Publishing Group UK (London )
                2045-2322
                24 January 2018
                24 January 2018
                2018
                : 8
                : 1538
                Affiliations
                [1 ]ISNI 0000000119578126, GRID grid.5515.4, Centro de Biología Molecular Severo Ochoa (UAM-CSIC), , Facultad de Ciencias, Universidad Autónoma de Madrid, ; 28049 Madrid, Spain
                [2 ]ISNI 0000 0001 2157 0406, GRID grid.7870.8, Instituto de Ingeniería Biológica y Médica, , Escuelas de Ingeniería, Medicina y Ciencias Biológicas, Departamento de Ingeniería Hidráulica y Ambiental, Escuela de Ingeniería, Pontificia Universidad Católica de Chile, Av Vicuña Mackenna, ; 4860 Santiago, Chile
                [3 ]Centro de Astrobiología (CSIC-INTA), Ctra de Ajalvir km 4, Torrejón de Ardoz, 28850 Madrid, Spain
                Article
                19903
                10.1038/s41598-018-19903-z
                5784017
                29367593
                7772ffb4-60a8-4cbd-8695-e9d7005749e7
                © The Author(s) 2018

                Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as 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 images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

                History
                : 13 November 2017
                : 8 January 2018
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