Microbial communities that thrive in subterranean consolidated sediments are largely unknown owing to the difficulty of extracting DNA. As this difficulty is often attributed to DNA binding onto the silica-bearing sediment matrix, we developed a DNA extraction method for consolidated sediment from the deep subsurface in which silica minerals were dissolved by being heated under alkaline conditions. NaOH concentrations (0.07 and 0.33 N), incubation temperatures (65 and 94 °C) and incubation times (30-90 min) before neutralization were evaluated based on the copy number of extracted prokaryotic DNA. Prokaryotic DNA was detected by quantitative PCR analysis after heating the sediment sample at 94 °C in 0.33 N NaOH solution for 50-80 min. Results of 16S rRNA gene sequence analysis of the extracted DNA were all consistent with regard to the dominant occurrence of the metallophilic bacterium, Cupriavidus metallidurans, and Pseudomonas spp. Mineralogical analysis revealed that the dissolution of a silica mineral (opal-CT) during alkaline treatment was maximized at 94 °C in 0.33 N NaOH solution for 50 min, which may have resulted in the release of DNA into solution. Because the optimized protocol for DNA extraction is applicable to subterranean consolidated sediments from a different locality, the method developed here has the potential to expand our understanding of the microbial community structure of the deep biosphere.
Understanding the behavior of uranium (U) in the environment is essential not only for the protection of aquifers from U contamination but also for predicting the fate of U and other actinides disposed of in deep geological settings. It has long been believed that the redox chemistry of U can be simply predicted by thermodynamics and that the development of a low redox potential is a sufficient condition for U reduction. However, recent studies have demonstrated that redox transformations of U are controlled by kinetic factors that are strongly influenced by microbial activity. Although abiological U oxidation proceeds efficiently under oxygenic conditions, abiological reduction of U is inhibited by the formation of negatively charged U(VI) CO 3 complexes that prevail in nature. Phylogenetically diverse microorganisms are capable of enzymatically reducing U(VI) CO 3 complexes to form U(IV) bearing minerals such as uraninite (UO 2+x . This complex is mainly produced by the microbial reduction of Fe(III) in natural systems. Thus, U(VI) reduction is controlled both directly and indirectly, at least in part, by microbial activity. Several mechanisms of U oxidation under anoxic conditions have been revealed recently by laboratory and field studies. U(IV) is abiologically oxidized by Fe(III) and Mn(IV) oxides. Microbial reduction of nitrate to molecular nitrogen, which occurs following the depletion of O 2 , produces nitrogen intermediates including nitrite (NO 2 -), nitrous oxide (NO), and nitric oxide (N 2 O). Although the nitrogen intermediates oxidize U(IV), poorly crystalline Fe(III) oxide minerals resulting from the oxidation of aqueous Fe(II) species by the nitrogen intermediates oxidize U(IV) more efficiently than the nitrogen intermediates alone. Remarkably, the formation of Ca U(VI) CO 3 complexes resulting from increased levels of Ca 2+ and/or HCO 3 -leads to the reoxidation of bioreduced U(IV) under reducing conditions. These geomicrobiological factors pose challenges in manipulating and/or predicting the mobility and fate of U in complex and heterogeneous environmental settings.
Understanding the behavior of uranium (U) in the environment is essential not only for the protection of aquifers from U contamination but also for predicting the fate of U and other actinides disposed of in deep geological settings. It has long been believed that the redox chemistry of U can be simply predicted by thermodynamics and that the development of a low redox potential is a sufficient condition for U reduction. However, recent studies have demonstrated that redox transformations of U are controlled by kinetic factors that are strongly influenced by microbial activity. Although abiological U oxidation proceeds efficiently under oxygenic conditions, abiological reduction of U is inhibited by the formation of negatively charged U(VI) CO 3 complexes that prevail in nature. Phylogenetically diverse microorganisms are capable of enzymatically reducing U(VI) CO 3 complexes to form U(IV) bearing minerals such as uraninite (UO 2+x ). The only abiological pathway currently known for the reduction of U(VI) CO 3 complexes involves the Fe(II) monohydroxo surface complex Fe III OFe II OH 0 . This complex is mainly produced by the microbial reduction of Fe(III) in natural systems. Thus, U(VI) reduction is controlled both directly and indirectly, at least in part, by microbial activity. Several mechanisms of U oxidation under anoxic conditions have been revealed recently by laboratory and field studies. U(IV) is abiologically oxidized by Fe(III) and Mn(IV) oxides. Microbial reduction of nitrate to molecular nitrogen, which occurs following the depletion of O 2 , produces nitrogen intermediates including nitrite (NO 2 -), nitrous oxide (NO), and nitric oxide (N 2 O). Although the nitrogen intermediates oxidize U(IV), poorly crystalline Fe(III) oxide minerals resulting from the oxidation of aqueous Fe(II) species by the nitrogen intermediates oxidize U(IV) more efficiently than the nitrogen intermediates alone. Remarkably, the formation of Ca U(VI) CO 3 complexes resulting from increased levels of Ca 2+ and/or HCO 3 -leads to the reoxidation of bioreduced U(IV) under reducing conditions. These geomicrobiological factors pose challenges in manipulating and/or predicting the mobility and fate of U in complex and heterogeneous environmental settings.
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