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Quantification of ratios of Bacteria and Archaea in methanogenic microbial community by fluorescence in situ hybridization and fluorescence spectrometry
Authors:Tay  Tiong-Lee Stephen  Ivanov  Volodymyr  Kim  In Soo  Feng  Lin  Tay  Joo-Hwa
Institution:(1) School of Civil and Structural Engineering, Nanyang Technological University, Block N1, 50 Nanyang Avenue, 639798 Singapore, Singapore;(2) Department of Environmental Science and Engineering, Kwangju Institute of Science and Technology, Puk-gu, Kwangju, Republic of Korea
Abstract:16S rRNA-targeted oligonucleotide probes for Bacteria (Eub338) and Archaea (Arc915) were used for whole-cell, fluorescence in situ hybridization (FISH) to quantify the ratio of these microbial groups in an anaerobic digester. The quantity of specifically bound (hybridized) probe was measured by fluorescence spectrometry and evaluated by analysing the dissociation curve of the hybrids, by the measurement of the binding with a nonsense probe, and by the competitive inhibition of the binding of the labelled probe by the corresponding unlabelled probe. Specific binding of oligonucleotide probes with the biomass of anaerobes was 40–50% of their total binding. The ratio of Arc915 and Eub338 probes hybridized with rRNAs of the cells in anaerobic sludge was 0.50. Measurement of FISH by fluorescence spectrometry appears to be a suitable method for quantification of the microbial community of anaerobes.
Keywords:Bacteria and Archaea  fluorescence in situ hybridization  fluorescence spectrometry  oligonucleotide probes
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