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Quantification of cell‐specific substrate uptake by probe‐defined bacteria under in situ conditions by microautoradiography and fluorescence in situ hybridization
Author(s) -
Nielsen Jeppe Lund,
Christensen Dinna,
Kloppenborg Marie,
Nielsen Per Halkjær
Publication year - 2003
Publication title -
environmental microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.954
H-Index - 188
eISSN - 1462-2920
pISSN - 1462-2912
DOI - 10.1046/j.1462-2920.2003.00402.x
Subject(s) - biology , bacteria , segmented filamentous bacteria , in situ , substrate (aquarium) , population , fluorescence in situ hybridization , biochemistry , incubation , fluorescence , candidatus , biophysics , microbiology and biotechnology , activated sludge , 16s ribosomal rna , chemistry , genetics , ecology , sewage treatment , demography , physics , organic chemistry , quantum mechanics , sociology , gene , engineering , chromosome , waste management
Summary A technique based on quantitative microautoradiography (QMAR) and fluorescence in situ hybridization (FISH) was developed and evaluated in order to determine the quantitative uptake of specific substrates in probe‐defined filamentous bacteria directly in a complex system. The technique, QMAR‐FISH, has a resolution of a single cell and is based on an improved fixation protocol and the use of an internal standard of bacteria with known specific radioactivity. The method was used to study the in situ ecophysiology of the filamentous bacteria ‘ Candidatus Meganema perideroedes’ and Thiothrix sp. directly in an activated sludge system. The cellular uptake rate of tritium‐labelled substrates revealed an average cell‐specific uptake rate of 4.1 ¥ 10 −15 mol of acetate cell−1 h −1 and 3.1 ¥ 10 −15 mol of acetate cell−1 h −1 for the two filamentous species respectively. The two filamentous species had very similar activity in all cells along each filament. Surprisingly, the filaments within both probe‐defined populations had threefold variation in activity between the different filaments, demonstrating a large variation in activity level within a single population in a complex system. The substrate affinity ( K s ) for uptake of acetate of the cells within the two filamentous bacteria was determined by incubation with variable concentrations of labelled acetate. The K s values of the ‘ Candidatus Meganema perideroedes’ and the Thiothrix filamentous bacteria were determined to be 1.8 µM and 2.4 µM acetate respectively.