Isotope Labeling and Microautoradiography of Active Heterotrophic Bacteria on the Basis of Assimilation of ¹⁴ CO ₂

Abstract

14 CO 2 assimilation. This is a metabolic feature that has not been described previously for filamentous bacteria. Such information could not have been derived by using the traditional MAR procedure, whereas the new HetCO 2 -MAR approach differentiates better between substrate uptake and substrate metabolism that result in growth. The HetCO 2 -MAR results were supported by stable isotope analysis of 13 C-labeled phospholipid fatty acids from activated sludge incubated under aerobic and anaerobic conditions in the presence of 13 CO 2 . In conclusion, the novel HetCO 2 -MAR approach expands the possibility for studies of the ecophysiology of uncultivated microorganisms.

Tracking heavy water (D 2 O) incorporation for identifying and sorting active microbial cells

“…Microbiologists have tested fluorescent probes and stains to quantify cellular rRNA or its precursors (18,19) and membrane potential (20) as activity measures, but cellular rRNA levels often do not correlate with activity (21) and membrane potential stains perform differently with different cell types (22). Recently, heterotrophic 14 CO 2 fixation was used to infer the general activity of heterotrophic microbes semiquantitatively (23), but the extent of heterotrophic CO 2 fixation can vary widely between different species and also depends on the heterotrophic substrate used for growth (24)(25)(26)(27). In addition, fluorescence-based methods have been developed for measuring single-cell biosynthesis of macromolecules (28)(29)(30), but the generalizability and usability of these new methods, which rely on the addition of substrates with modified functional groups, are still either largely untested in complex ecosystems or have known biases across different species.…”

High bicarbonate assimilation in the dark by Arctic bacteria

“…This process was found to be widespread and tightly coupled to cell growth and has been used as a measure of bacterial metabolic activity (Romanenko, 1964;Roslev et al, 2004;Hesselsoe et al, 2005). However, its use as a proxy for bacterial heterotrophic production was ruled out due to the large uncertainty in the ratio of inorganic C fixed to total C assimilated (Overbeck and Daley, 1973).…”

Who eats what, where and when? Isotope-labelling experiments are coming of age

“…As with SIP, synthesizing complex organic compounds is challenging and requires safety precautions, because of the radioactive label. Alternatively, heterotrophic growth can be studied by incubating environmental samples with an unlabelled substrate in the presence of 14 CO 2 (Hesselsoe et al, 2005). Because heterotrophic organisms assimilate CO 2 during growth under aerobic and anaerobic conditions, this approach (HetCO 2 -MAR) is affordable and has the convenience of using the same labelled compound for assessing growth on multiple substrates.…”

“…Because heterotrophic organisms assimilate CO 2 during growth under aerobic and anaerobic conditions, this approach (HetCO 2 -MAR) is affordable and has the convenience of using the same labelled compound for assessing growth on multiple substrates. Such an approach has been demonstrated for pure cultures and filamentous 'Candidatus Microthrix parvicella' in activated sludge under different electron donor/ acceptor combinations (Hesselsoe et al, 2005). If HetCO 2 -MAR is used as a discovery tool, it is important to remember that not only heterotrophic substrate-consuming but also all active autotrophic microorganisms will be labelled.…”