Extensive physiological analyses of different microbial community members in many samples are difficult because of the restricted number of target populations that can be investigated in reasonable time by standard substrate-mediated isotope-labeling techniques. The diversity and ecophysiology of Rhodocyclales in activated sludge from a full-scale wastewater treatment plant were analyzed following a holistic strategy based on the isotope array approach, which allows for a parallel functional probing of different phylogenetic groups. Initial diagnostic microarray, comparative 16S rRNA gene sequence, and quantitative fluorescence in situ hybridization surveys indicated the presence of a diverse community, consisting of an estimated number of 27 operational taxonomic units that grouped in at least seven main Rhodocyclales lineages. Substrate utilization profiles of probe-defined populations were determined by radioactive isotope array analysis and microautoradiography-fluorescence in situ hybridization of activated sludge samples that were briefly exposed to different substrates under oxic and anoxic, nitrate-reducing conditions. Most detected Rhodocyclales groups were actively involved in nitrogen transformation, but varied in their consumption of propionate, butyrate, or toluene, and thus in their ability to use different carbon sources in activated sludge. This indicates that the functional redundancy of nitrate reduction and the functional versatility of substrate usage are important factors governing niche overlap and differentiation of diverse Rhodocyclales members in this activated sludge.

/pdf/isotope-array-analysis-of-rhodocyclales-uncovers-functional-5ef3m4yayd.pdf

Isotope array analysis of Rhodocyclales uncovers functional redundancy and versatility in an activated sludge

Isotope array analysis of Rhodocyclales uncovers functional redundancy and versatility in an activated sludge.

Most heterotrophic bacteria assimilate CO(2) in various carboxylation reactions during biosynthesis. In this study, assimilation of (14)CO(2) by heterotrophic bacteria was used for isotope labeling of active microorganisms in pure cultures and environmental samples. Labeled cells were visualized by microautoradiography (MAR) combined with fluorescence in situ hybridization (FISH) to obtain simultaneous information about activity and identity. Cultures of Escherichia coli and Pseudomonas putida assimilated sufficient (14)CO(2) during growth on various organic substrates to obtain positive MAR signals. The MAR signals were comparable with the traditional MAR approach based on uptake of (14)C-labeled organic substrates. Experiments with E. coli showed that (14)CO(2) was assimilated during both fermentation and aerobic and anaerobic respiration. The new MAR approach, HetCO(2)-MAR, was evaluated by targeting metabolic active filamentous bacteria, including "Candidatus Microthrix parvicella" in activated sludge. "Ca. Microthrix parvicella" was able to take up oleic acid under anaerobic conditions, as shown by the traditional MAR approach with [(14)C]oleic acid. However, the new HetCO(2)-MAR approach indicated that "Ca. Microthrix parvicella," did not significantly grow on oleic acid under anaerobic conditions with or without addition of NO(2)(-), whereas the addition of O(2) or NO(3)(-) initiated growth, as indicated by detectable (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.

Isotope Labeling and Microautoradiography of Active Heterotrophic Bacteria on the Basis of Assimilation of 14CO2

In this study we used the assimilation of isotope labeled CO2 to measure the substrate preferences by two different bioaugmentation mixtures proposed for bioremediation of diesel oil contamination. All active microorganisms assimilate CO2 in various carboxylation processes involved in growth. The CO2 assimilation by the two mixtures was measured upon addition of glucose, diesel oil or specific compounds present in diesel oil (naphthalene, toluene, hexadecane, and octane). It was shown that within short term incubations with diesel oil (<5 h), one bioaugmentation mixture was superior to the other regarding the assimilation of CO2. This observation was confirmed in a labor-intensive long term microcosm study (60 days). The applied method open various possibilities for fast pre-testing of substrate-preferences by microbial-bioaugmentation mixtures without microcosm experiments, onsite tests, and complicated chemical analysis. This study also demonstrates the possibility to obtain further information on the substrate preferences at a single cell level of phylogenetically defined microbial subgroups in bioaugmentation mixtures, based on combined analyses of microautoradiography and fluorescence in situ hybridization.

H A Barker

Papers

The Chemistry and Metabolism of Bacteria