In lakes, seasonal phytoplankton blooms and allochthonous plant debris intensify particulate organic carbon fluxes to the lakebed. Microbes associated with these particles likely vary with organic substrate lability and redox conditions. To explore microbial compositional responses to these variables, we analyzed particle-associated and free-living assemblages in the permanently redox-stratified Fayetteville Green Lake using 16 S rRNA amplicon sequencing during the peak and end of cyanobacterial and photoautotrophic sulfur bacterial blooms. Assemblage compositions were strongly influenced by redox conditions and particle association. Assemblage compositions varied seasonally above the lower oxycline boundary (summer-generalist heterotrophs; autumn-iron reducers and specialist heterotrophs), but not in the anoxic region below. Particle-associated assemblages were less diverse than free-living assemblages and were dominated by heterotrophs that putatively metabolize complex organic substrates, purple sulfur bacteria, sulfur-cycling Desulfocapsa, and eukaryotic algae. The least diverse particle-associated assemblages occurred near the lower oxycline boundary, where microbial activities and abundances were highest, and anoxygenic photoautotrophs were enriched. The low-diversity particle-associated heterotrophs likely remineralize complex organic substrates, releasing simpler organic substrates to free-living assemblages during transit, thereby influencing surrounding microbial diversity and function. Our results challenge the paradigm that phytoplankton from the shallow photic zone are the primary contributor to the vertical flux. We suggest that photoautotrophic prokaryotes from the deep photic zone contribute significantly to deep-water carbon in this environment, and possibly in other oxygen-deficient waters with sulfidic photic zones. Furthermore, results suggest that seasonally variable terrestrial carbon and metal inputs also influence microbial diversity and function in similar systems.In permanently redox-stratified water bodies, biogenic particles sink through redox zones, and each zone has distinctive free-living planktonic microbial assemblages (Fuchsman et al. 2011;Suter et al. 2018). The interplay between particle-associated microbes and those free-living in surrounding waters likely differs among redox zones. Redox conditions can vary at macroscales with lake depth and at microscales within particles themselves (Wright et al. 2012).

BackgroundOur knowledge of the phylogeny and diversity of aquatic protists is rapidly increasing due to molecular surveys and next-generation sequencing approaches. This has led to a considerable discrepancy between the taxa known from cultures and those known from environmental 18S rRNA gene sequences. Hence, it is generally difficult to assign ecological functions to new taxa detected by culture-independent molecular approaches.Methodology/Principal FindingsA combination of unamended dark incubations and 18S rRNA sequencing was chosen to link molecular diversity data of uncultured protists with heterotrophic, presumably bacterivorous, growth. The incubations, conducted with Baltic Sea brackish water, resulted in a consistent shift from a protistan community dominated by phototrophs to one in which heterotrophs predominated. This was determined on the basis of cell abundance and 18S rRNA sequences derived from fingerprint analysis and clone libraries. The bulk of enriched phylotypes after incubation were related to hitherto uncultured marine taxa within chrysophytes, ochrophytes, choanoflagellates, cercozoans, and picobiliphytes, mostly represented in recently established or here defined environmental clades. Their growth in the dark, together with coinciding results from studies with a similar objective, provides evidence that these uncultured taxa represent heterotrophic or mixotrophic species.Conclusions/SignificanceThese findings shed some light into the trophic role of diverse uncultured protists especially within functionally heterogeneous groups (e.g., chrysophytes, ochrophytes) and groups that appear to be puzzling with regard to their nutrition (picobiliphytes). Additionally, our results indicate that the heterotrophic flagellate community in the southwestern Baltic Sea is dominated by species of marine origin. The combination of unamended incubations with molecular diversity analysis is thus confirmed as a promising approach to explore the trophic mode of environmentally relevant protist taxa for which only sequence data are currently available.

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The suitability of stable isotope probing (SIP) and Raman microspectroscopy to measure growth rates of heterotrophic bacteria at the single-cell level was evaluated. Label assimilation into E. coli biomass during growth on a complex 13 C-labeled carbon source was monitored in time course experiments. 13 C-incorporation into various biomolecules was measured by spectral “red shifts” of Raman-scattered emissions. The 13 C- and 12 C-isotopologues of the amino acid phenylalanine (Phe) proved to be a quantitatively accurate reporter molecules of cellular isotopic fractional abundances ( f cell ). Values of f cell determined by Raman microspectroscopy and independently by isotope-ratio mass spectrometry (IRMS) over a range of isotopic enrichments were statistically indistinguishable. Progressive labeling of Phe in E. coli cells among a range of 13 C/ 12 C organic substrate admixtures occurred predictably through time. Relative isotopologue abundances of Phe determined by Raman spectral analysis enabled accurate calculation of bacterial growth rates as confirmed independently by optical density (OD) measurements. Results demonstrate that combining stable isotope probing (SIP) and Raman microspectroscopy can be a powerful tool for studying bacterial growth at the single-cell level when grown on defined or complex organic 13 C-carbon sources even in mixed microbial assemblages. Importance: Population growth dynamics and individual cell growth rates are the ultimate expressions of a microorganism’s fitness to its environmental conditions, whether natural or engineered. Natural habitats and many industrial settings harbor complex microbial assemblages. Their heterogeneity in growth responses to existing and changing conditions is often difficult to grasp by standard methodologies. In this proof of concept study, we tested whether Raman microspectroscopy can reliably quantify assimilation of isotopically-labeled nutrients into E. coli cells and enable determination of individual growth rates among heterotrophic bacteria. Raman-derived growth rate estimates were statistically indistinguishable from those derived by standard optical density measurements of the same cultures. Raman microspectroscopy also can be combined with methods for phylogenetic identification. We report development of Raman-based techniques that enable researchers to directly link genetic identity to functional traits and rate measurements of single cells within mixed microbial assemblages, currently a major technical challenge in microbiological research.

The deep basins of the central Baltic Sea are characterized by anoxic and sulfidic bottom water and steep vertical pelagic redox gradients. The highly active prokaryotic assemblages of this and other redox transition zones have been intensely studied, while knowledge on the protistan communities remains fragmentary. Thus, we conducted a multi-annual microscopybased study, combined in one year with 18S rRNA gene and transcript-based DGGE fingerprints to identify the dominant protist taxa and to assess their vertical distribution. Both approaches, applied in high vertical resolution, demonstrated strong stratifications of the protist community composition along the redox gradient. The suboxic zone was dominated by dinoflagellates and oligotrichous ciliates related to Strombidium, whereas the interface and upper sulfidic zone were dominated by ciliates of the genera Mesodinium and Metacystis. Several flagellate taxa within the jakobids, euglenozoans and choanoflagellates occurred exclusively in sulfidic water. Our morphological approach indicates that the pelagic redoxclines of the central Baltic Sea are inhabited by a stable and characteristic protist community. Incongruously, certain taxa (e.g. Mesodinium and Metacystis sp.) which remained undetected by the molecular fingerprinting technique could be identified and enumerated by microscopic observations, whereas small and virtually amorphous protists (especially flagellates) were detected only by sequencing DGGE bands. Fine-scaled assessment of dominant protists in distinct redox strata is a crucial step in understanding their impact and interactions with the prokaryotic world and the biogeochemical processes they mediate in these zones.