Dynamic Phaeodactylum tricornutum exometabolites shape surrounding bacterial communities.

Roles of different ecological classes of algal exometabolites in regulating microbial community composition are not well understood. Here, we identify exometabolites from the model diatom Phaeodactylum tricornutum and demonstrate their potential to influence bacterial abundances. We profiled exometabolites across a time course of axenic algal growth using liquid chromatography-tandem mass spectrometry. We then investigated growth of 12 bacterial isolates on individual-identified exometabolites. Lastly, we compared responses of a P. tricornutum-adapted enrichment community to additions of two contrasting metabolites: selective growth substrate 4-hydroxybenzoic acid and putative signaling/facilitator molecule lumichrome. We identified 50 P. tricornutum metabolites and found distinct temporal accumulation patterns. Two exometabolites (of 12 tested) supported growth of distinct subsets of bacterial isolates. While algal exudates and algal presence drove similar changes in community composition compared with controls, exogenous 4-hydroxybenzoic acid addition promoted increased abundances of taxa that utilized it in isolation, and also revealed the importance of factors relating to algal presence in regulating community composition. This work demonstrates that secretion of selective bacterial growth substrates represents one mechanism by which algal exometabolites can influence bacterial community composition and illustrates how the algal exometabolome has the potential to modulate bacterial communities as a function of algal growth.

Label-Free Multiphoton Imaging of Microbes in Root, Mineral, and Soil Matrices with Time-Gated Coherent Raman and Fluorescence Lifetime Imaging.

Imaging biogeochemical interactions in complex microbial systems─such as those at the soil-root interface─is crucial to studies of climate, agriculture, and environmental health but complicated by the three-dimensional (3D) juxtaposition of materials with a wide range of optical properties. We developed a label-free multiphoton nonlinear imaging approach to provide contrast and chemical information for soil microorganisms in roots and minerals with epi-illumination by simultaneously imaging two-photon excitation fluorescence (TPEF), coherent anti-Stokes Raman scattering (CARS), second-harmonic generation (SHG), and sum-frequency mixing (SFM). We used fluorescence lifetime imaging (FLIM) and time gating to correct CARS for the autofluorescence background native to soil particles and fungal hyphae (TG-CARS) using time-correlated single-photon counting (TCSPC). We combined TPEF, TG-CARS, and FLIM to maximize image contrast for live fungi and bacteria in roots and soil matrices without fluorescence labeling. Using this instrument, we imaged symbiotic arbuscular mycorrhizal fungi (AMF) structures within unstained plant roots in 3D to 60 µm depth. High-quality imaging was possible at up to 30 µm depth in a clay particle matrix and at 15 µm in complex soil preparation. TG-CARS allowed us to identify previously unknown lipid droplets in the symbiotic fungus, Serendipita bescii. We also visualized unstained putative bacteria associated with the roots of Brachypodium distachyon in a soil microcosm. Our results show that this multimodal approach holds significant promise for rhizosphere and soil science research.

Attachment between heterotrophic bacteria and microalgae influences symbiotic microscale interactions.

The surface and surroundings of microalgal cells (phycosphere) are critical interaction zones but have been difficult to functionally interrogate due to methodological limitations. We examined effects of phycosphere-associated bacteria for two biofuel-relevant microalgal species (Phaeodactylum tricornutum and Nannochloropsis salina) using stable isotope tracing and high spatial resolution mass spectrometry imaging (NanoSIMS) to quantify elemental exchanges at the single-cell level. Each algal species responded differently to bacterial attachment. In P. tricornutum, a high percentage of cells had attached bacteria (92%-98%, up to eight bacteria per alga) and fixed 64% more carbon with attached bacteria compared to axenic cells. In contrast, N. salina cells were less commonly associated with bacteria (42%-63%), harboured fewer bacteria per alga, and fixed 10% more carbon without attached bacteria compared to axenic cells. An uncultivated bacterium related to Haliscomenobacter sp. was identified as an effective mutualist; it increased carbon fixation when attached to P. tricornutum and incorporated 71% more algal-fixed carbon relative to other bacteria. Our results illustrate how phylogenetic identity and physical location of bacteria and algae facilitate diverse metabolic responses. Phycosphere-mediated, mutualistic chemical exchanges between autotrophs and heterotrophs may be a fruitful means to increase microalgal productivity for applied engineering efforts.

Increasing aggregate size reduces single-cell organic carbon incorporation by hydrogel-embedded wetland microbes.

Microbial degradation of organic carbon in sediments is impacted by the availability of oxygen and substrates for growth. To better understand how particle size and redox zonation impact microbial organic carbon incorporation, techniques that maintain spatial information are necessary to quantify elemental cycling at the microscale. In this study, we produced hydrogel microspheres of various diameters (100, 250, and 500 µm) and inoculated them with an aerobic heterotrophic bacterium isolated from a freshwater wetland (Flavobacterium sp.), and in a second experiment with a microbial community from an urban lacustrine wetland. The hydrogel-embedded microbial populations were incubated with 13C-labeled substrates to quantify organic carbon incorporation into biomass via nanoSIMS. Additionally, luminescent nanosensors enabled spatially explicit measurements of oxygen concentrations inside the microspheres. The experimental data were then incorporated into a reactive-transport model to project long-term steady-state conditions. Smaller (100 µm) particles exhibited the highest microbial cell-specific growth per volume, but also showed higher absolute activity near the surface compared to the larger particles (250 and 500 µm). The experimental results and computational models demonstrate that organic carbon availability was not high enough to allow steep oxygen gradients and as a result, all particle sizes remained well-oxygenated. Our study provides a foundational framework for future studies investigating spatially dependent microbial activity in aggregates using isotopically labeled substrates to quantify growth.

Microbiome processing of organic nitrogen input supports growth and cyanotoxin production of Microcystis aeruginosa cultures.

Nutrient-induced blooms of the globally abundant freshwater toxic cyanobacterium Microcystis cause worldwide public and ecosystem health concerns. The response of Microcystis growth and toxin production to new and recycled nitrogen (N) inputs and the impact of heterotrophic bacteria in the Microcystis phycosphere on these processes are not well understood. Here, using microbiome transplant experiments, cyanotoxin analysis, and nanometer-scale stable isotope probing to measure N incorporation and exchange at single cell resolution, we monitored the growth, cyanotoxin production, and microbiome community structure of several Microcystis strains grown on amino acids or proteins as the sole N source. We demonstrate that the type of organic N available shaped the microbial community associated with Microcystis, and external organic N input led to decreased bacterial colonization of Microcystis colonies. Our data also suggest that certain Microcystis strains could directly uptake amino acids, but with lower rates than heterotrophic bacteria. Toxin analysis showed that biomass-specific microcystin production was not impacted by N source (i.e. nitrate, amino acids, or protein) but rather by total N availability. Single-cell isotope incorporation revealed that some bacterial communities competed with Microcystis for organic N, but other communities promoted increased N uptake by Microcystis, likely through ammonification or organic N modification. Our laboratory culture data suggest that organic N input could support Microcystis blooms and toxin production in nature, and Microcystis-associated microbial communities likely play critical roles in this process by influencing cyanobacterial succession through either decreasing (via competition) or increasing (via biotransformation) N availability, especially under inorganic N scarcity.

Single-cell isotope tracing reveals functional guilds of bacteria associated with the diatom Phaeodactylum tricornutum.

Bacterial remineralization of algal organic matter fuels algal growth but is rarely quantified. Consequently, we cannot currently predict whether some bacterial taxa may provide more remineralized nutrients to algae than others. Here, we quantified bacterial incorporation of algal-derived complex dissolved organic carbon and nitrogen and algal incorporation of remineralized carbon and nitrogen in fifteen bacterial co-cultures growing with the diatom Phaeodactylum tricornutum at the single-cell level using isotope tracing and nanoSIMS. We found unexpected strain-to-strain and cell-to-cell variability in net carbon and nitrogen incorporation, including non-ubiquitous complex organic nitrogen utilization and remineralization. We used these data to identify three distinct functional guilds of metabolic interactions, which we termed macromolecule remineralizers, macromolecule users, and small-molecule users, the latter exhibiting efficient growth under low carbon availability. The functional guilds were not linked to phylogeny and could not be elucidated strictly from metabolic capacity as predicted by comparative genomics, highlighting the need for direct activity-based measurements in ecological studies of microbial metabolic interactions.

The V-type ATPase enhances photosynthesis in marine phytoplankton and further links phagocytosis to symbiogenesis.

Diatoms, dinoflagellates, and coccolithophores are dominant groups of marine eukaryotic phytoplankton that are collectively responsible for the majority of primary production in the ocean.1 These phytoplankton contain additional intracellular membranes around their chloroplasts, which are derived from ancestral engulfment of red microalgae by unicellular heterotrophic eukaryotes that led to secondary and tertiary endosymbiosis.2 However, the selectable evolutionary advantage of these membranes and the physiological significance for extant phytoplankton remain poorly understood. Since intracellular digestive vacuoles are ubiquitously acidified by V-type H+-ATPase (VHA),3 proton pumps were proposed to acidify the microenvironment around secondary chloroplasts to promote the dehydration of dissolved inorganic carbon (DIC) into CO2, thus enhancing photosynthesis.4,5 We report that VHA is localized around the chloroplasts of centric diatoms and that VHA significantly contributes to their photosynthesis across a wide range of oceanic irradiances. Similar results in a pennate diatom, dinoflagellate, and coccolithophore, but not green or red microalgae, imply the co-option of phagocytic VHA activity into a carbon-concentrating mechanism (CCM) is common to secondary endosymbiotic phytoplankton. Furthermore, analogous mechanisms in extant photosymbiotic marine invertebrates6,7,8 provide functional evidence for an adaptive advantage throughout the transition from endosymbiosis to symbiogenesis. Based on the contribution of diatoms to ocean biogeochemical cycles, VHA-mediated enhancement of photosynthesis contributes at least 3.5 Gtons of fixed carbon per year (or 7% of primary production in the ocean), providing an example of a symbiosis-derived evolutionary innovation with global environmental implications.

Microbial community structure and function on sinking particles in the North Pacific Subtropical Gyre.

Sinking particles mediate the transport of carbon and energy to the deep-sea, yet the specific microbes associated with sedimenting particles in the ocean's interior remain largely uncharacterized. In this study, we used particle interceptor traps (PITs) to assess the nature of particle-associated microbial communities collected at a variety of depths in the North Pacific Subtropical Gyre. Comparative metagenomics was used to assess differences in microbial taxa and functional gene repertoires in PITs containing a preservative (poisoned traps) compared to preservative-free traps where growth was allowed to continue in situ (live traps). Live trap microbial communities shared taxonomic and functional similarities with bacteria previously reported to be enriched in dissolved organic matter (DOM) microcosms (e.g., Alteromonas and Methylophaga), in addition to other particle and eukaryote-associated bacteria (e.g., Flavobacteriales and Pseudoalteromonas). Poisoned trap microbial assemblages were enriched in Vibrio and Campylobacterales likely associated with eukaryotic surfaces and intestinal tracts as symbionts, pathogens, or saprophytes. The functional gene content of microbial assemblages in poisoned traps included a variety of genes involved in virulence, anaerobic metabolism, attachment to chitinaceaous surfaces, and chitin degradation. The presence of chitinaceaous surfaces was also accompanied by the co-existence of bacteria which encoded the capacity to attach to, transport and metabolize chitin and its derivatives. Distinctly different microbial assemblages predominated in live traps, which were largely represented by copiotrophs and eukaryote-associated bacterial communities. Predominant sediment trap-assocaited eukaryotic phyla included Dinoflagellata, Metazoa (mostly copepods), Protalveolata, Retaria, and Stramenopiles. These data indicate the central role of eukaryotic taxa in structuring sinking particle microbial assemblages, as well as the rapid responses of indigenous microbial species in the degradation of marine particulate organic matter (POM) in situ in the ocean's interior.

High-resolution imaging of pelagic bacteria by Atomic Force Microscopy and implications for carbon cycling.

In microbial oceanography, cell size, volume and carbon (C) content of pelagic bacteria and archaea ('bacteria') are critical parameters in addressing the in situ physiology and functions of bacteria, and their role in the food web and C cycle. However, because of the diminutive size of most pelagic bacteria and errors caused by sample fixation and processing, an accurate measurement of the size and volume has been challenging. We used atomic force microscopy (AFM) to obtain high-resolution images of pelagic bacteria and Synechococcus. We measured the length, width and height of live and formalin-fixed pelagic bacteria, and computed individual cell volumes. AFM-based measurements were compared with those by epifluorescence microscopy (EFM) using 4',6-diamidino-2-phenylindole (DAPI). The ability to measure cell height by AFM provides methodological advantage and ecophysiological insight. For the samples examined, EFM (DAPI)-based average cell volume was in good agreement (1.1-fold) with live sample AFM. However, the agreement may be a fortuitous balance between cell shrinkage due to fixation/drying (threefold) and Z-overestimation (as EFM does not account for cell flattening caused by sample processing and assumes that height=width). The two methods showed major differences in cell volume and cell C frequency distributions. This study refines the methodology for quantifying bacteria-mediated C fluxes and the role of bacteria in marine ecosystems, and suggests the potential of AFM for individual cell physiological interrogations in natural marine assemblages.

Major role of microbes in carbon fluxes during Austral winter in the Southern Drake Passage.

Carbon cycling in Southern Ocean is a major issue in climate change, hence the need to understand the role of biota in the regulation of carbon fixation and cycling. Southern Ocean is a heterogeneous system, characterized by a strong seasonality, due to long dark winter. Yet, currently little is known about biogeochemical dynamics during this season, particularly in the deeper part of the ocean. We studied bacterial communities and processes in summer and winter cruises in the southern Drake Passage. Here we show that in winter, when the primary production is greatly reduced, Bacteria and Archaea become the major producers of biogenic particles, at the expense of dissolved organic carbon drawdown. Heterotrophic production and chemoautotrophic CO(2) fixation rates were substantial, also in deep water, and bacterial populations were controlled by protists and viruses. A dynamic food web is also consistent with the observed temporal and spatial variations in archaeal and bacterial communities that might exploit various niches. Thus, Southern Ocean microbial loop may substantially maintain a wintertime food web and system respiration at the expense of summer produced DOC as well as regenerate nutrients and iron. Our findings have important implications for Southern Ocean ecosystem functioning and carbon cycle and its manipulation by iron enrichment to achieve net sequestration of atmospheric CO(2).