Simultaneous quantification of active carbon- and nitrogen-fixing communities and estimation of fixation rates using fluorescence in situ hybridization and flow cytometry.

Understanding the interconnectivity of oceanic carbon and nitrogen cycles, specifically carbon and nitrogen fixation, is essential in elucidating the fate and distribution of carbon in the ocean. Traditional techniques measure either organism abundance or biochemical rates. As such, measurements are performed on separate samples and on different time scales. Here, we developed a method to simultaneously quantify organisms while estimating rates of fixation across time and space for both carbon and nitrogen. Tyramide signal amplification fluorescence in situ hybridization (TSA-FISH) of mRNA for functionally specific oligonucleotide probes for rbcL (ribulose-1,5-bisphosphate carboxylase/oxygenase; carbon fixation) and nifH (nitrogenase; nitrogen fixation) was combined with flow cytometry to measure abundance and estimate activity. Cultured samples representing a diversity of phytoplankton (cyanobacteria, coccolithophores, chlorophytes, diatoms, and dinoflagellates), as well as environmental samples from the open ocean (Gulf of Mexico, USA, and southeastern Indian Ocean, Australia) and an estuary (Galveston Bay, Texas, USA), were successfully hybridized. Strong correlations between positively tagged community abundance and (14)C/(15)N measurements are presented. We propose that these methods can be used to estimate carbon and nitrogen fixation in environmental communities. The utilization of mRNA TSA-FISH to detect multiple active microbial functions within the same sample will offer increased understanding of important biogeochemical cycles in the ocean.

Reduction of the Powerful Greenhouse Gas N2O in the South-Eastern Indian Ocean.

Nitrous oxide (N2O) is a powerful greenhouse gas and a key catalyst of stratospheric ozone depletion. Yet, little data exist about the sink and source terms of the production and reduction of N2O outside the well-known oxygen minimum zones (OMZ). Here we show the presence of functional marker genes for the reduction of N2O in the last step of the denitrification process (nitrous oxide reductase genes; nosZ) in oxygenated surface waters (180-250 O2 µmol.kg(-1)) in the south-eastern Indian Ocean. Overall copy numbers indicated that nosZ genes represented a significant proportion of the microbial community, which is unexpected in these oxygenated waters. Our data show strong temperature sensitivity for nosZ genes and reaction rates along a vast latitudinal gradient (32°S-12°S). These data suggest a large N2O sink in the warmer Tropical waters of the south-eastern Indian Ocean. Clone sequencing from PCR products revealed that most denitrification genes belonged to Rhodobacteraceae. Our work highlights the need to investigate the feedback and tight linkages between nitrification and denitrification (both sources of N2O, but the latter also a source of bioavailable N losses) in the understudied yet strategic Indian Ocean and other oligotrophic systems.

Nutrient uplift in a cyclonic eddy increases diversity, primary productivity and iron demand of microbial communities relative to a western boundary current.

The intensification of western boundary currents in the global ocean will potentially influence meso-scale eddy generation, and redistribute microbes and their associated ecological and biogeochemical functions. To understand eddy-induced changes in microbial community composition as well as how they control growth, we targeted the East Australian Current (EAC) region to sample microbes in a cyclonic (cold-core) eddy (CCE) and the adjacent EAC. Phototrophic and diazotrophic microbes were more diverse (2-10 times greater Shannon index) in the CCE relative to the EAC, and the cell size distribution in the CCE was dominated (67%) by larger micro-plankton [Formula: see text], as opposed to pico- and nano-sized cells in the EAC. Nutrient addition experiments determined that nitrogen was the principal nutrient limiting growth in the EAC, while iron was a secondary limiting nutrient in the CCE. Among the diazotrophic community, heterotrophic NifH gene sequences dominated in the EAC and were attributable to members of the gamma-, beta-, and delta-proteobacteria, while the CCE contained both phototrophic and heterotrophic diazotrophs, including Trichodesmium, UCYN-A and gamma-proteobacteria. Daily sampling of incubation bottles following nutrient amendment captured a cascade of effects at the cellular, population and community level, indicating taxon-specific differences in the speed of response of microbes to nutrient supply. Nitrogen addition to the CCE community increased picoeukaryote chlorophyll a quotas within 24 h, suggesting that nutrient uplift by eddies causes a 'greening' effect as well as an increase in phytoplankton biomass. After three days in both the EAC and CCE, diatoms increased in abundance with macronutrient (N, P, Si) and iron amendment, whereas haptophytes and phototrophic dinoflagellates declined. Our results indicate that cyclonic eddies increase delivery of nitrogen to the upper ocean to potentially mitigate the negative consequences of increased stratification due to ocean warming, but also increase the biological demand for iron that is necessary to sustain the growth of large-celled phototrophs and potentially support the diversity of diazotrophs over longer time-scales.

Changes in Microbial Plankton Assemblages Induced by Mesoscale Oceanographic Features in the Northern Gulf of Mexico.

Mesoscale circulation generated by the Loop Current in the Northern Gulf of Mexico (NGOM) delivers growth-limiting nutrients to the microbial plankton of the euphotic zone. Consequences of physicochemically driven community shifts on higher order consumers and subsequent impacts on the biological carbon pump remain poorly understood. This study evaluates microbial plankton <10 µm abundance and community structure across both cyclonic and anti-cyclonic circulation features in the NGOM using flow cytometry (SYBR Green I and autofluorescence parameters). Non-parametric multivariate hierarchical cluster analyses indicated that significant spatial variability in community structure exists such that stations that clustered together were defined as having a specific 'microbial signature' (i.e. statistically homogeneous community structure profiles based on relative abundance of microbial groups). Salinity and a combination of sea surface height anomaly and sea surface temperature were determined by distance based linear modeling to be abiotic predictor variables significantly correlated to changes in microbial signatures. Correlations between increased microbial abundance and availability of nitrogen suggest nitrogen-limitation of microbial plankton in this open ocean area. Regions of combined coastal water entrainment and mesoscale convergence corresponded to increased heterotrophic prokaryote abundance relative to autotrophic plankton. The results provide an initial assessment of how mesoscale circulation potentially influences microbial plankton abundance and community structure in the NGOM.

Towards an Understanding of the Interactions between Freshwater Inflows and Phytoplankton Communities in a Subtropical Estuary in the Gulf of Mexico.

Subtropical estuaries worldwide face increased pressure on their ecosystem health and services due to increasing human population growth and associated land use/land cover changes, expansion of ports, and climate change. We investigated freshwater inflows (river discharge) and the physico-chemical characteristics of Galveston Bay (Texas, USA) as mechanisms driving variability in phytoplankton biomass and community composition between February 2008 and December 2009. Results of multivariate analyses (hierarchical cluster analysis, PERMANOVA, Mantel test, and nMDS ordination coupled to environmental vector fitting) revealed that temporal and spatial differences in phytoplankton community structure correlate to differences in hydrographic and water quality parameters. Spatially, phytoplankton biomass and community composition responded to nutrient loading from the San Jacinto River in the northwest region of the bay (consistent with nutrient limitation) while hydraulic displacement (and perhaps other processes) resulted in overall lower biomass in the Trinity River delta (northeast region). The influence of inflows on phytoplankton diminished along a north to south gradient in the bay. Temporally, temperature and variables associated with freshwater inflow (discharge volume, salinity, inorganic nitrogen and phosphorus concentrations) were major influences on phytoplankton dynamics. Dissolved inorganic nitrogen: phosphorus (DIN:DIP) ratios suggest that phytoplankton communities will be predominately nitrogen limited. Diatoms dominated during periods of moderate to high freshwater inflows in winter/spring and were more abundant in the upper bay while cyanobacteria dominated during summer/fall when inflow was low. Given the differential influences of freshwater inflow on the phytoplankton communities of Galveston Bay, alterations upstream (magnitude, timing, frequency) will likely have a profound effect on downstream ecological processes and corresponding ecosystem services.