Widespread detection of Candidatus Accumulibacter phosphatis, a polyphosphate-accumulating organism, in sediments of the Columbia River estuary.

Enhanced biological phosphorus removal (EBPR) exploits the metabolism of polyphosphate-accumulating organisms (PAOs) to remove excess phosphorus (P) from wastewater treatment. Candidatus Accumulibacter phosphatis (Accumulibacter) is the most abundant and well-studied PAO in EBPR systems. In a previous study, we detected polyphosphates throughout peripheral bay sediments, and hypothesized that an estuary is an ideal setting to evaluate PAOs in a natural system, given that estuaries are characterized by dynamic dissolved oxygen fluctuations that potentially favour PAO metabolism. We detected nucleotide sequences attributable to Accumulibacter (16S rRNA, ppk1) in sediments within three peripheral bays of the Columbia River estuary at abundances rivalling those observed in conventional wastewater treatment plants (0.01%-2.6%). Most of the sequences attributable to Accumulibacter were Type I rather than Type II, despite the fact that the estuary does not have particularly high nutrient concentrations. The highest diversity of Accumulibacter was observed in oligohaline peripheral bays, while the greatest abundances were observed at the mouth of the estuary in mesohaline sediments in the spring and summer. In addition, an approximately 70% increase in polyphosphate concentrations observed at one of the sites between dawn and dusk suggests that PAOs may play an important role in P cycling in estuary sediments.

Effect of metformin exposure on growth and photosynthetic performance in the unicellular freshwater chlorophyte, Chlorella vulgaris.

Many pharmaceuticals have negative effects on biota when released into the environment. For example, recent work has shown that the commonly prescribed antidiabetic drug, metformin (N,N-dimethylbiguanide), has endocrine disrupting effects on fish. However, effects of metformin on aquatic primary producers are poorly known. We exposed cultured isolates of a freshwater chlorophyte, Chlorella vulgaris, to a range of metformin concentrations (0-767.9 mg L-1) to test the hypothesis that exposure negatively affects photosynthesis and growth. A cessation of growth, increase in non-photochemical quenching (NPQ, NPQmax), and reduced electron transport rate (ETR) were observed 24 h after exposure to a metformin concentration of 767.8 mg L-1 (4.6 mM). By 48 h, photosynthetic efficiency of photosystem II (Fv/Fm), α, the initial slope of the ETR-irradiance curve, and Ek (minimum irradiance required to saturate photosynthesis) were reduced. At a lower concentration (76.8 mg L-1), negative effects on photosynthesis (increase in NPQ, decrease in ETR) were delayed, occurring between 72 and 96 h. No negative effects on photosynthesis were observed at an exposure concentration of 1.5 mg L-1. It is likely that metformin impairs photosynthesis either through downstream effects from inhibition of complex I of the electron transport chain or via activation of the enzyme, SnRK1 (sucrose non-fermenting-related kinase 1), which acts as a cellular energy regulator in plants and algae and is an ortholog of the mammalian target of metformin, AMPK (5' adenosine monophosphate-activated protein kinase).

Determination of intracellular pH in phytoplankton using the fluorescent probe, SNARF, with detection by fluorescence spectroscopy.

The maintenance of pH homeostasis is critical for a variety of cellular metabolic processes. Although ocean acidification is likely to influence cellular metabolism and energy balance, the degree to which intracellular pH in phytoplankton differs from the external environment under varying environmental pH levels is not well characterized. While there are numerous existing methods for the determination of intracellular pH in the form of single peak emission (e.g., BCECF) and radioisotopic (e.g., 14C-DMO) indicators for use with phytoplankton, the fluorescent pH indicator seminaphtharhodafluor (SNARF) has not been established as a robust method for measuring in vivo pH in phytoplankton. SNARF has superior accuracy and sensitivity since it exhibits dual emission peaks from a single excitation wavelength and the ratio of the two are related to pH. The use of a ratio limiting variations in fluorescence due to dye loading, photobleaching, and instrument variation; moreover, like other fluorescence-based assays, it does not require the specialized equipment and permits that radioisotopic methods do. As a first step, we tested the performance of SNARF for measuring intracellular pH in vivo in a number of phytoplankton taxa. SNARF detection was accomplished using fluorescence spectroscopy (FS) and laser scanning microscopy (LSM). Since SNARF fluorescence is activated by cleavage of an ester group from the core fluorophore by non-specific esterases, we measured esterase activity using fluorescein diacetate (FDA) to characterize variability in esterase activity among phytoplankton taxa, with a view towards its influence on assay performance. Esterase activity cell volume; however, there was no indication that enzyme specificity and differences in individual esterase profiles adversely affected SNARF performance in phytoplankton. Assays of intracellular pH using SNARF were comparable to those made with 14C-labeled DMO, an accepted standard method. Thus, SNARF provides robust measurements of intracellular pH in phytoplankton, constituting a useful tool in investigations of the effects of ocean acidification and fluctuations in environmental pH on cellular physiology.

Divergent gene expression among phytoplankton taxa in response to upwelling.

Frequent blooms of phytoplankton occur in coastal upwelling zones creating hotspots of biological productivity in the ocean. As cold, nutrient-rich water is brought up to sunlit layers from depth, phytoplankton are also transported upwards to seed surface blooms that are often dominated by diatoms. The physiological response of phytoplankton to this process, commonly referred to as shift-up, is characterized by increases in nitrate assimilation and rapid growth rates. To examine the molecular underpinnings behind this phenomenon, metatranscriptomics was applied to a simulated upwelling experiment using natural phytoplankton communities from the California Upwelling Zone. An increase in diatom growth following 5 days of incubation was attributed to the genera Chaetoceros and Pseudo-nitzschia. Here, we show that certain bloom-forming diatoms exhibit a distinct transcriptional response that coordinates shift-up where diatoms exhibited the greatest transcriptional change following upwelling; however, comparison of co-expressed genes exposed overrepresentation of distinct sets within each of the dominant phytoplankton groups. The analysis revealed that diatoms frontload genes involved in nitrogen assimilation likely in order to outcompete other groups for available nitrogen during upwelling events. We speculate that the evolutionary success of diatoms may be due, in part, to this proactive response to frequently encountered changes in their environment.

Graphical coding data and operational guidance for implementation or modification of a LabVIEW®-based pHstat system for the cultivation of microalgae.

The influence of pH on phytoplankton physiology is an important facet of the body of research on ocean acidification. We provide data developed during the design and implementation of a novel pHstat system capable of maintaining both static and dynamic pH environments in a laboratory setting. These data both help improve functionality of the system, and provide specific coding blocks for controlling the pHstat using a LabVIEW® virtual instrument (VI). The data in this paper support the research article "Development of an economical, autonomous pHstat system for culturing phytoplankton under steady state or dynamic conditions" (Golda et al. [2]). These data will be of interest to researchers studying the effects of changing pH on phytoplankton in a laboratory context, and to those desiring to build their own pHstat system(s). These data can also be used to facilitate modification of the pHstat system to control salinity, temperature, or other environmental factors.

Use of Highly Specific Molecular Markers Reveals Positive Correlation between Abundances of Mesodinium cf. major and Its Preferred Prey, Teleaulax amphioxeia, During Red Water Blooms in the Columbia River Estuary.

In a previous study, Teleaulax amphioxeia-the preferred prey of Mesodinium in the Columbia River estuary-were undetectable within intense annual blooms, suggesting blooms are prey-limited or prey are acquired outside of bloom patches. We used a novel molecular approach specifically targeting the prey (i.e., Unique Sequence Element [USE] within the ribosomal RNA 28S D2 regions of T. amphioxeia nucleus and nucleomorph) in estuarine water samples acquired autonomously with an Environmental Sample Processor integrated within a monitoring network (ESP-SATURN). This new approach allowed for both more specific detection of the prey and better constraint of sample variability. A positive correlation was observed between abundances of M. cf. major and T. amphioxeia during bloom periods. The correlation was stronger at depth (> 8.2 m) and weak or nonexistent in the surface, suggesting that predator-prey dynamics become uncoupled when stratification is strong. We confirmed exclusive selectivity for T. amphioxeia by M. cf. major and observed the incorporation of the prey nucleus into a 4-nuclei complex, where it remained functionally active. The specific biomarker for T. amphioxeia was also recovered in M. cf. major samples from a Namibian coastal bloom, suggesting that a specific predator-prey relationship might be widespread between M. cf. major and T. amphioxeia.

Development of an economical, autonomous pHstat system for culturing phytoplankton under steady state or dynamic conditions.

Laboratory investigations of physiological processes in phytoplankton require precise control of experimental conditions. Chemostats customized to control and maintain stable pH levels (pHstats) are ideally suited for investigations of the effects of pH on phytoplankton physiology, for example in context of ocean acidification. Here we designed and constructed a simple, flexible pHstat system and demonstrated its operational capabilities under laboratory culture conditions. In particular, the system is useful for simulating natural cyclic pH variability within aquatic ecosystems, such as diel fluctuations that result from metabolic activity or tidal mixing in estuaries. The pHstat system operates in two modes: (1) static/set point pH, which maintains pH at a constant level, or (2) dynamic pH, which generates regular, sinusoidal pH fluctuations by systematically varying pH according to user-defined parameters. The pHstat is self-regulating through the use of interchangeable electronically controlled reagent or gas-mediated pH-modification manifolds, both of which feature flow regulation by solenoid valves. Although effective pH control was achieved using both liquid reagent additions and gas-mediated methods, the liquid manifold exhibited tighter control (±0.03pH units) of the desired pH than the gas manifold (±0.10pH units). The precise control provided by this pHstat system, as well as its operational flexibility will facilitate studies that examine responses by marine microbiota to fluctuations in pH in aquatic ecosystems.

Enumeration of Parasitic Chytrid Zoospores in the Columbia River via Quantitative PCR.

UNLABELLED: Through lethal infection, fungal parasites of phytoplankton ("chytrids") repackage organic material from the large, effectively inedible, colonial diatoms they infect into much smaller zoospores, which are easier for zooplankton to consume. However, their small size and lack of distinguishing morphological features render it difficult to distinguish zoospores from other small flagellates in mixed assemblages in the natural environment. In this study, we developed and tested a method to quantify chytrid zoospores in field studies using quantitative PCR (qPCR) targeting the internal transcribed spacer 2 (ITS2) region within the rRNA gene cluster. To achieve accurate quantification, the assay was designed to be highly specific for a parasite (Rhizophydium planktonicum) of the diatom Asterionella formosa; however, the approach is applicable to additional host-parasite systems. Parasitic zoospores were detected and quantified in the freshwater reaches of the lower Columbia River, as well as in the salt-influenced estuary and river plume. The coincidence between zoospore abundances and a prevalence of small zooplankton during blooms of large, colonial diatoms in the spring suggests that Columbia River zooplankton may be poised to benefit nutritionally from chytrid zoospores, thus providing a mechanism to retain organic carbon within the system and reduce losses to downstream export. We estimate that ∼15% of the carbon biomass tied up in blooms of the dominant diatom species is transformed into zoospores through the parasitic shunt during spring. IMPORTANCE: The small size of the parasitic fungi that infect phytoplankton makes it difficult to identify and quantify them in natural systems. We developed and tested a method to quantify these organisms (chytrid zoospores) using a molecular technique that targets the internal transcribed spacer region within the rRNA gene cluster. Using this method, we quantified the abundance of the motile stage of a specific parasite in the freshwater and saltwater-influenced regions of the Columbia River in the U.S. Pacific Northwest. Parasitic chytrid zoospores were found to be present throughout the year and at higher abundances during the spring, when phytoplankton blooms occur. The presence of these organisms indicates not only that they may be responsible for the death of host phytoplankton cells but that they may also provide a readily available food source to small consumers (zooplankton) in the food web of the Columbia River.

Evaluation of Daphnid Grazing on Microscopic Zoosporic Fungi by Using Comparative Threshold Cycle Quantitative PCR.

UNLABELLED: Lethal parasitism of large phytoplankton by chytrids (microscopic zoosporic fungi) may play an important role in organic matter and nutrient cycling in aquatic environments by shunting carbon away from hosts and into much smaller zoospores, which are more readily consumed by zooplankton. This pathway provides a mechanism to more efficiently retain carbon within food webs and reduce export losses. However, challenges in accurate identification and quantification of chytrids have prevented a robust assessment of the relative importance of parasitism for carbon and energy flows within aquatic systems. The use of molecular techniques has greatly advanced our ability to detect small, nondescript microorganisms in aquatic environments in recent years, including chytrids. We used quantitative PCR (qPCR) to quantify the consumption of zoospores by Daphnia in laboratory experiments using a culture-based comparative threshold cycle (CT) method. We successfully quantified the reduction of zoospores in water samples during Daphnia grazing and confirmed the presence of chytrid DNA inside the daphnid gut. We demonstrate that comparative CT qPCR is a robust and effective method to quantify zoospores and evaluate zoospore grazing by zooplankton and will aid in better understanding how chytrids contribute to organic matter cycling and trophic energy transfer within food webs. IMPORTANCE: The study of aquatic fungi is often complicated by the fact that they possess complex life cycles that include a variety of morphological forms. Studies that rely on morphological characteristics to quantify the abundances of all stages of the fungal life cycle face the challenge of correctly identifying and enumerating the nondescript zoospores. These zoospores, however, provide an important trophic link between large colonial phytoplankton and zooplankton: that is, once the carbon is liberated from phytoplankton into the parasitic zoospores, the latter are consumed by zooplankton and carbon is retained in the aquatic food web rather than exported from the system. This study provides a tool to quantify zoospores and evaluate the consumption of zoospores by zooplankton in order to further our understanding of their role in food web dynamics.

Discovery of a Katablepharis sp. in the Columbia River estuary that is abundant during the spring and bears a unique large ribosomal subunit sequence element.

Heterotrophic protists play significant roles in pelagic food webs as bacterivorous and herbivorous consumers. However, heterotrophic protists-unlike autotrophic ones-are often difficult to track since they tend to lack features such as photosynthetic pigments that allow for remote sensing or for bulk characterization. Difficulty in the identification of heterotrophic protists has often resulted in lumping them into broad groups, but there is a strong need to develop methods that increase the spatial and temporal resolution of observations applied to particular organisms in order to discover the drivers of population structure and ecological function. In surveys of small subunit rRNA, gene (SSU) sequences of microbial eukaryotes from the Columbia River to the Pacific Ocean, the heterotrophic flagellate Katablepharis sp. were found to dominate protist assemblages (including autotrophic and heterotrophic fractions) in the spring, prior to the freshet. We discovered a 332 base pair unique sequence element (USE) insertion in the large subunit rRNA gene (28S) that is not present in other katablepharids or in any other eukaryote. Using this USE, we were able to detect Katablepharis within mixed assemblages in river, estuarine, and oceanic samples and determine spatial and temporal patterns in absolute abundance through quantitative PCR and fluorescence in situ hybridization. Given their high abundance and repeatable temporal patterns of occurrence, we hypothesize that the Columbia River Estuary Katablepharis (Katablepharis CRE) plays an important role in estuarine biogeochemical and ecosystem function.

Disinfection of ballast water with iron activated persulfate.

The treatment of ballast water carried onboard ships is critical to reduce the spread of nonindigenous aquatic organisms that potentially include noxious and harmful taxa. We tested the efficacy of persulfate (peroxydisulfate, S2O8(2-), PS) activated with zerovalent iron (Fe(0)) as a chemical biocide against two taxa of marine phytoplankton grown in bench-scale, batch cultures: the diatom, Pseudonitzshia delicatissima and the green alga, Dunaliella tertiolecta . After testing a range of PS concentrations (0-4 mM activated PS) and exposure times (1-7 days), we determined that a dosage of 4 mM of activated PS was required to inactivate cells from both species, as indicated by reduced or halted growth and a reduction in photosynthetic performance. Longer exposure times were required to fully inactivate D. tertiolecta (7 days) compared to P. delicatissima (5 days). Under these conditions, no recovery was observed upon placing cells from the exposed cultures into fresh media lacking biocide. The results demonstrate that activated PS is an effective chemical biocide against species of marine phytoplankton. The lack of harmful byproducts produced during application makes PS an attractive alternative to other biocides currently in use for ballast water treatments and merits further testing at a larger scale.

Determining indicator taxa across spatial and seasonal gradients in the Columbia River coastal margin.

Bacterioplankton communities are deeply diverse and highly variable across space and time, but several recent studies demonstrate repeatable and predictable patterns in this diversity. We expanded on previous studies by determining patterns of variability in both individual taxa and bacterial communities across coastal environmental gradients. We surveyed bacterioplankton diversity across the Columbia River coastal margin, USA, using amplicon pyrosequencing of 16S rRNA genes from 596 water samples collected from 2007 to 2010. Our results showed seasonal shifts and annual reassembly of bacterioplankton communities in the freshwater-influenced Columbia River, estuary, and plume, and identified indicator taxa, including species from freshwater SAR11, Oceanospirillales, and Flavobacteria groups, that characterize the changing seasonal conditions in these environments. In the river and estuary, Actinobacteria and Betaproteobacteria indicator taxa correlated strongly with seasonal fluctuations in particulate organic carbon (ρ=-0.664) and residence time (ρ=0.512), respectively. In contrast, seasonal change in communities was not detected in the coastal ocean and varied more with the spatial variability of environmental factors including temperature and dissolved oxygen. Indicator taxa of coastal ocean environments included SAR406 and SUP05 taxa from the deep ocean, and Prochlorococcus and SAR11 taxa from the upper water column. We found that in the Columbia River coastal margin, freshwater-influenced environments were consistent and predictable, whereas coastal ocean community variability was difficult to interpret due to complex physical conditions. This study moves beyond beta-diversity patterns to focus on the occurrence of specific taxa and lends insight into the potential ecological roles these taxa have in coastal ocean environments.

Seasonal changes in bacterial and archaeal gene expression patterns across salinity gradients in the Columbia River coastal margin.

Through their metabolic activities, microbial populations mediate the impact of high gradient regions on ecological function and productivity of the highly dynamic Columbia River coastal margin (CRCM). A 2226-probe oligonucleotide DNA microarray was developed to investigate expression patterns for microbial genes involved in nitrogen and carbon metabolism in the CRCM. Initial experiments with the environmental microarrays were directed toward validation of the platform and yielded high reproducibility in multiple tests. Bioinformatic and experimental validation also indicated that >85% of the microarray probes were specific for their corresponding target genes and for a few homologs within the same microbial family. The validated probe set was used to query gene expression responses by microbial assemblages to environmental variability. Sixty-four samples from the river, estuary, plume, and adjacent ocean were collected in different seasons and analyzed to correlate the measured variability in chemical, physical and biological water parameters to differences in global gene expression profiles. The method produced robust seasonal profiles corresponding to pre-freshet spring (April) and late summer (August). Overall relative gene expression was high in both seasons and was consistent with high microbial abundance measured by total RNA, heterotrophic bacterial production, and chlorophyll a. Both seasonal patterns involved large numbers of genes that were highly expressed relative to background, yet each produced very different gene expression profiles. April patterns revealed high differential gene expression in the coastal margin samples (estuary, plume and adjacent ocean) relative to freshwater, while little differential gene expression was observed along the river-to-ocean transition in August. Microbial gene expression profiles appeared to relate, in part, to seasonal differences in nutrient availability and potential resource competition. Furthermore, our results suggest that highly-active particle-attached microbiota in the Columbia River water column may perform dissimilatory nitrate reduction (both dentrification and DNRA) within anoxic particle microniches.