Seth G. John

A simple and efficient method for concentration of ocean viruses by chemical flocculation

Ocean viruses alter ecosystems through host mortality, horizontal gene transfer and by facilitating remineralization of limiting nutrients. However, the study of wild viral populations is limited by inefficient and unreliable concentration techniques. Here, we develop a new technique to recover viruses from natural waters using iron-based flocculation and large-pore-size filtration, followed by resuspension of virus-containing precipitates in a pH 6 buffer. Recovered viruses are amenable to gene sequencing, and a variable proportion of phages, depending upon the phage, retain their infectivity when recovered. This Fe-based virus flocculation, filtration and resuspension method (FFR) is efficient (> 90% recovery), reliable, inexpensive and adaptable to many aspects of marine viral ecology and genomics research.

A new method for precise determination of iron, zinc and cadmium stable isotope ratios in seawater by double-spike mass spectrometry.

The study of Fe, Zn and Cd stable isotopes (δ(56)Fe, δ(66)Zn and δ(114)Cd) in seawater is a new field, which promises to elucidate the marine cycling of these bioactive trace metals. However, the analytical challenges posed by the low concentration of these metals in seawater has meant that previous studies have typically required large sample volumes, highly limiting data collection in the oceans. Here, we present the first simultaneous method for the determination of these three isotope systems in seawater, using Nobias PA-1 chelating resin to extract metals from seawater, purification by anion exchange chromatography, and analysis by double spike MC-ICPMS. This method is designed for use on only a single litre of seawater and has blanks of 0.3, 0.06 and <0.03 ng for Fe, Zn and Cd respectively, representing a 1-20 fold reduction in sample size and a 4-130 decrease in blank compared to previously reported methods. The procedure yields data with high precision for all three elements (typically 0.02-0.2‰; 1σ internal precision), allowing us to distinguish natural variability in the oceans, which spans 1-3‰ for all three isotope systems. Simultaneous extraction and purification of three metals makes this method ideal for high-resolution, large-scale endeavours such as the GEOTRACES program.

Distinct iron isotopic signatures and supply from marine sediment dissolution

Oceanic iron inputs must be traced and quantified to learn how they affect primary productivity and climate. Chemical reduction of iron in continental margin sediments provides a substantial dissolved flux to the oceans, which is isotopically lighter than the crust, and so may be distinguished in seawater from other sources, such as wind-blown dust. However, heavy iron isotopes measured in seawater have recently led to the proposition of another source of dissolved iron from ‘non-reductive’ dissolution of continental margins. Here we present the first pore water iron isotope data from a passive-tectonic and semi-arid ocean margin (South Africa), which reveals a smaller and isotopically heavier flux of dissolved iron to seawater than active-tectonic and dysoxic continental margins. These data provide in situ evidence of non-reductive iron dissolution from a continental margin, and further show that geological and hydro-climatic factors may affect the amount and isotopic composition of iron entering the ocean.

Undocumented water column sink for cadmium in open ocean oxygen-deficient zones

Significance Cadmium (Cd), a trace nutrient for marine algae, has a marine distribution that closely matches the macronutrients nitrate and phosphate. Sedimentary microfossil records of Cd provide reconstructions of past ocean nutrient distributions that facilitate understanding the role of the oceans in the carbon cycle and climate change. However, incomplete knowledge of processes that control the addition and removal of Cd in the ocean, and Cd’s variability relative to major nutrients, limit use of the paleoceanographic proxy. We present coupled data of Cd concentration and isotopic composition in seawater and suspended marine particles, indicating direct removal of Cd via coprecipitation with sulfide in oxygen-deficient waters. Thus, the marine Cd cycle may be highly sensitive to the extent of global oceanic oxygen depletion. Cadmium (Cd) is a micronutrient and a tracer of biological productivity and circulation in the ocean. The correlation between dissolved Cd and the major algal nutrients in seawater has led to the use of Cd preserved in microfossils to constrain past ocean nutrient distributions. However, linking Cd to marine biological processes requires constraints on marine sources and sinks of Cd. Here, we show a decoupling between Cd and major nutrients within oxygen-deficient zones (ODZs) in both the Northeast Pacific and North Atlantic Oceans, which we attribute to Cd sulfide (CdS) precipitation in euxinic microenvironments around sinking biological particles. We find that dissolved Cd correlates well with dissolved phosphate in oxygenated waters, but is depleted compared with phosphate in ODZs. Additionally, suspended particles from the North Atlantic show high Cd content and light Cd stable isotope ratios within the ODZ, indicative of CdS precipitation. Globally, we calculate that CdS precipitation in ODZs is an important, and to our knowledge a previously undocumented marine sink of Cd. Our results suggest that water column oxygen depletion has a substantial impact on Cd biogeochemical cycling, impacting the global relationship between Cd and major nutrients and suggesting that Cd may be a previously unidentified tracer for water column oxygen deficiency on geological timescales. Similar depletions of copper and zinc in the Northeast Pacific indicate that sulfide precipitation in ODZs may also have an influence on the global distribution of other trace metals.

The biogeochemical cycling of zinc and zinc isotopes in the North Atlantic Ocean

Zinc (Zn) is a marine micronutrient, with an overall oceanic distribution mirroring the major macronutrients, especially silicate. Seawater Zn isotope ratios (δ66Zn) are a relatively new oceanographic parameter which may offer insights into the biogeochemical cycling of Zn. To date, the handful of published studies of seawater δ66Zn show the global deep ocean to be both remarkably homogeneous (approximately +0.5‰) and isotopically heavier than the marine sources of Zn (+0.1 to +0.3‰). Here we present the first high-resolution oceanic section of δ66Zn, from the U.S. GEOTRACES GA03 North Atlantic Transect, from Lisbon to Woods Hole. Throughout the surface ocean, biological uptake and release of isotopically light Zn, together with scavenging of heavier Zn, leads to large variability in δ66Zn. In the ocean below 1000 m, δ66Zn is generally homogeneous (+0.50 ± 0.14‰; 2 SD), though deviations from +0.5‰ allow us to identify specific sources of Zn. The Mediterranean Outflow is characterized by δ66Zn of +0.1 to +0.3‰, while margin sediments are a source of isotopically light Zn (−0.5 to −0.8‰), which we attribute to release of nonregenerated biogenic Zn. Mid-Atlantic Ridge hydrothermal vents are also a source of light Zn (close to −0.5‰), though Zn is not transported far from the vents. Understanding the biogeochemical cycling of Zn in the modern ocean begins to address the imbalance between the light δ66Zn signature of marine sources and the globally homogeneous deep oceans (δ66Zn of +0.5‰) on long timescales, with overall patterns pointing to sediments as an important sink for isotopically light Zn throughout the oceans.

The vertical distribution of iron stable isotopes in the North Atlantic near Bermuda

Seawater dissolved iron isotope ratios (δ^(56)Fe) have been measured in the North Atlantic near Bermuda. In a full-depth profile, seawater dissolved δ^(56)Fe is isotopically heavy compared to crustal values throughout the water column (δ^(56)Fe_(IRMM-014) = +0.30‰ to +0.71‰). Iron isotope ratios are relatively homogenous in the upper water column (between +0.30‰ to +0.45‰ above 1500 m), and δ^(56)Fe increases below this to a maximum of +0.71‰ at 2500 m, decreasing again to +0.35‰ at 4200 m. The δ^(56)Fe profile is very different from the iron concentration profile; in the upper water column [Fe] is variable while δ^(56)Fe is relatively constant, and in the deeper water column δ^(56)Fe varies while [Fe] remains relatively constant. The δ^(56)Fe profile is also not well correlated with other hydrographic tracers in the North Atlantic such as temperature, salinity, or the concentrations of oxygen, phosphate, silica, and CFC-11. The dissimilarity between δ^(56)Fe profiles and profiles of [Fe] and other hydrographic tracers shows that Fe isotope ratios provide a unique sort of information about ocean chemistry, and they suggest that Fe isotopes may therefore be a valuable new tool for tracing the global sources, sinks, and biogeochemical cycling of Fe.

Optimizing sample and spike concentrations for isotopic analysis by double-spike ICPMS

Double spike techniques are widely used for measuring the isotopic composition of natural samples. In order to achieve the most accurate results by double spike analysis, it is important to choose an appropriate double-spike composition, analyte concentration, and spike to natural ratio (Cspk/Cnat) where Cnat is the concentration of a sample or standard with a natural abundance of the isotopes and Cspk is the concentration of an added spike with an unnatural isotope composition. Here, the effect of varying these parameters is explored using a Monte Carlo technique which simulates error from counting statistics, Johnson noise, and isobaric interferences. Typically, optimal spike composition and Cspk/Cnat are calculated under the constraint that total concentration of spike plus sample (Cspk + Cnat) must remain constant, so that as the amount of double spike is increased, the amount of sample is decreased. In practice, there is no reason for Cspk + Cnat to be held constant, because an analyst with a fixed quantity of sample may add any amount of spike to this sample as long as detector limits are not exceeded. Therefore, here, double spikes are here optimized while allowing Cspk and Cnat to vary independently. For thirty three different elements, this new approach of allowing Cspk and Cnat to vary independently led to a decrease in theoretical error of up to ∼30% in the absence of isobaric interferences. In the presence of isobaric interferences, this approach can deliver even larger improvements in accuracy and precision. Theoretical error is then compared to observed error both for δ56Fe standards and for δ56Fe, δ66Zn, and δ114Cd measured in seawater. Theoretical error and measured error for real seawater samples are highly correlated, with 78%, 85%, and 96% of observed error in δ56Fe, δ66Zn, and δ114Cd, respectively, accounted for using an error model which includes only Johnson noise and counting statistics. This confirms that such models, which minimize theoretical error, can be used to optimize spike composition, Cspk, and Cnat in order to increase accuracy and precision for analysis of natural samples.

Divergent responses of Atlantic coastal and oceanic Synechococcus to iron limitation

Significance Conventional knowledge suggests that coastal phytoplankton are less able to adapt to Fe limitation than open ocean species. Here we show that in contrast to the established paradigm, coastal Synechococcus from the New England shelf is capable of dynamic, multitiered Fe adaptation that allows it to thrive over a broad range of Fe concentrations by partitioning Fe among different uptake and storage proteins. This protein-based response is beneficial in high nitrogen (N) waters with low and variable Fe:N ratios. An oceanic Atlantic Synechococcus isolate lacks this adaptive response, suggesting the small yet significant N cost of retaining Fe response proteins offsets the benefit of Fe adaptability in the southern Sargasso Sea, where N is chronically scarce and Fe:N ratios are high. Marine Synechococcus are some of the most diverse and ubiquitous phytoplankton, and iron (Fe) is an essential micronutrient that limits productivity in many parts of the ocean. To investigate how coastal and oceanic Atlantic Synechococcus strains acclimate to Fe availability, we compared the growth, photophysiology, and quantitative proteomics of two Synechococcus strains from different Fe regimes. Synechococcus strain WH8102, from a region in the southern Sargasso Sea that receives substantial dust deposition, showed impaired growth and photophysiology as Fe declined, yet used few acclimation responses. Coastal WH8020, from the dynamic, seasonally variable New England shelf, displayed a multitiered, hierarchical cascade of acclimation responses with different Fe thresholds. The multitiered response included changes in Fe acquisition, storage, and photosynthetic proteins, substitution of flavodoxin for ferredoxin, and modified photophysiology, all while maintaining remarkably stable growth rates over a range of Fe concentrations. Modulation of two distinct ferric uptake regulator (Fur) proteins that coincided with the multitiered proteome response was found, implying the coastal strain has different regulatory threshold responses to low Fe availability. Low nitrogen (N) and phosphorus (P) availability in the open ocean may favor the loss of Fe response genes when Fe availability is consistent over time, whereas these genes are retained in dynamic environments where Fe availability fluctuates and N and P are more abundant.

Dissolved iron and iron isotopes in the southeastern Pacific Ocean

The Southeast Pacific Ocean is a severely understudied yet dynamic region for trace metals such as iron, since it experiences steep redox and productivity gradients in upper waters and strong hydrothermal iron inputs to deep waters. In this study, we report the dissolved iron (dFe) distribution from seven stations and Fe isotope ratios (δ56Fe) from three of these stations across a near-zonal transect from 20 to 27°S. We found elevated dFe concentrations associated with the oxygen-deficient zone (ODZ), with light δ56Fe implicating porewater fluxes of reduced Fe. However, temporal dFe variability and rapid δ56Fe shifts with depth suggest gradients in ODZ Fe source and/or redox processes vary over short-depth/spatial scales. The dFe concentrations decreased rapidly offshore, and in the upper ocean dFe was controlled by biological processes, resulting in an Fe:C ratio of 4.2 µmol/mol. Calculated vertical diffusive Fe fluxes were greater than published dust inputs to surface waters, but both were orders of magnitude lower than horizontal diffusive fluxes, which dominate dFe delivery to the gyre. The δ56Fe data in the deep sea showed evidence for a −0.2‰ Antarctic Intermediate Water end-member and a heavy δ56Fe of +0.55‰ for distally transported hydrothermal dissolved Fe from the East Pacific Rise. These heavy δ56Fe values were contrasted with the near-crustal δ56Fe recorded in the hydrothermal plume reaching Station ALOHA in the North Pacific. The heavy hydrothermal δ56Fe precludes a nanopyrite composition of hydrothermal dFe and instead suggests the presence of oxides or, more likely, binding of hydrothermal dFe by organic ligands in the distal plume.

An Inexpensive, Accurate, and Precise Wet-Mount Method for Enumerating Aquatic Viruses

ABSTRACT Viruses affect biogeochemical cycling, microbial mortality, gene flow, and metabolic functions in diverse environments through infection and lysis of microorganisms. Fundamental to quantitatively investigating these roles is the determination of viral abundance in both field and laboratory samples. One current, widely used method to accomplish this with aquatic samples is the “filter mount” method, in which samples are filtered onto costly 0.02-μm-pore-size ceramic filters for enumeration of viruses by epifluorescence microscopy. Here we describe a cost-effective (ca. 500-fold-lower materials cost) alternative virus enumeration method in which fluorescently stained samples are wet mounted directly onto slides, after optional chemical flocculation of viruses in samples with viral concentrations of <5 × 107 viruses ml−1. The concentration of viruses in the sample is then determined from the ratio of viruses to a known concentration of added microsphere beads via epifluorescence microscopy. Virus concentrations obtained by using this wet-mount method, with and without chemical flocculation, were significantly correlated with, and had precision equivalent to, those obtained by the filter mount method across concentrations ranging from 2.17 × 106 to 1.37 × 108 viruses ml−1 when tested by using cultivated viral isolates and natural samples from marine and freshwater environments. In summary, the wet-mount method is significantly less expensive than the filter mount method and is appropriate for rapid, precise, and accurate enumeration of aquatic viruses over a wide range of viral concentrations (≥1 × 106 viruses ml−1) encountered in field and laboratory samples.