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Wan, B, Huang RX, Diaz JM, Tang YZ.  2019.  Polyphosphate adsorption and hydrolysis on aluminum oxides. Environmental Science & Technology. 53:9542-9552.   10.1021/acs.est.9b01876   AbstractWebsite

The geochemical behaviors of phosphate-containing species at mineral-water interfaces are of fundamental importance for controlling phosphorus mobility, fate, and bioavailability. This study investigates the sorption and hydrolysis of polyphosphate (a group of important long-chained phosphate molecules) on aluminum oxides in the presence of divalent metal cations (Ca2+, Cu-,(2+) Mg2+, Mn2+, and Zn2+) at pH 6-8. gamma-Al2O3 with three particle sizes (5, 35, and 70 nm) was used as an analogue of natural aluminum oxides to investigate the particle size effect. All metal cations enhanced polyphosphate hydrolysis at different levels, with Ca2+ showing the most significant enhancement, and the difference in the enhancement might be due to the intrinsic affinity of metal cations to polyphosphate. In the presence of Ca2+, the hydrolysis rate decreased with increasing mineral particle size. Solid-state P-31 nuclear magnetic resonance spectroscopy (NMR) revealed the main surface P species to be amorphous calcium phosphate precipitates, phosphate groups in polyphosphate that formed direct bonds with the mineral surface as inner-sphere complexes, and phosphate groups in polyphosphate that were not directly bonded to the mineral surfaces. Our results reveal the critical roles of mineral water interface processes and divalent metal cations on controlling polyphosphate speciation and transformation and phosphorus cycling.

Diaz, JM, Steffen R, Sanders JG, Tang YZ, Duhamel S.  2019.  Preferential utilization of inorganic polyphosphate over other bioavailable phosphorus sources by the model diatoms Thalassiosira spp. Environmental Microbiology. 21:2415-2425.   10.1111/1462-2920.14630   AbstractWebsite

Polyphosphates and phosphomonoesters are dominant components of marine dissolved organic phosphorus (DOP). Collectively, DOP represents an important nutritional phosphorus (P) source for phytoplankton growth in the ocean, but the contribution of specific DOP sources to microbial community P demand is not fully understood. In a prior study, it was reported that inorganic polyphosphate was not bioavailable to the model diatoms Thalassiosira weissflogii and Thalassiosira pseudonana. However, in this study, we show that the previous finding was a misinterpretation based on a technical artefact of media preparation and that inorganic polyphosphate is actually widely bioavailable to Thalassiosira spp. In fact, orthophosphate, inorganic tripolyphosphate (3polyP), adenosine triphosphate (ATP) and adenosine monophosphate supported equivalent growth rates and final growth yields within each of four strains of Thalassiosira spp. However, enzyme activity assays revealed in all cultures that cell-associated hydrolysis rates of 3polyP were typically more than similar to 10-fold higher than degradation of ATP and the model phosphomonoester compound 4-methylumbelliferyl phosphate. These results build on prior work, which showed the preferential utilization of polyphosphates in the cell-free exudates of Thalassiosira spp., and suggest that inorganic polyphosphates may be a key bioavailable source of P for marine phytoplankton.

Sutherland, KM, Coe A, Gast RJ, Plummer S, Suffridge CP, Diaz JM, Bowman JS, Wankel SD, Hansel CM.  2019.  Extracellular superoxide production by key microbes in the global ocean. Limnology and Oceanography.   10.1002/lno.11247   AbstractWebsite

Bacteria and eukaryotes produce the reactive oxygen species superoxide both within and outside the cell. Although superoxide is typically associated with the detrimental and sometimes fatal effects of oxidative stress, it has also been shown to be involved in a range of essential biochemical processes, including cell signaling, growth, differentiation, and defense. Light-independent extracellular superoxide production has been shown to be widespread among many marine heterotrophs and phytoplankton, but the extent to which this trait is relevant to marine microbial physiology and ecology throughout the global ocean is unknown. Here, we investigate the dark extracellular superoxide production of five groups of organisms that are geographically widespread and represent some of the most abundant organisms in the global ocean. These include Prochlorococcus, Synechococcus, Pelagibacter, Phaeocystis, and Geminigera. Cell-normalized net extracellular superoxide production rates ranged seven orders of magnitude, from undetectable to 14,830 amol cell(-1) h(-1), with the cyanobacterium Prochlorococcus being the lowest producer and the cryptophyte Geminigera being the most prolific producer. Extracellular superoxide production exhibited a strong inverse relationship with cell number, pointing to a potential role in cell signaling. We demonstrate that rapid, cell-number-dependent changes in the net superoxide production rate by Synechococcus and Pelagibacter arose primarily from changes in gross production of extracellular superoxide, not decay. These results expand the relevance of dark extracellular superoxide production to key marine microbes of the global ocean, suggesting that superoxide production in marine waters is regulated by a diverse suite of marine organisms in both dark and sunlit waters.

Diaz, JM, Plummer S, Hansel CM, Andeer PF, Saito MA, McIlvin MR.  2019.  NADPH-dependent extracellular superoxide production is vital to photophysiology in the marine diatom Thalassiosira oceanica.. PNAS. 116(33):16448-16453.   10.1073/pnas.1821233116  
Diaz, JM, Holland A, Sanders JG, Bulski K, Mollett D, Chou C-W, Phillips D, Tang Y, Duhamel S.  2018.  Dissolved organic phosphorus utilization by phytoplankton reveals preferential degradation of polyphosphates over phosphomonoesters. Frontiers in Marine Science. 5   10.3389/fmars.2018.00380   Abstract

The nutritionally available pool of dissolved organic phosphorus (DOP) supports marine primary productivity in a range of ocean ecosystems but remains poorly resolved. Here, the relative lability of model phosphorous (P) compounds representing the major P(V) bond classes of marine DOP – phosphomonoesters (P-O-C) and phosphoanhydrides (P-O-P) – was assessed in diatom cultures of the genus Thalassiosira, as well as coastal field sites of the western North Atlantic. In diatom samples, maximum enzymatic hydrolysis rates revealed that the P-anhydride bonds of inorganic tripolyphosphate (3poly-P), followed by the P-anhydride bonds of adenosine 5’-triphosphate (ATP), were preferentially degraded relative to the P-monoesters adenosine 5’-monophosphate (AMP) and 4-methylumbelliferone phosphate (MUF-P). Consistent with these rate measurements, targeted proteomics analysis demonstrated that the underlying phosphatase diversity present in diatom samples was dominated by P-anhydride degrading enzymes (inorganic pyrophosphatases and nucleoside triphosphatases). Furthermore, biomass-normalized rates of ATP degradation were always suppressed under P-replete conditions in diatom cultures, but the effect of overall P availability on 3poly-P degradation was inconsistent among diatom strains, suggesting that inorganic polyphosphate (poly-P) degradation may persist irrespective of prevailing P levels in the marine environment. Indeed, the majority of field sites examined in the P-replete coastal western North Atlantic exhibited significantly higher maximum rates of inorganic poly-P hydrolysis relative to P-monoester hydrolysis, which was largely driven by phytoplankton dynamics. Based on these results, the possibility that P-anhydride utilization may contribute comparably or even more substantially than P-esters to community-level P demand, phytoplankton growth, and primary productivity should be considered.

Huang, R, Wan B, Hultz M, Diaz JM, Tang Y.  2018.  Phosphatase-mediated hydrolysis of linear polyphosphates. Environmental Science & Technology. 52:1183-1190.: American Chemical Society   10.1021/acs.est.7b04553   AbstractWebsite

Polyphosphates are a group of phosphorus (P) containing molecules that are produced by a wide range of microorganisms and human activities. Although polyphosphates are ubiquitous in aquatic environments and are of environmental significance, little is known about their transformation and cycling. This study characterized the polyphopshate-hydrolysis mechanisms of several representative phosphatase enzymes and evaluated the effects of polyphosphate chain length, light condition, and calcium (Ca2+). 31P nuclear magnetic resonance (NMR) spectroscopy was used to monitor the dynamic changes of P molecular configuration during polyphosphate hydrolysis and suggested a terminal-only degradation pathway by the enzymes. Such mechanism enabled the quantification of the hydrolysis rates by measuring orthophosphate production over time. At the same initial concentration of polyphosphate molecules, the hydrolysis rates were independent of chain length. The hydrolysis of polyphosphate was also unaffected by light condition, but was reduced by the presence of Ca2+. The released orthophosphates formed Ca-phosphate precipitates in the presence of Ca2+, likely in amorphous phases. Results from this study lay the foundation for better understanding the chemical processes governing polyphosphate transport and transformation in various environmental settings.

Diaz, JM, Plummer S.  2018.  Production of extracellular reactive oxygen species by phytoplankton: past and future directions. Journal of Plankton Research. 40(6):655-666.   10.1093/plankt/fby039  
Diaz, JM, Plummer S, Tomas C, Alves-de-Souza C.  2018.  Production of extracellular superoxide and hydrogen peroxide by five marine species of harmful bloom-forming algae. Journal of Plankton Research. 40(6):667-677.   10.1093/plankt/fby043  
Diaz, JM, Hansel CM, Apprill A, Brighi C, Zhang T, Weber L, McNally S, Xun L.  2016.  Species-specific control of external superoxide levels by the coral holobiont during a natural bleaching event. Nature Communications. 7:13801.: The Author(s)   10.1038/ncomms13801   Abstract

The reactive oxygen species superoxide (O2·−) is both beneficial and detrimental to life. Within corals, superoxide may contribute to pathogen resistance but also bleaching, the loss of essential algal symbionts. Yet, the role of superoxide in coral health and physiology is not completely understood owing to a lack of direct in situ observations. By conducting field measurements of superoxide produced by corals during a bleaching event, we show substantial species-specific variation in external superoxide levels, which reflect the balance of production and degradation processes. Extracellular superoxide concentrations are independent of light, algal symbiont abundance and bleaching status, but depend on coral species and bacterial community composition. Furthermore, coral-derived superoxide concentrations ranged from levels below bulk seawater up to ∼120 nM, some of the highest superoxide concentrations observed in marine systems. Overall, these results unveil the ability of corals and/or their microbiomes to regulate superoxide in their immediate surroundings, which suggests species-specific roles of superoxide in coral health and physiology.

Zhang, T, Diaz JM, Brighi C, Parsons RJ, McNally S, Apprill A, Hansel CM.  2016.  Dark production of extracellular superoxide by the coral Porites astreoides and representative symbionts. Frontiers in Marine Science. 3   10.3389/fmars.2016.00232   AbstractWebsite

The reactive oxygen species (ROS) superoxide has been implicated in both beneficial and detrimental processes in coral biology, ranging from pathogenic disease resistance to coral bleaching. Despite the critical role of ROS in coral health, there is a distinct lack of ROS measurements and thus an incomplete understanding of underpinning ROS sources and production mechanisms within coral systems. Here, we quantified in situ extracellular superoxide concentrations at the surfaces of aquaria-hosted Porites astreoides during a diel cycle. High concentrations of superoxide (~10’s of nM) were present at coral surfaces, and these levels did not change significantly as a function of time of day. These results indicate that the coral holobiont produces extracellular superoxide in the dark, independent of photosynthesis. As a short-lived anion at physiological pH, superoxide has a limited ability to cross intact biological membranes. Further, removing surface mucus layers from the P. astreoides colonies did not impact external superoxide concentrations. We therefore attribute external superoxide derived from the coral holobiont under these conditions to the activity of the coral host epithelium, rather than mucus-derived epibionts or internal sources such as endosymbionts (e.g., Symbiodinium). However, endosymbionts likely contribute to internal ROS levels via extracellular superoxide production. Indeed, common coral symbionts, including multiple strains of Symbiodinium (clades A to D) and the bacterium Endozoicomonas montiporae LMG 24815, produced extracellular superoxide in the dark and at low light levels. Further, representative P. astreoides symbionts, Symbiodinium CCMP2456 (clade A) and E. montiporae, produced similar concentrations of superoxide alone and in combination with each other, in the dark and low light, and regardless of time of day. Overall, these results indicate that healthy, non-stressed P. astreoides and representative symbionts produce superoxide externally, which is decoupled from photosynthetic activity and circadian control. Corals may therefore produce extracellular superoxide constitutively, highlighting an unclear yet potentially beneficial role for superoxide in coral physiology and health.

Hansel, CM, Buchwald C, Diaz JM, Ossolinski JE, Dyhrman ST, Van Mooy BAS, Polyviou D.  2016.  Dynamics of extracellular superoxide production by Trichodesmium colonies from the Sargasso Sea. Limnology and Oceanography. 61:1188-1200.   10.1002/lno.10266   Abstract

Reactive oxygen species (ROS) are key players in the health and biogeochemistry of the ocean and its inhabitants. The vital contribution of microorganisms to marine ROS levels, particularly superoxide, has only recently come to light, and thus the specific biological sources and pathways involved in ROS production are largely unknown. To better understand the biogenic controls on ROS levels in tropical oligotrophic systems, we determined rates of superoxide production under various conditions by natural populations of the nitrogen-fixing diazotroph Trichodesmium obtained from various surface waters in the Sargasso Sea. Trichodesmium colonies collected from eight different stations all produced extracellular superoxide at high rates in both the dark and light. Colony density and light had a variable impact on extracellular superoxide production depending on the morphology of the Trichodesmium colonies. Raft morphotypes showed a rapid increase in superoxide production in response to even low levels of light, which was not observed for puff colonies. In contrast, superoxide production rates per colony decreased with increasing colony density for puff morphotypes but not for rafts. These findings point to Trichodesmium as a likely key source of ROS to the surface oligotrophic ocean. The physiological and/or ecological factors underpinning morphology-dependent controls on superoxide production need to be unveiled to better understand and predict superoxide production by Trichodesmium and ROS dynamics within marine systems.

Diaz, JM, Björkman KM, Haley ST, Ingall ED, Karl DM, Longo AF, Dyhrman ST.  2016.  Polyphosphate dynamics at Station ALOHA, North Pacific subtropical gyre. Limnology and Oceanography. 61:227-239.   10.1002/lno.10206   Abstract

Polyphosphate (polyP) was examined within the upper water column (≤ 150 m) of Station ALOHA (22° 45′N, 158° 00′W) during two cruises conducted in May–June 2013 and September 2013. Phosphorus molar ratios of particulate polyP to total particulate phosphorus (TPP) were relatively low, similar to previously reported values from the temperate western North Atlantic, and did not exhibit strong vertical gradients, reflecting a lack of polyP recycling relative to other forms of TPP with depth. Furthermore, relationships among polyP:TPP, soluble reactive phosphorus (SRP), and alkaline phosphatase activity (APA) were also consistent with previous observations from the Atlantic Ocean. To ascertain potential mechanisms of biological polyP production and utilization, surface seawater was incubated following nutrient additions. Results were consistent with polyP:TPP enrichment under opposite extremes of APA, suggesting diverse polyP accumulation/retention mechanisms. Addition of exogenous polyP (45 ± 5 P atoms) to field incubations did not increase chlorophyll content relative to controls, suggesting that polyP was not bioavailable to phytoplankton at Station ALOHA. To clarify this result, phytoplankton cultures were screened for the ability to utilize exogenous polyP. PolyP bioavailability was variable among model diatoms of the genus Thalassiosira, yet chain length did not influence polyP bioavailability. Thus, microbial community composition may influence polyP dynamics in the ocean, and vice versa.

Saad, EM, Longo AF, Chambers LR, Huang R, Benitez-Nelson C, Dyhrman ST, Diaz JM, Tang Y, Ingall ED.  2016.  Understanding marine dissolved organic matter production: Compositional insights from axenic cultures of Thalassiosira pseudonana. Limnology and Oceanography. 61:2222-2233.   10.1002/lno.10367   Abstract

Marine dissolved organic matter (DOM) is a key source of carbon and nutrients to microbial life in the oceans, but rapid biological utilization of labile DOM confounds its compositional characterization. In order to characterize potentially bioavailable DOM produced by phytoplankton, DOM from axenic cultures of Thalassiosira pseudonana cultivated in phosphorus (P) replete and low P conditions was extracted using high-recovery electrodialysis (ED) techniques, which resulted in an average dissolved organic carbon (DOC) recovery of 76% ± 7% from all cultures. Low P concentrations resulted in greater cell-normalized production of DOC relative to P replete culture controls at the same growth phase. Despite the different nutrient conditions, DOC composition and DOM molar ratios of carbon to nitrogen (C : N) were similar in all cultures. In contrast, low P concentrations influenced DOM molar carbon to phosphorus (C : P) ratios and dissolved organic phosphorus (DOP) composition. Under P replete and low P conditions, DOM C : P ratios were 130 (± 22) and 2446 (± 519), respectively. 31P Nuclear Magnetic Resonance (NMR) spectroscopy identified P esters (> 90% of DOP) as the dominant P species in DOM produced under P replete conditions, with small or negligible contributions from phosphonates or glycerol P and polyphosphates. However, based on direct fluorometric analysis, DOP from low P cultures was greater than 8 times enriched in dissolved polyphosphate compared to DOP from replete cultures, which is consistent with the growing evidence that polyphosphate is a dynamic component of total P in low P ocean regions.

Marsico, RM, Schneider RJ, Voelker BM, Zhang T, Diaz JM, Hansel CM, Ushijima S.  2015.  Spatial and temporal variability of widespread dark production and decay of hydrogen peroxide in freshwater. Aquatic Sciences. 77:523-533.   10.1007/s00027-015-0399-2   Abstract

Hydrogen peroxide (H2O2) is an oxidant and reductant of redox active metals and a potential source of strong oxidants such as the hydroxyl radical (·OH). H2O2 production in freshwater has been largely attributed to photo-oxidation of chromophoric dissolved organic matter, while its decay has been linked to enzymatic processes as well as to chemical reactions with metals. More recently, however, microorganisms were postulated as a significant source as well as a sink of H2O2 in freshwater. In this study, we examined the spatial and temporal variability of dark H2O2 production rates ($${\text{P}}_{{{\text{H}}_{ 2} {\text{O}}_{ 2} }}$$PH2O2) and pseudo-first order dark decay rate coefficients ($${\text{k}}_{{{\text{loss,H}}_{ 2} {\text{O}}_{ 2} }}$$kloss,H2O2) in incubations of water samples from sites with a range of trophic states in Colorado (CO) and Massachusetts (MA). Observed values of $${\text{P}}_{{{\text{H}}_{ 2} {\text{O}}_{ 2} }}$$PH2O2and $${\text{k}}_{{{\text{loss,H}}_{ 2} {\text{O}}_{ 2} }}$$kloss,H2O2ranged from 3 to 259 nM h−1 and 0.02 to 8.87 h−1, respectively. Microbial cell numbers and chlorophyll content correlated strongly with $${\text{k}}_{{{\text{loss,H}}_{ 2} {\text{O}}_{ 2} }}$$kloss,H2O2while filtering the freshwater samples removed the majority of $${\text{k}}_{{{\text{loss,H}}_{ 2} {\text{O}}_{ 2} }}$$kloss,H2O2, indicating breakdown by biota as the major sink of H2O2. Dark production of H2O2 was also ubiquitous, but $${\text{P}}_{{{\text{H}}_{ 2} {\text{O}}_{ 2} }}$$PH2O2was not well correlated with indicators of microbial abundance. For instance, several oligotrophic sites (with low $${\text{k}}_{{{\text{loss,H}}_{ 2} {\text{O}}_{ 2} }}$$kloss,H2O2) exhibited moderately high $${\text{P}}_{{{\text{H}}_{ 2} {\text{O}}_{ 2} }}$$PH2O2, while a sample with unusually high chlorophyll content (and a correspondingly high $${\text{k}}_{{{\text{loss,H}}_{ 2} {\text{O}}_{ 2} }}$$kloss,H2O2) had a relatively low $${\text{P}}_{{{\text{H}}_{ 2} {\text{O}}_{ 2} }}$$PH2O2. One possible explanation for this phenomenon is that the ability to break down H2O2 is similar among different microorganisms, but the ability to produce H2O2 differs with microbial composition.

De Santiago, A, Longo AF, Ingall ED, Diaz JM, King LE, Lai B, Weber RJ, Russell AG, Oakes M.  2014.  Characterization of selenium in ambient aerosols and primary emission sources. Environmental Science & Technology. 48:8988-8994.: American Chemical Society   10.1021/es500379y   Abstract

Atmospheric selenium (Se) in aerosols was investigated using X-ray absorption near-edge structure (XANES) spectroscopy and X-ray fluorescence (XRF) microscopy. These techniques were used to determine the oxidation state and elemental associations of Se in common primary emission sources and ambient aerosols collected from the greater Atlanta area. In the majority of ambient aerosol and primary emission source samples, the spectroscopic patterns as well as the absence of elemental correlations suggest Se is in an elemental, organic, or oxide form. XRF microscopy revealed numerous Se-rich particles, or hotspots, accounting on average for ∼16% of the total Se in ambient aerosols. Hotspots contained primarily Se0/Se(−II). However, larger, bulk spectroscopic characterizations revealed Se(IV) as the dominant oxidation state in ambient aerosol, followed by Se0/Se(−II) and Se(VI). Se(IV) was the only observed oxidation state in gasoline, diesel, and coal fly ash, while biomass burning contained a combination of Se0/Se(−II) and Se(IV). Although the majority of Se in aerosols was in the most toxic form, the Se concentration is well below the California Environmental Protection Agency chronic exposure limit (∼20000 ng/m3).

Longo, AF, Ingall ED, Diaz JM, Oakes M, King LE, Nenes A, Mihalopoulos N, Violaki K, Avila A, Benitez-Nelson CR, Brandes J, McNulty I, Vine DJ.  2014.  P-NEXFS analysis of aerosol phosphorus delivered to the Mediterranean Sea. Geophysical Research Letters. 41:4043-4049.   10.1002/2014GL060555   Abstract

Biological productivity in many ocean regions is controlled by the availability of the nutrient phosphorus. In the Mediterranean Sea, aerosol deposition is a key source of phosphorus and understanding its composition is critical for determining its potential bioavailability. Aerosol phosphorus was investigated in European and North African air masses using phosphorus near-edge X-ray fluorescence spectroscopy (P-NEXFS). These air masses are the main source of aerosol deposition to the Mediterranean Sea. We show that European aerosols are a significant source of soluble phosphorus to the Mediterranean Sea. European aerosols deliver on average 3.5 times more soluble phosphorus than North African aerosols and furthermore are dominated by organic phosphorus compounds. The ultimate source of organic phosphorus does not stem from common primary emission sources. Rather, phosphorus associated with bacteria best explains the presence of organic phosphorus in Mediterranean aerosols.

Ingall, ED, Diaz JM, Longo AF, Oakes M, Finney L, Vogt S, Lai B, Yager PL, Twining BS, Brandes JA.  2013.  Role of biogenic silica in the removal of iron from the Antarctic seas. Nature Communications. 4:1981.: Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved.   10.1038/ncomms2981   Abstract

The upper basin of Effingham Inlet possesses permanently anoxic bottom waters, with a water column redox transition zone typically occurring at least 40 m above the sediment‐water interface. During our sampling campaign in April and July 2007, this redox transition zone was associated with sharp peaks in a variety of parameters, including soluble reactive phosphorus (SRP) and total particulate phosphorus (TPP). Based on sequential extraction results, TPP maxima exhibited preferential accumulation of an operationally defined class of loosely adsorbed organic phosphorus (P), which may contain a substantial fraction of polyphosphate (poly‐P). This poly‐P may furthermore be involved in the redox‐dependent remobilization of SRP. For example, direct fluorometric analysis of poly‐P content revealed that particulate inorganic poly‐P was present at concentrations ranging from 1 to 9 nM P within and several meters above the TPP maximum. Below the depth of 1% oxygen saturation, however, particulate inorganic poly‐P was undetectable (<0.8 nM in situ). Assuming this concentration profile reflects the remineralization of inorganic poly‐P to SRP across the redox transition, inorganic poly‐P degradation accounted for as much as 4 ± 3% (average ± standard deviation) to 9 ± 8% of the vertical turbulent diffusive SRP flux. This finding is a conservative estimate due in part to sample storage effects associated with our analysis of poly‐P content. By comparison, iron‐linked P cycling accounted for at most 65 ± 33% of the diffusive SRP flux, leaving ∼25% unaccounted for. Thus, while redox‐sensitive poly‐P remineralization in Effingham Inlet appears modest based on our direct conservative estimate, it may be higher from a mass balance viewpoint. Poly‐P cycling may therefore be an overlooked mechanism for the redox‐sensitive cycling of P in some hypoxic/anoxic boundaries, especially iron‐poor marine oxygen minimum zones.

Diaz, JM, Hansel CM, Voelker BM, Mendes CM, Andeer PF, Zhang T.  2013.  Widespread production of extracellular superoxide by heterotrophic bacteria. Science. 340:1223-1226.   10.1126/science.1237331   Abstract

The global imprint of biological activity in aquatic environments is often considered a consequence of enzyme-mediated redox reactions that support metabolic activity, such as reducing oxygen during respiration. But some organisms also release redox-active reactive oxygen species (ROS) into the environment—to acquire trace metals or to prevent viral infections—which can influence global processes like nutrient availability and contaminant transport. Photosynthetic organisms like phytoplankton are thought to be the primary biological source of ROS in freshwater and marine environments. However, Diaz et al. (p. 1223, published online 2 May; see the Perspective by Shaked and Rose) now show that a broad range of ecologically and phylogenetically diverse heterotrophic bacteria also produce large quantities of superoxide. Production rates vary widely across 30 common bacterial isolates but in some cases were greater than production rates of phytoplankton. Because these bacteria do not require light to grow, they may be the dominant source of ROS in dark environments like the deep ocean, terrestrial soils, or lake sediments.Superoxide and other reactive oxygen species (ROS) originate from several natural sources and profoundly influence numerous elemental cycles, including carbon and trace metals. In the deep ocean, the permanent absence of light precludes currently known ROS sources, yet ROS production mysteriously occurs. Here, we show that taxonomically and ecologically diverse heterotrophic bacteria from aquatic and terrestrial environments are a vast, unrecognized, and light-independent source of superoxide, and perhaps other ROS derived from superoxide. Superoxide production by a model bacterium within the ubiquitous Roseobacter clade involves an extracellular oxidoreductase that is stimulated by the reduced form of nicotinamide adenine dinucleotide (NADH), suggesting a surprising homology with eukaryotic organisms. The consequences of ROS cycling in immense aphotic zones representing key sites of nutrient regeneration and carbon export must now be considered, including potential control of carbon remineralization and metal bioavailability.

Mitchell, K, Mason PRD, Van Cappellen P, Johnson TM, Gill BC, Owens JD, Diaz J, Ingall ED, Reichart G-J, Lyons TW.  2012.  Selenium as paleo-oceanographic proxy: A first assessment. Geochimica et Cosmochimica Acta. 89:302-317.   10.1016/j.gca.2012.03.038   AbstractWebsite

Selenium (Se) is an essential trace element, which, with multiple oxidation states and six stable isotopes, has been suggested as a potentially powerful paleoenvironmental proxy. In this study, bulk Se concentrations and isotopic compositions were analyzed in a suite of about 120 samples of fine-grained marine sedimentary rocks and sediments spanning the entire Phanerozoic. While the Se concentrations vary greatly (0.22–72ppm), the δ82/76Se values fall in a fairly narrow range from −1 to +1‰ (relative to NIST SRM3149), with the exception of laminated black shales from the New Albany Shale formation (Late Devonian), which have δ82/76Se values of up to +2.20‰. Black Sea sediments (Holocene) and sedimentary rocks from the Alum Shale formation (Late Cambrian) have Se to total organic carbon ratios (Se/TOC) and δ82/76Se values close to those found in modern marine plankton (1.72±0.15×10−6mol/mol and 0.42±0.22‰). For the other sedimentary sequences and sediments, the Se/TOC ratios show Se enrichment relative to modern marine plankton. Additional input of isotopically light terrigenous Se may explain the Se/TOC and δ82/76Se data measured in recent Arabian Sea sediments (Pleistocene). The very high Se concentrations in sedimentary sequences that include the Cenomanian–Turonian Oceanic Anoxic Event (OAE) 2 may reflect an enhanced input of volcanogenic Se to the oceans. As the latter has an isotopic composition not greatly different from marine plankton, the volcanogenic source does not impart a distinct signature to the sedimentary Se isotope record. The lowest average δ82/76Se values are observed in the OAE2 samples from Demerara Rise and Cape Verde Basin cores (δ82/76Se=−0.14±0.45‰) and could reflect fractionation associated with microbial or chemical reduction of Se oxyanions in the euxinic water column. In contrast, a limiting availability of seawater Se during periods of increased organic matter production and burial may be responsible for the elevated δ82/76Se values and low Se/TOC ratios in the black shales of the New Albany Shale formation. Overall, our results indicate that to unlock the full proxy potential of marine sedimentary Se records, we need to gain a much more detailed understanding of the sources, chemical speciation, isotopic fractionations and cycling of Se in the marine environment.

Diaz, JM, Ingall ED, Snow SD, Benitez-Nelson CR, Taillefert M, Brandes JA.  2012.  Potential role of inorganic polyphosphate in the cycling of phosphorus within the hypoxic water column of Effingham Inlet, British Columbia. Global Biogeochemical Cycles. 26   10.1029/2011GB004226   Abstract

The upper basin of Effingham Inlet possesses permanently anoxic bottom waters, with a water column redox transition zone typically occurring at least 40 m above the sediment-water interface. During our sampling campaign in April and July 2007, this redox transition zone was associated with sharp peaks in a variety of parameters, including soluble reactive phosphorus (SRP) and total particulate phosphorus (TPP). Based on sequential extraction results, TPP maxima exhibited preferential accumulation of an operationally defined class of loosely adsorbed organic phosphorus (P), which may contain a substantial fraction of polyphosphate (poly-P). This poly-P may furthermore be involved in the redox-dependent remobilization of SRP. For example, direct fluorometric analysis of poly-P content revealed that particulate inorganic poly-P was present at concentrations ranging from 1 to 9 nM P within and several meters above the TPP maximum. Below the depth of 1% oxygen saturation, however, particulate inorganic poly-P was undetectable (<0.8 nM in situ). Assuming this concentration profile reflects the remineralization of inorganic poly-P to SRP across the redox transition, inorganic poly-P degradation accounted for as much as 4 ± 3% (average ± standard deviation) to 9 ± 8% of the vertical turbulent diffusive SRP flux. This finding is a conservative estimate due in part to sample storage effects associated with our analysis of poly-P content. By comparison, iron-linked P cycling accounted for at most 65 ± 33% of the diffusive SRP flux, leaving ∼25% unaccounted for. Thus, while redox-sensitive poly-P remineralization in Effingham Inlet appears modest based on our direct conservative estimate, it may be higher from a mass balance viewpoint. Poly-P cycling may therefore be an overlooked mechanism for the redox-sensitive cycling of P in some hypoxic/anoxic boundaries, especially iron-poor marine oxygen minimum zones.

Ingall, Ellery D., Brandes, Jay A., Diaz, Julia M., de Jonge, Martin D., Paterson, David, McNulty, Ian, Elliott, W. Crawford, Northrup, Paul.  2011.  Phosphorus K-edge XANES spectroscopy of mineral standards. Journal of Synchrotron Radiation. 18:189-197.   10.1107/S0909049510045322   Abstract

Phosphorus K-edge X-ray absorption near-edge structure (XANES) spectroscopy was performed on phosphate mineral specimens including (a) twelve specimens from the apatite group covering a range of compositional variation and crystallinity; (b) six non-apatite calcium-rich phosphate minerals; (c) 15 aluminium-rich phosphate minerals; (d) ten phosphate minerals rich in either reduced iron or manganese; (e) four phosphate minerals rich in either oxidized iron or manganese; (f) eight phosphate minerals rich in either magnesium, copper, lead, zinc or rare-earth elements; and (g) four uranium phosphate minerals. The identity of all minerals examined in this study was independently confirmed using X-ray powder diffraction. Minerals were distinguished using XANES spectra with a combination of pre-edge features, edge position, peak shapes and post-edge features. Shared spectral features were observed in minerals with compositions dominated by the same specific cation. Analyses of apatite-group minerals indicate that XANES spectral patterns are not strongly affected by variations in composition and crystallinity typical of natural mineral specimens.

Jiang, L-Q, Cai W-J, Wang Y, Diaz J, Yager PL, Hu X.  2010.  Pelagic community respiration on the continental shelf off Georgia, USA. Biogeochemistry. 98:101-113.   10.1007/s10533-009-9379-8   AbstractWebsite

The South Atlantic Bight (SAB) has been a focus for the study of continental shelf ecosystem respiration during the past two decades. However, two questions concerning respiration in this area have yet to be answered. First, why do previous estimates of respiration in the SAB exceed measured carbon fixation rates by almost an order of magnitude? Second, considering that bacteria are responsible for most of the pelagic community respiration in the SAB, why is respiration almost uniform from the coastline to the shelf break, while bacterial production estimates decrease offshore? This study addresses these critical questions by presenting new pelagic community respiration data that were collected across the entire width of the continental shelf off Georgia, USA from June 2003 to May 2006. The respiration was calculated as in vitro changes of dissolved oxygen and dissolved inorganic carbon concentrations during deck incubations. The measured respiration rates ranged from 0.3(±0.1) to 21.2(±1.4) mmol m−3 day−1. They followed a clear seasonal pattern, being lowest over the entire shelf in winter and reaching maxima in summer. Summertime respiration rates were highest on the inner shelf and decreased with distance offshore. Consistent with this trend, bacterial abundance measurements taken during the sampling month of July 2005 followed a pattern of seaward decline. The SAB organic carbon fluxes calculated from the respiration data are close to the estimates for primary production, which resolves a long-standing mystery regarding perceived carbon imbalance in the SAB.

Diaz, JM, Ingall ED.  2010.  Fluorometric Quantification of Natural Inorganic Polyphosphate. Environmental Science & Technology. 44:4665-4671.: American Chemical Society   10.1021/es100191h   AbstractWebsite

Polyphosphate, a linear polymer of orthophosphate, is abundant in the environment and a key component in wastewater treatment and many bioremediation processes. Despite the broad relevance of polyphosphate, current methods to quantify it possess significant disadvantages. Here, we describe a new approach for the direct quantification of inorganic polyphosphate in complex natural samples. The protocol relies on the interaction between the fluorochrome 4′,6-diamidino-2-phenylindole (DAPI) and dissolved polyphosphate. With the DAPI-based approach we describe, polyphosphate can be quantified at concentrations ranging from 0.5−3 μM P in a neutral-buffered freshwater matrix with an accuracy of ±0.03 μM P. The patterns of polyphosphate concentration versus fluorescence yielded by standards exhibit no chain length dependence across polyphosphates ranging from 15−130 phosphorus units in size. Shorter length polyphosphate molecules (e.g., polyphosphate of three and five phosphorus units in length) contribute little to no signal in this approach, as these molecules react only slightly or not at all with DAPI in the concentration range tested. The presence of salt suppresses fluorescence from intermediate polyphosphate chain lengths (e.g., 15 phosphorus units) at polyphosphate concentrations ranging from 0.5−3 μM P. For longer chain lengths (e.g., 45−130 phosphorus units), this salt interference is not evident at conductivities up to ∼10mS/cm. Our results indicate that standard polyphosphates should be stored frozen for no longer than 10−15 days to avoid inconsistent results associated with standard degradation. We have applied the fluorometric protocol to the analysis of five well-characterized natural samples to demonstrate the use of the method.

de Jonge, MD, Holzner C, Baines SB, Twining BS, Ignatyev K, Diaz J, Howard DL, Legnini D, Miceli A, McNulty I, Jacobsen CJ, Vogt S.  2010.  Quantitative 3D elemental microtomography of Cyclotella meneghiniana at 400-nm resolution. Proceedings of the National Academy of Sciences. 107:15676-15680. Abstract

X-ray fluorescence tomography promises to map elemental distributions in unstained and unfixed biological specimens in three dimensions at high resolution and sensitivity, offering unparalleled insight in medical, biological, and environmental sciences. X-ray fluorescence tomography of biological specimens has been viewed as impractical—and perhaps even impossible for routine application—due to the large time required for scanning tomography and significant radiation dose delivered to the specimen during the imaging process. Here, we demonstrate submicron resolution X-ray fluorescence tomography of a whole unstained biological specimen, quantifying three-dimensional distributions of the elements Si, P, S, Cl, K, Ca, Mn, Fe, Cu, and Zn in the freshwater diatom Cyclotella meneghiniana with 400-nm resolution, improving the spatial resolution by over an order of magnitude. The resulting maps faithfully reproduce cellular structure revealing unexpected patterns that may elucidate the role of metals in diatom biology and of diatoms in global element cycles. With anticipated improvements in data acquisition and detector sensitivity, such measurements could become routine in the near future.

Diaz, J, Ingall E, Vogt S, de Jonge MD, Paterson D, Rau C, Brandes JA.  2009.  Characterization of phosphorus, calcium, iron, and other elements in organisms at sub-micron resolution using X-ray fluorescence spectromicroscopy. Limnology and Oceanography: Methods. 7:42-51.   10.4319/lom.2009.7.42   Abstract

X-ray spectromicroscopy (combined X-ray spectroscopy and microscopy) is uniquely capable of determining sub-micron scale elemental content and chemical speciation in minimally-prepared particulate samples. The high spatial resolutions achievable with this technique have enabled the close examination of important microscale processes relevant to the cycling of biogeochemically important elements. Here, we demonstrate the value of X-ray microscopy to environmental and biological research by examining the phosphorus and metal chemistry of complete individual cells from the algal genera Chlamydomonas sp. and Chlorella sp. X-ray analysis revealed that both genera store substantial intracellular phosphorus as distinct, heterogeneously distributed granules whose X-ray fluorescence spectra are consistent with that of polyphosphate. Polyphosphate inclusions ranged in size from 0.3-1.4 µm in diameter and exhibited a nonspecies-specific average phosphorus concentration of 6.87 ± 1.86 µg cm−2, which was significantly higher than the average concentration of phosphorus measured in the total cell, at 3.14 ± 0.98 µg cm−2 (95% confidence). Polyphosphate was consistently associated with calcium and iron, exhibiting average P:cation molar ratios of 8.31 ± 2.00 and 108 ± 34, respectively (95% confidence). In some cells, polyphosphate was also associated with potassium, zinc, manganese, and titanium. Based on our results, X-ray spectromicroscopy can provide high-resolution elemental data on minimally prepared, unsectioned cells that are unattainable through alternative microscopic methods and conventional bulk chemical techniques currently available in many fields of marine chemistry.