An optimized method for extraction and purification of inorganic phosphate from plant material for oxygen isotope ratio analysis.

Compound-specific stable isotope ratio analysis of oxygen isotopes in inorganic phosphate can be used to study biological phosphorus cycling and the transformation processes controlling the fate of phosphorus. However, methods for extraction of inorganic phosphate from plant tissue for oxygen isotope ratio analysis are not consistent. Further, the purification into solid silver phosphate can be challenging and laborious. In this work, a detailed and optimized method to provide a more consistent, easily implementable and reproducible extraction using trichloroacetic acid and subsequent purification of inorganic phosphate from plant material for oxygen isotope ratio analysis is presented. Key focus points were: uniform extraction of inorganic phosphate from barley leaves, removal of dissolved organic material, flexibility in regards to the amount of inorganic phosphate extracted for the purification into silver phosphate, reduced use of chemicals and, removal of co-precipitated oxygen-bearing compounds before analysis. Most notable optimizations to the method and associated effects were:•Drying of plant material before inorganic phosphate extraction increases the method applicability to a broader range of plant sample types.•Removal of dissolved organic matter improves inorganic phosphate purification.•Sample volume adjustment according to inorganic phosphate content is vital for effective and quantitative precipitations.

Mechanism of methylphosphonic acid photo-degradation based on phosphate oxygen isotopes and density functional theory.

Methylphosphonic acid (MPn) is an intermediate in the synthesis of the phosphorus-containing nerve agents, such as sarin and VX, and a biosynthesis product of marine microbes with ramifications to global climate change and eutrophication. Here, we applied the multi-labeled water isotope probing (MLWIP) approach to investigate the C-P bond cleavage mechanism of MPn under UV irradiation and density functional theory (DFT) to simulate the photo-oxidation reaction process involving reactive oxygen species (ROS). The results contrasted with those of the addition of the ROS-quenching compounds, 2-propanol and NaN3. The degradation kinetics results indicated that the extent of MPn degradation was more under alkaline conditions and that the degradation process was more rapid at the initial stage of the reaction. The phosphate oxygen isotope data confirmed that one exogenous oxygen atom was incorporated into the product orthophosphate (PO4) following the C-P bond cleavage, and the oxygen isotopic composition of this free PO4 was found to vary with pH. The combined results of the ROS-quenching experiments and DFT indicate that the C-P bond was cleaved by OH-/˙OH and not by other reactive oxygen species. Based on these results, we have established a mechanistic model for the photolysis of MPn, which provides new insights into the fate of MPn and other phosphonate/organophosphate compounds in the environment.

Phosphate oxygen isotope evidence for methylphosphonate sources of methane and dissolved inorganic phosphate.

The ocean is an important source of methane, however, the sources of oceanic methane and mechanisms of its release to the atmosphere have only recently begun to be understood. Recent studies have identified methylphosphonate (MPn) as a previously unknown and likely source of methane in the aerobic ocean (Karl et al., 2008), as well as shown the biosynthesis of methylphosphonic acid to be a widespread trait in marine microbes (Metcalf et al., 2012). The mechanisms and reaction pathways from MPn to free methane, however, have not been well studied. Here we present results of laboratory studies on the photo-degradation of MPn, a likely mechanism of methane release to the atmosphere and phosphate release to the surface oceans. Phosphonoacetic acid was also studied as an additional model compound for comparison. We used the multi-labeled water isotope probing (MLWIP) approach, involving 18O-labeled waters to probe the photolytic mechanism of CP bond cleavage in phosphates through analysis of P released from MPn as PO4. These studies identified distinct reaction pathways involving phosphates compared with other common organophosphorus compounds (e.g., phosphoesters), as well as suggest the involvement of both ambient water and atmospheric oxygen in CP bond cleavage. There is only a small amount of water oxygen incorporated into product PO4 after cleavage of the CP bond in MPn, suggesting atmospheric O2 or radicals formed from O2 under Ultra Violet Radiation (UVR), as the primary source of O that replaces C in the CP bond of MPn. Model calculations suggest that the δ18OP signature of phosphate released via UV-degradation of phosphates is largely (75%) inherited from the original phosphate substrate. This opens up the possibility of tracing and differentiating specific phosphate sources of dissolved phosphate from other organophosphorus (Porg) sources (e.g., phosphoesters) used in primary production, as well as for tracing specific MPn sources of atmospheric methane.

Archean phosphorus liberation induced by iron redox geochemistry.

The element phosphorus (P) is central to ecosystem growth and is proposed to be a limiting nutrient for life. The Archean ocean may have been strongly phosphorus-limited due to the selective binding of phosphate to iron oxyhydroxide. Here we report a new route to solubilizing phosphorus in the ancient oceans: reduction of phosphate to phosphite by iron(II) at low (<200 °C) diagenetic temperatures. Reduction of phosphate to phosphite was likely widespread in the Archean, as the reaction occurs rapidly and is demonstrated from thermochemical modeling, experimental analogs, and detection of phosphite in early Archean rocks. We further demonstrate that the higher solubility of phosphite compared to phosphate results in the liberation of phosphorus from ferruginous sediments. This phosphite is relatively stable after its formation, allowing its accumulation in the early oceans. As such, phosphorus, not as phosphate but as phosphite, could have been a major nutrient in early pre-oxygenated oceans.

Probing the metabolic water contribution to intracellular water using oxygen isotope ratios of PO4.

Knowledge of the relative contributions of different water sources to intracellular fluids and body water is important for many fields of study, ranging from animal physiology to paleoclimate. The intracellular fluid environment of cells is challenging to study due to the difficulties of accessing and sampling the contents of intact cells. Previous studies of multicelled organisms, mostly mammals, have estimated body water composition-including metabolic water produced as a byproduct of metabolism-based on indirect measurements of fluids averaged over the whole organism (e.g., blood) combined with modeling calculations. In microbial cells and aquatic organisms, metabolic water is not generally considered to be a significant component of intracellular water, due to the assumed unimpeded diffusion of water across cell membranes. Here we show that the (18)O/(16)O ratio of PO4 in intracellular biomolecules (e.g., DNA) directly reflects the O isotopic composition of intracellular water and thus may serve as a probe allowing direct sampling of the intracellular environment. We present two independent lines of evidence showing a significant contribution of metabolic water to the intracellular water of three environmentally diverse strains of bacteria. Our results indicate that ∼30-40% of O in PO4 comprising DNA/biomass in early stationary phase cells is derived from metabolic water, which bolsters previous results and also further suggests a constant metabolic water value for cells grown under similar conditions. These results suggest that previous studies assuming identical isotopic compositions for intracellular/extracellular water may need to be reconsidered.

Oxygen isotope studies of phosphite oxidation: purification and analysis of reactants and products by high-temperature conversion elemental analyzer/isotope ratio mass spectrometry.

RATIONALE: Increased attention has been recently focused on the origin and reactions of reduced-P oxyanions such as phosphite [PO3 (III)] in terrestrial and biological systems. We present new methods for studying O-isotopic reactions between PO3 (III) and other oxygen sources during oxidation of PO3 (III) to PO4 (V). METHODS: Na2 (HPO3 )·5H2 O, used as a PO3 (III) source, contains structural water due to its hygroscopic nature; thus, we developed a method for determining the δ(18) O value of PO3 (III) after the removal of structural water. Next, we tested two techniques for purifying PO4 (V) from aqueous PO3 (III)/PO4 (V) mixtures: (1) precipitation of PO4 (V) as ammonium phosphomolybdate (APM); and (2) precipitation of PO4 (V) as magnesium ammonium phosphate (MAP). The O-isotope compositions, (18) O:(16) O (δ(18) O values), of Na2 (HPO3 ) and Ag3 PO4 were analyzed by TC/EA/IRMS. RESULTS: Structural water was removed from Na2 (HPO3 )·5H2 O after drying at 100 °C under vacuum and the δ(18) O value of PO3 (III) was obtained. The δ(18) O values of PO4 (V), which was extracted from (18) O-labeled PO3 (III)/PO4 (V) mixtures by APM and MAP precipitations, were not altered by the precipitation process. This result confirms that PO3 (III) is not converted into PO4 (V) by oxidation or hydrolysis under either strong acidic/oxidizing (APM) or alkaline (MAP) conditions for up to a 24-h period. CONCLUSIONS: We conclude that both APM and MAP precipitation are reliable and effective methods for the separation and purification of PO4 (V) from aqueous PO3 (III)/PO4 (V) mixtures. The methods described here will permit the study of the isotopic evolution of various pathways of geochemical as well as biological PO3 (III) oxidation.

Oxygen isotope signature of UV degradation of glyphosate and phosphonoacetate: tracing sources and cycling of phosphonates.

The degradation of phosphonates in the natural environment constitutes a major route by which orthophosphate (Pi) is regenerated from organic phosphorus and recently implicated in marine methane production, with ramifications to environmental pollution issues and global climate change concerns. This work explores the application of stable oxygen isotope analysis in elucidating the CP bond cleavage mechanism(s) of phosphonates by UV photo-oxidation and for tracing their sources in the environment. The two model phosphonates used, glyphosate and phosphonoacetic acid were effectively degraded after exposure to UV irradiation. The isotope results indicate the involvement of both ambient water and atmospheric oxygen in the CP bond cleavage and generally consistent with previously posited mechanisms of UV-photon excitation reactions. A model developed to calculate the oxygen isotopic composition of the original phosphonate P-moiety, shows both synthetic phosphonates having distinctly lower values compared to naturally derived organophosphorus compounds. Such mechanistic models, based on O-isotope probing, are useful for tracing the sources and reactions of phosphonates in the environment.

Phosphate oxygen isotopic evidence for a temperate and biologically active Archaean ocean.

Oxygen and silicon isotope compositions of cherts and studies of protein evolution have been interpreted to reflect ocean temperatures of 55-85 degrees C during the early Palaeoarchaean era ( approximately 3.5 billion years ago). A recent study combining oxygen and hydrogen isotope compositions of cherts, however, makes a case for Archaean ocean temperatures being no greater than 40 degrees C (ref. 5). Ocean temperature can also be assessed using the oxygen isotope composition of phosphate. Recent studies show that (18)O:(16)O ratios of dissolved inorganic phosphate (delta(18)O(P)) reflect ambient seawater temperature as well as biological processing that dominates marine phosphorus cycling at low temperature. All forms of life require and concentrate phosphorus, and as a result of biological processing, modern marine phosphates have delta(18)O(P) values typically between 19-26 per thousand (VSMOW), highly evolved from presumed source values of approximately 6-8 per thousand that are characteristic of apatite in igneous rocks and meteorites. Here we report oxygen isotope compositions of phosphates in sediments from the 3.2-3.5-billion-year-old Barberton Greenstone Belt in South Africa. We find that delta(18)O(P) values range from 9.3 per thousand to 19.9 per thousand and include the highest values reported for Archaean rocks. The temperatures calculated from our highest delta(18)O(P) values and assuming equilibrium with sea water with delta(18)O = 0 per thousand (ref. 12) range from 26 degrees C to 35 degrees C. The higher delta(18)O(P) values are similar to those of modern marine phosphate and suggest a well-developed phosphorus cycle and evolved biologic activity on the Archaean Earth.