Solution 31P Nuclear Magnetic Resonance Research Articles

Internal phosphorus loading in a chain of eutrophic hardwater lakes in Saskatchewan, Canada

Sediments can act as a source or sink of phosphorus (P) for the water column of lakes. In iron (Fe)-rich softwater lakes, redox processes are important contributors to sediment P flux. However, the contribution of redox processes to P flux in hardwater lakes, with high pH and high concentrations of redox-insensitive calcium (Ca) is unknown. Intact sediment cores, collected in different seasons (summer or fall) from a chain of eutrophic hardwater lakes in southeastern Saskatchewan, Canada, were used to quantify sediment P fluxes in laboratory incubations under hypoxic or oxic conditions at temperatures consistent with the season of sample collection. Geochemical analyses determined concentrations of sediment total (TP) and organic P (TPo), organic matter (OM), total Ca and magnesium, and total and extractable manganese, Fe and aluminum. Sediment P pools were determined using sequential fractionation and solution 31P nuclear magnetic resonance spectroscopy. Sediment P fluxes were significantly higher in sediment cores incubated under hypoxic conditions (−24.4 to 28.5 mg P m−2 d−1) than oxic conditions (−60.3 to 14.2 mg P m−2 d−1). There were significant seasonal and lake differences for TP, TPo and cation concentrations, with Ca the dominant cation in all but one lake. Phosphate bound in the redox-sensitive pool was the only sediment P fraction that significantly differed among the lakes (0.10 to 0.18 mg P g−1 d.w.; 9 to 16 % of TP), with an inverse relationship to sediment P flux. Principal component analysis suggests that high concentrations of internally-generated TPo forms and OM in surface sediments play a key role in internal P loading in these lakes. However, sediment Ca appears to have an overriding effect on sediment P, partially masking the impact of redox control on internal P loading in these hardwater prairie lakes.

Intensive agricultural management-induced subsurface accumulation of water-extractable colloidal P in a Vertisol

Abstract. Long-term excessive application of mineral fertilizer leads to phosphorus (P) accumulation, increasing the risk of P migration and loss from the soil profile. The colloids in the soil profile are important carriers for P migration due to their high P adsorption and transport capacity. It is not clearly understood how colloidal P (CP) is distributed in subsoils (<1.2 m) of a Vertisol, contributing to subsurface P loss. Understanding the depth sequence distribution and speciation of colloidal P in the soil profile is critical for a comprehensive assessment of P loss. In this study, water-extractable colloids (WECs) with the size of 0.35–2 µm were obtained from a 0–120 cm soil profile by a sedimentation and centrifugation scheme. The dissolved reactive P (DRP) and dissolved total P (DTP) in soil supernatant with particle sizes <0.35 µm were measured by molybdate blue colorimetry. Solution 31P nuclear magnetic resonance (NMR) and P K-edge XANES (X-ray absorption near-edge structure) were used to characterize the species and distribution of CP in the soil profile of fertilized farmland. Total and available P in bulk soil and colloids decreased with soil depth. The organic P (OP) contained 97–344 mg kg−1 per bulk soil and 110–630 mg kg−1 per WEC. The OP in soil profile consists of orthophosphate mono-esters and diesters primarily according to NMR results. It suggested that OP in WECs from subsoils might be affected by the translocation of CP from surface soils, probably due to soil acidification and preferential flow caused by swelling–shrinkage clays, including montmorillonite and nontronite detected by X-ray powder diffractometer (XRD) results. Additionally, the more negative zeta potential of surface soil colloids suggests the high mobility of colloidal P towards the subsoils. The CP concentration for <2 µm was about 38–93 mg P kg−1 per bulk soil, which is 6–37 times that of DRP, suggesting that CP plays a dominant role in P transport within the soil profile. The relatively small fraction of orthophosphate diesters suggests limited P assimilation by microorganisms for the accumulation of WECs containing organically bound P in subsoils. The P K-edge XANES results indicated that the proportions of Al-P, Fe-P, and inositol hexakisphosphate (IHP) of WECs decreased, but hydroxyapatite (HAP) increased with soil depth. This study showed that inorganic and organic P migrated from the surface to deeper layers along the soil profile, with soil colloids having a significant effect on P migration from both surface and subsurface layers. The findings have an important significance for soil P migration evaluation and agricultural non-point source pollution control in Vertisols.

Migration of phosphorus in pig manure during pyrolysis process and slow-release mechanism of biochar in hydroponic application

Pyrolysis is an effective method for treating of livestock and poultry manure developed in recent years. It can completely decompose pathogens and antibiotics, stabilize heavy metals, and enrich phosphorus (P) in biochar. To elucidate the P migration mechanism under different pig manure pyrolysis temperatures, sequential fractionation, solution 31P nuclear magnetic resonance, X-ray photoelectron spectroscopy, X-ray diffraction, and K-edge X-ray absorption near-edge structure techniques were used to analyze the P species in pig manure biochar (PMB). The results indicated that most of the organic P in the pig manure was converted to inorganic P during pyrolysis. Moreover, the transformation to different P groups pathways was clarified. The phase transition from amorphous to crystalline calcium phosphate was promoted when the temperature was above 600 °C. The content of P extracted by hydrochloric acid, which was the long-term available P for plant uptake, increased significantly. PMB pyrolyzed at 600 °C can be used as a highly effective substitute for P source. It provides the necessary P species (e.g. water-soluble P.) and metal elements for the growth of water spinach plants, and which are slow-release comparing with the Hogland nutrient solution.

31P Nuclear Magnetic Resonance Spectroscopy as a Probe of Thorium-Phosphorus Bond Covalency: Correlating Phosphorus Chemical Shift to Metal-Phosphorus Bond Order.

We report the use of solution and solid-state 31P Nuclear Magnetic Resonance (NMR) spectroscopy combined with Density Functional Theory calculations to benchmark the covalency of actinide-phosphorus bonds, thus introducing 31P NMR spectroscopy to the investigation of molecular f-element chemical bond covalency. The 31P NMR data for [Th(PH2)(TrenTIPS)] (1, TrenTIPS = {N(CH2CH2NSiPri3)3}3-), [Th(PH)(TrenTIPS)][Na(12C4)2] (2, 12C4 = 12-crown-4 ether), [{Th(TrenTIPS)}2(μ-PH)] (3), and [{Th(TrenTIPS)}2(μ-P)][Na(12C4)2] (4) demonstrate a chemical shift anisotropy (CSA) ordering of (μ-P)3- > (═PH)2- > (μ-PH)2- > (-PH2)1- and for 4 the largest CSA for any bridging phosphido unit. The B3LYP functional with 50% Hartree-Fock mixing produced spin-orbit δiso values that closely match the experimental data, providing experimentally benchmarked quantification of the nature and extent of covalency in the Th-P linkages in 1-4 via Natural Bond Orbital and Natural Localized Molecular Orbital analyses. Shielding analysis revealed that the 31P δiso values are essentially only due to the nature of the Th-P bonds in 1-4, with largely invariant diamagnetic but variable paramagnetic and spin-orbit shieldings that reflect the Th-P bond multiplicities and s-orbital mediated transmission of spin-orbit effects from Th to P. This study has permitted correlation of Th-P δiso values to Mayer bond orders, revealing qualitative correlations generally, but which should be examined with respect to specific ancillary ligand families rather than generally to be quantitative, reflecting that 31P δiso values are a very sensitive reporter due to phosphorus being a soft donor that responds to the rest of the ligand field much more than stronger, harder donors like nitrogen.

Effects of different chemical agents on changes in sediment phosphorus composition and the response of sediment microbial community

Controlling the release of sediment phosphorus (P) using chemical agents is a promising method for controlling internal P in eutrophic lakes. However, mineral P formation and changes in the organic P composition after sediment amendment with P-inactivation agents remain poorly understood. Furthermore, little is known about the changes in the sediment microbial community composition after remediation. Here, various ratios of poly aluminum chloride (PAC) and lanthanum-modified bentonite (LMB) were added to nutrient-rich sediments and incubated. Sequential P extraction, solution/solid-state 31P nuclear magnetic resonance (NMR), and microbial analyses were periodically performed on the inactivated sediments. The results indicate that PAC and LMB effectively reduced sediment iron-bound P and organic P, respectively, markedly increasing the content of aluminum- and calcium-bound P in the sediment, respectively. Solid-state 31P NMR results confirmed the formation of rhabdophane (LaPO4. nH2O) in the LMB-amended sediment. Solution 31P NMR results showed that PAC preferentially reduced the organic P fractions of pyrophosphate, whereas LMB efficiently reduced the organic P fractions of orthophosphate, monoesters, and diesters in the sediment. Compared with the control sediment, PAC addition can cause short-term negative effects on sediment microbes at high doses, whereas LMB addition can increase bacterial diversity or richness in the sediment. These results provide a deeper understanding of the differences between PAC and LMB in internal sediment P control.

Accumulation of Labile P Forms and Promotion of Microbial Community Diversity in Mollisol with Long-Term Manure Fertilization

Soil phosphorus (P) can be divided into inorganic P (Pi) and organic P (Po). Microorganisms play essential roles in soil P transformation. However, there are many ways to detect P transformation, and the relationship between P forms and microorganisms under long-term fertilization is largely unclear. In this study, soil P forms were analyzed by a chemical sequential fractionation method and solution 31P nuclear magnetic resonance (31P-NMR) technique. Phospholipid fatty acid (PLFA) contents were measured by gas chromatography as the characterization of soil microbial community structures. The objective was to determine the changes of soil P forms and associated microbial community composition in mollisol with long-term fertilization. We sampled soil from a field experiment with 26-year-old continuous maize (Zea mays L.) cropping in Northeastern China. Three fertilization treatments were selected as chemical fertilization (NPK), NPK with crop straw (NPKS), and NPK with manure (NPKM). As shown in 31P-NMR spectra, orthophosphate accounted for 62.8–85.8% of total extract P. Comparison to NPK and NPKS treatments, NPKM application notably increased the concentrations of Po, Olsen-P, orthophosphate, orthophosphate monoester, and total P. Soil P fractions including resin-Pi, NaHCO3-P, NaOH-P, and HCl-P, especially Pi fractions, were enhanced by NPKM. The amounts of total PLFAs and PLFAs in bacteria, Gram-positive (G+) and Gram-negative (G−) bacteria, actinomycetes, and fungi were high in NPKM-treated soil. The percentages of PLFAs in bacteria and fungi in total soil PLFAs were 56.8% and 9.7%, respectively, which did not show any significant difference among the treatments. NPKM increased the proportions (%) of PLFAs in G+ bacteria, and NPKS increased the proportions (%) of G− bacteria in total PLFAs. The composition of soil microbial community was found to be significantly affected by soil total carbon and pH. There was a close relationship between HCl-Pi, NaHCO3-Po, orthophosphate, and pyrophosphate with anaerobe, aerobes, and G+. Manure addition directly increased soil available P concentrations, and indirectly acted through the alterations of anaerobe, aerobes, and G+. It is concluded that long-term NPKM application would lead to the accumulation of labile P and moderately labile P in mollisol through the activity of soil microbes.

Phosphorus application promoted the sequestration of orthophosphate within soil microorganisms and regulated the soil solution P supply in a temperate grassland in northern China: A 31P NMR study

Anthropogenic activities have made phosphorus (P) a new sustainability issue globally. To better understand the mechanisms of soil P dynamics responding to inorganic P fertilization (0–12.5 g P m−2yr−1), we conducted a 2-year field experiment in a temperate grassland in northern China. The P fertilizer was added once per year before the growth period as NaH2PO4. We randomly collected topsoil samples (0–10 cm) and analyzed the soil properties, microbial community, and extracellular enzyme activities, and quantified the soil P fractions using solution 31P nuclear magnetic resonance (31P NMR) spectroscopy. P application promoted transformation from organic P to inorganic P driven by microbial carbon (C) demand and regulated by altered soil properties and microbial characteristics. P addition decreased orthophosphate diesters, especially DNA, due to increased hydrolysis under increased soil pH. This may increase hydrolysates such as α-glycerophosphate and choline phosphate. Orthophosphate monoesters, especially myo-inositol hexaphosphate, also decreased following increased P availability. This could be explained by increased alkaline phosphatase activity and reduced sorption due to a decreased metal (Ca, Al, and Fe) content. Furthermore, P fertilization increased soil NH4+-N, thereby increasing the growth of phosphonate-solubilizing microorganisms (Bacillus, Streptomyces, Bradyrhizobium, Mesorhizobium, Penicillium, Aspergillus, and Fusarium). Their increased growth also contributed to inorganic P solubilization and organic P mineralization due to their P-solubilizing specificity. Together, these factors increased inorganic P, especially orthophosphate, which was temporarily immobilized in microbial cells, to regulate the soil-solution P supply, and created a potentially available P sink in the grassland.

Phosphorus speciation along a soil to kettle hole transect: Sequential P fractionation, P XANES, and 31P NMR spectroscopy

Phosphorus (P) is an essential element for all living organisms but can also be an important factor in the eutrophication of aquatic/marine ecosystems. Kettle holes are often situated in regions under intense agricultural land use where there is a high risk of nutrient transfer to larger waterbodies. The chemical speciation of soil P influences its rate of transfer from arable fields to aquatic environments. However, there is a paucity of information on the P speciation in kettle holes and their effect on the P cycle. Thus, we investigated the P composition of arable soils and kettle hole sediments in surface and subsurface layers along a transect of an agricultural field. Multiple P analyses were carried out including modified sequential Hedley P fractionation, P K-edge X-ray absorption near edge structure (XANES) spectroscopy, and 31P nuclear magnetic resonance (NMR) spectroscopy. The Pt concentrations ranged from 680 to 1123 mg kg−1 in the soils and 797 to 2591 mg kg−1 in the sediments. A predominance of the residual-P fraction, characterized as not extractable P, occluded P or stable forms of organic P (Po) was determined by sequential fractionation, ranging from 37 to 67 % followed by 3 to 38 % H2SO4-Pi and 3 to 16 % NaOH-Po of total P (Pt) in the soils and sediments. Analyses with P K–edge XANES spectroscopy assigned 61 to 100 % Fe- and Al-P, 0 to 14 % Ca-P, and 0 to 39 % Po in the arable soils and 46 to 74 % Fe- and Al-P, 14 to 43 % Ca-P, and 0 – 38 % Po in the adjoining kettle hole sediments. Solution 31P NMR spectra on alkaline extracts revealed a broad signal in the phosphomonoester region, which was the most abundant form of organic P across all samples. Besides, the proportion of ‘complex’ phosphomonesters (broad signal) to that of total extractable P was about 2-fold greater in the kettle hole sediments than in soils. Complex forms of organic P are known to be strongly associated with organic matter (OM) and appear to accumulate on a decadel timeframe. Therefore, it is desirable that kettle hole sediments are conserving P (and carbon), which will help reduce the transfer of P from agricultural fields to other, more vulnerable aquatic/marine ecosystems.

Molecular-Level Insights into Phosphorus Transformation Mechanisms in Entisol Soils under Multiple Long-Term Fertilization Regimes

Improving phosphorus (P) fertilization strategies benefits mitigating future global P shortage and reducing legacy P loss risk in agricultural lands. In this study, the molecular transformation mechanisms of P in Entisol soils under multiple long-term fertilization regimes including PK; NK; NPK; NPK with pig manure (NPKM); and NPK with rice straw return (NPKS) were investigated by sequential fractionation (SF), synchrotron-based P K-edge X-ray absorption near-edge structure (P-XANES) and solution 31P nuclear magnetic resonance (P-NMR) spectroscopy. Compared with conventional NPK fertilization, a higher accumulation of total P occurred in soils under the PK, NPKM and NPKS treatments. By SF, there were relatively higher contents of NaHCO3-extracted inorganic P (Pi) fractions for the soils under PK (52.5 mg/kg) and NPKM (35.5 mg/kg) fertilization relative to the NPK (23.3 mg/kg) treatment. Consistently, P-XANES analysis revealed that there was a higher proportion of brushite, as a bioavailable P form, in soil under the PK and NPKM treatments compared with the NPK treatment, indicating higher P availability in the Entisol soils under PK and NPKM fertilizations. By P-NMR, long-term PK fertilization resulted in relatively a higher ratio of orthophosphate diesters to orthophosphate monoesters after correction compared with the NPK fertilization, which strongly suggested that N deficiency enhanced the biodegradability of soil organic P (Po) pools, thus providing new molecular-level insights into soil Po transformation. Collectively, these results, obtained from a long-term experimental study, facilitates the comprehensive understanding of P availability and transformation mechanisms in Entisol soils under multiple fertilization regimes, and thus benefits the improvement of fertilization strategies in agricultural soils.

Phosphorus species in sequentially extracted soil organic matter fractions

The majority of organic P (Porg) in soil is considered to be part of soil organic matter (SOM) associations, but its chemical nature is largely ‘unresolved’. In this study, we investigated the Porg composition in different SOM fractions of a Gleysol soil using the Humeomics sequential chemical fractionation (SCF) procedure combined with nuclear magnetic resonance (NMR) spectroscopy.In summary, SCF procedure with subsequent NaOH-EDTA extraction of the soil residue extracted a total of 1769 mgP/kgsoil compared to 1682 mgP/kgsoil of a single-step NaOH-EDTA extraction. Approximately 38 % of the extracted Porg was present in the form of the unresolved Porg pool, which was represented by one or two underlying broad signals in the phosphomonoester region of solution 31P NMR spectra. The SCF revealed that phosphomonoesters were recovered in each fraction: 47 % of the unresolved phosphomonoesters were associated with the SOM fraction released by breaking ester bonds (40 %) and ether bonds (7 %), whereas about 30 % of this unresolved Porg pool appeared in the SOM fraction closely associated with the soil mineral phase. Furthermore, the extractability of inositol phosphates (IP) was increased from 312 mgP/kgsoil to 534 mgP/kgsoil (factor 1.7) using the SCF procedure compared to a single-step NaOH-EDTA extraction. Previous studies have reported the presence of IP in molecular size fractions greater than 10 kDa. Our findings on the removal of IP with the fractionation of the SOM could explain the presence of IP in these large associations.We demonstrate that major pools of Porg are closely associated with SOM structures, comprising a diverse array of chemical species and bonding types. These results forward our understanding of Porg stabilisation, P transformation, and P cycling in terrestrial ecosystems towards an association point of view.

Characteristics and controls of inorganic and organic phosphorus transformation during long-term paddy soil evolution

Information on phosphorus (P) transformation during long-term paddy soil evolution is important for maintaining rice yield and nutrient management. This study was aimed to reveal the characteristics and controls of inorganic and organic P transformation during paddy soil evolution over a millennial time scale using both sequential chemical extraction and solution 31P nuclear magnetic resonance spectroscopy. Results show that the rate and trajectory of P evolution in the topsoil differ markedly from that of the illuvial horizon. The amount of apatite P, non-occluded P, occluded P and total P in the topsoils increased rapidly during the early stage of paddy soil evolution (< 50 yrs) due to anthropogenic P additions, but declined in the older paddy soils due to the leaching loss of P sorbents (CaCO3 and Fe/Al oxides); thus, the added P cannot be efficiently retained by the older paddy soils. Total organic P including phosphate monoester in the topsoils reached maximum in the 300-yr paddy soil and declined thereafter. However, the phosphate diester pool (including DNA) and its proportion in the topsoil increased continuously across the paddy soil chronosequence, suggesting that long-term paddy cultivation promoted labile organic P accumulation. In the deeper soil horizons with less human impacts, the temporal trends in inorganic and organic P across the paddy soil chronosequence can be fully predicted by previous established P transformation models during natural pedogenesis, although the rates of P changes differ among different sites. Our study demonstrates that long-term paddy cultivation alters the rate and trajectory of inorganic and organic P transformation in the topsoils over a millennial time scale, and highlights the importance of labile organic P activation for nutrient supply in the older paddy soils.

Fuel from within: Can suspended phosphorus maintain algal blooms in Lake Dianchi

Extensive algal bloom in the surface water is a pressing issue in Lake Dianchi that causes lake restoration to be difficult owing to complex and variable phosphorus (P) sources in the water column. P released from algae, suspended particles (SS), and sediment can provide sustainable P sources for algal blooms. However, little is known regarding the dynamic of P speciation in these substances from different sources. In this study, solution 31P nuclear magnetic resonance (31P NMR) and chemical sequential extraction were employed to identify P speciation in algae, SS, and sediment during different periods. Results showed that dissolved inorganic P (Pi) directly accumulated in algae in the form of orthophosphate (ortho-P) and pyrophosphate (pyro-P). Algae preferentially utilized Pi, followed by organic P (Po) in the water column when the Pi was insufficient during growth and reproduction. The 31P NMR spectra demonstrated that ortho-P, orthophosphate monoesters (mono-P), orthophosphate diesters (diester-P), and pyro-P dominated the P compounds across the samples tested. Increasing remineralization of SS mono-P driven by intense alkaline phosphatase activities was caused by increasing P needs of algae and pressure of P supply in the water column. The higher ratios of diester-P to mono-P in sediment (mean 0.55) than those in algae (mean 0.07) and SS (mean 0.11 in surface water, 0.14 in bottom water) suggested that the degradation and regeneration occurred within these P compounds during or after sedimentation. Pi content in algae during growth and reproduction was controlled by its P absorption and utilization strategies. Results of this study provide insights into the dynamic cycling of P in algae, SS, and sediment, explaining the reason for algal blooms in the surface water with low concentrations of dissolved P.

Coordination of Palladium(II) and Platinum(II) Complexes to One Vacant Site in an α-Keggin-Type Polyoxotungstate.

A palladium-platinum bimetal complex containing a mono-lacunary α-Keggin-type polyoxotungstate is demonstrated. The bimetallic complex Cs2.5{PdII(bpy)}0.25[α-PW11O39{PdII(bpy)}{cis-PtII(NH3)2}]·12H2O·2CH3CN (bpy = 2,2'-bipyridine) (PdPt) was synthesized by the reaction of Cs4.9K0.1[α-PW11O39{cis-PtII(NH3)2}]·10H2O (Pt1) with PdII(bpy)Cl2 in aqueous solution. The compound PdPt was characterized via elemental analysis, thermogravimetric/differential thermal analysis, and Fourier transform infrared, ultraviolet-visible, cold-spray ionization mass, and solution (1H, 13C, and 31P) nuclear magnetic resonance spectroscopies. The coordination of {cis-PtII(NH3)2} and {PdII(bpy)} moieties to the mono-vacant site of [PW11O39]7- resulted in an asymmetric, monomeric compound. A unique exchange reaction between the counter cation [PdII(bpy)]2+ and {cis-PtII(NH3)2} moiety in [α-PW11O39{PdII(bpy)}{cis-PtII(NH3)2}]3- in a dimethylsulfoxide solution was also observed.

Long-term fertilization impacts on soil phosphorus forms using XANES and NMR spectroscopy

ABSTRACT A 32-year fertilizer experiment was conducted to investigate mineral and organic fertilization impacts on soil P forms by P K-edge X-ray absorption near edge structure and solution 31P nuclear magnetic resonance spectroscopy. The experiment included six treatments: control without fertilization, mineral fertilizers (NPK), pig manure at 30,000 (M1) and 60,000 kg ha–1 (M2), M1 plus NPK, and M2 plus NPK. The predominant P compounds were hydroxyapatite (up to 51.3% of soil total P) and orthophosphate and orthophosphate monoesters (up to 85.7% and 57.0% of soil NaOH-EDTA extractable total P, respectively). Compared with the control, the concentrations and proportions of monocalcium phosphate and orthophosphate increased following mineral fertilizers alone. Manure application, especially high rate manure combined with mineral fertilizers, further increased the concentrations and proportions of monocalcium phosphate and orthophosphate relative to mineral fertilizers alone. Moreover, manure amendment also generally increased the concentrations of other inorganic and organic P compounds. Manure application improve soil P availability by adding high concentrations of bioavailable inorganic P compounds to soil, similar to the addition of mineral fertilizers. This study contributes to the understanding of applied fertilizers and residual soil P dynamics and provides insights for sustainable P management in agricultural ecosystems.

Potential Phosphorus Uptake Mechanisms in the Deep Sedimentary Biosphere

Our understanding of phosphorus (P) dynamics in the deep subseafloor environment remains limited. Here we investigate potential microbial P uptake mechanisms in oligotrophic marine sediments beneath the North Atlantic Gyre and their effects on the relative distribution of organic P compounds as a function of burial depth and changing redox conditions. We use metagenomic analyses to determine the presence of microbial functional genes pertaining to P uptake and metabolism, and solution 31P nuclear magnetic resonance spectroscopy (31P NMR) to characterize and quantify P substrates. Phosphorus compounds or compound classes identified with 31P NMR include inorganic P compounds (orthophosphate, pyrophosphate, polyphosphate), phosphonates, orthophosphate monoesters (including inositol hexakisphosphate stereoisomers) and orthophosphate diesters (including DNA and phospholipid degradation products). Some of the genes identified include genes related to phosphate transport, phosphonate and polyphosphate metabolism, as well as phosphite uptake. Our findings suggest that the deep sedimentary biosphere may have adapted to take advantage of a wide array of P substrates and could play a role in the gradual breakdown of inositol and sugar phosphates, as well as reduced P compounds and polyphosphates.

The molecular size continuum of soil organic phosphorus and its chemical associations

The chemical nature of most organic P (Porg) in soil remains ‘unresolved’ but is accounted for by a broad signal in the phosphomonoester region of solution 31P nuclear magnetic resonance (NMR) spectra. The molecular size range of this broad NMR signal and its molecular structure remain unclear. The aim of this study was to elucidate the chemical nature of Porg with increasing molecular size in soil extracts combining size exclusion chromatography (SEC) with solution 31P NMR spectroscopy. Gel-filtration SEC was carried out on NaOH-EDTA extracts of four soils (range 238–1135 mg Porg/kgsoil) to collect fractions with molecular sizes of < 5, 5–10, 10–20, 20–50, 50–70, and > 70 kDa. These were then analysed by NMR spectroscopy.Organic P was detected across the entire molecular size continuum from < 5 to > 70 kDa. Concentrations of Porg in the > 10 kDa fraction ranged from 107 to 427 mg P/kgsoil and exhibited on average three to four broad signals in the phosphomonoester region of NMR spectra. These broad signals were most prominent in the 10–20 and 20–50 kDa fractions, accounting for on average 77% and 74% of total phosphomonoesters, respectively. Our study demonstrates that the broad signals are present in all investigated molecular size fractions and comprise on average three to four components of varying NMR peak line width (20 to 250 Hz).The stereoisomers myo- and scyllo-inositol hexakisphosphates (IP6) were also present across multiple molecular size ranges but were predominant in the 5–10 kDa fraction. The proportion of IP associated with large molecular size fractions > 10 kDa was on average 23% (SD = 39%) of total IP across all soils. These findings suggest that stabilisation of IP in soil includes processes associated with the organic phase.

Soil phosphomonoesters in large molecular weight material comprise multiple components

AbstractThe majority of soil organic P is typically that of “unresolved” phosphomonoesters, which are associated with organic P (Po) in large molecular weight (MW) fractions. However, the composition of this pool of Po remains unclear and could contain more than one component. Our aim was to identify the number and nature of components in soil extracts of different MW using solution 31P nuclear magnetic resonance (NMR) spectroscopy and spectral deconvolution fitting. Three surface soils were collected from native sites, which were extracted for Po using 0.25 M NaOH + 0.05 M Na2–EDTA (unfractionated), followed by size separation using ultrafiltration, resulting in four MW fractions (&gt;10 kDa, &lt;10 kDa, &lt;10 kDa and &gt;3 kDa, and &lt;3 kDa). The NMR analyses revealed a diversity of Po species in all MW fractions across the three soils. In particular, the phosphomonoester region of the &gt;10‐kDa fraction comprised up to 10 components of varying linewidth (0.5–18.6 Hz) and a broad signal (228 Hz). This broad signal was also present in the &lt;3‐kDa fraction, albeit at a lower concentration (average was 29% of that in the unfractionated extracts). Furthermore, solution 31P NMR spectra on the &lt;3‐kDa fraction revealed a diversity of sharp signals (up to 28) in the phosphomonoester region. Overall, our findings show that soil phosphomonoesters in large MW fractions comprise multiple components of varying linewidth.

Combining diffusive gradients in thin‐films (DGT) and 31P NMR spectroscopy to determine phosphorus species in soil

AbstractThe diffusive gradients in thin‐films (DGT) technique shows in many publications a superior correlation to the amount of plant‐available phosphorus (P) in soil. However, this technique cannot give information on the plant‐available P species in soil. Therefore, we combined DGT with solution 31P nuclear magnetic resonance (NMR) spectroscopy. This was achieved by using a modified DGT device in which the diffusive layer had a larger pore size, the binding layer incorporated an adsorption material with a higher capacity, and the device had a larger exposure area. The spectroscopic investigation was undertaken after elution of the deployed DGT binding layer in a NaOH solution. Adsorption tests using solutions of known organic P compounds showed that a sufficient amount of these compounds could be adsorbed on the binding layer in order for them to be analyzed by solution 31P NMR spectroscopy. Furthermore, various intermediates of the hydrolysis of trimetaphosphate in soil could be also analyzed over time.Core Ideas We show a promising combination of DGT technique and solution 31P NMR spectroscopy. This technique combination can identify organic phosphorus species in solutions. Modified DGT device can analyze the hydrolysis of trimetaphosphate in soil.

Growth-phase dependence of bacterial membrane lipid profile and labeling for in-cell solid-state NMR applications

Cell labeling is a preliminary step in multiple biophysical approaches, including the solid-state nuclear magnetic resonance (NMR) study of bacteria in vivo. Deuterium solid-state NMR has been used in the past years to probe bacterial membranes and their interactions with antimicrobial peptides, following a standard labeling protocol. Recent results from our laboratory on a slow-growing bacterium has shown the need to optimize this protocol, especially the bacterial growth time before harvest and the concentration of exogenous labeled fatty acids to be used for both Escherichia coli and Bacillus subtilis. It is also essential for the protocol to remain harmless to cells while providing optimal labeling. We have therefore developed a fast and facile approach to monitor the lipid composition of bacterial membranes under various growth conditions, combining solution 31P NMR and GCMS. Using this approach, the optimized labeling conditions of Escherichia coli and Bacillus subtilis with deuterated palmitic acid were determined. Our results show a modification of B. subtilis phospholipid profile as a function of the growth stage, as opposed to E. coli. Our protocol recommends low concentrations of exogenous palmitic acid in the growth medium, and bacteria harvest after the exponential phase.

Intracellular polyphosphate length characterization in polyphosphate accumulating microorganisms (PAOs): Implications in PAO phenotypic diversity and enhanced biological phosphorus removal performance

Polyphosphate (polyP) accumulating organisms (PAOs) are the key agent to perform enhanced biological phosphorus removal (EBPR) activity, and intracellular polyP plays a key role in this process. Potential associations between EBPR performance and the polyP structure have been suggested, but are yet to be extensively investigated, mainly due to the lack of established methods for polyP characterization in the EBPR system. In this study, we explored and demonstrated that single-cell Raman spectroscopy (SCRS) can be employed for characterizing intracellular polyPs of PAOs in complex environmental samples such as EBPR systems. The results, for the first time, revealed distinct distribution patterns of polyP length (as Raman peak position) in PAOs in lab-scale EBPR reactors that were dominated with different PAO types, as well as among different full-scale EBPR systems with varying configurations. Furthermore, SCRS revealed distinctive polyP composition/features among PAO phenotypic sub-groups, which are likely associated with phylogenetic and/or phenotypic diversity in EBPR communities, highlighting the possible resolving power of SCRS at the microdiversity level. To validate the observed polyP length variations via SCRS, we also performed and compared bulk polyP length characteristics in EBPR biomass using conventional polyacrylamide gel electrophoresis (PAGE) and solution 31P nuclear magnetic resonance (31P-NMR) methods. The results are consistent with the SCRS findings and confirmed the variations in the polyP lengths among different EBPR systems. Compared to conventional methods, SCRS exhibited advantages as compared to conventional methods, including the ability to characterize in situ the intracellular polyPs at subcellular resolution in a label-free and non-destructive way, and the capability to capture subtle and detailed biochemical fingerprints of cells for phenotypic classification. SCRS also has recognized limitations in comparison with 31P-NMR and PAGE, such as the inability to quantitatively detect the average polyP chain length and its distribution. The results provided initial evidence for the potential of SCRS-enabled polyP characterization as an alternative and complementary microbial community phenotyping method to facilitate the phenotype-function (performance) relationship deduction in EBPR systems.