Добірка наукової літератури з теми "³¹P nuclear magnetic resonance (NMR) spectroscopy; organic phosphorus; myo-inositol hexakisphosphate (phytate)"

Abstract. Phosphorus (P) species in colloidal and dissolved soil fractions may have different distributions. To understand which P species are potentially involved, we obtained water extracts from the surface soils of a gradient from Cambisol, Stagnic Cambisol to Stagnosol from temperate grassland in Germany. These were filtered to < 450 nm, and divided into three procedurally defined fractions: small-sized colloids (20–450 nm), nano-sized colloids (1–20 nm), and dissolved P (< 1 nm), using asymmetric flow field-flow fractionation (AF4), as well as filtration for solution 31P-nuclear magnetic resonance (NMR) spectroscopy. The total P of soil water extracts increased in the order Cambisol < Stagnic Cambisol < Stagnosol due to increasing contributions from the dissolved P fraction. Associations of C–Fe/Al–PO43−/pyrophosphate were absent in nano-sized (1–20 nm) colloids from the Cambisol but not in the Stagnosol. The 31P-NMR results indicated that this was accompanied by elevated portions of organic P in the order Cambisol > Stagnic Cambisol > Stagnosol. Across all soil types, elevated proportions of inositol hexakisphosphate (IHP) species (e.g., myo-, scyllo- and D-chiro-IHP) were associated with soil mineral particles (i.e., bulk soil and small-sized soil colloids), whereas other orthophosphate monoesters and phosphonates were found in the dissolved P fraction. We conclude that P species composition varies among colloidal and dissolved soil fractions after characterization using advanced techniques, i.e., AF4 and NMR. Furthermore, stagnic properties affect P speciation and availability by potentially releasing dissolved inorganic and ester-bound P forms as well as nano-sized organic matter–Fe/Al–P colloids.

Few studies have considered the influence of climate on organic phosphorus (P) speciation in soils. We used sodium hydroxide–ethylenediaminetetra-acetic acid (NaOH–EDTA) soil extractions and solution 31P nuclear magnetic resonance spectroscopy to investigate the soil P composition of five alpine and sub-alpine soils. The aim was to compare the P speciation of this set of soils with those of soils typically reported in the literature from other cold and wet locations, as well as those of other Australian soils from warmer and drier environments. For all alpine and sub-alpine soils, the majority of P detected was in an organic form (54–66% of total NaOH–EDTA extractable P). Phosphomonoesters comprised the largest pool of extractable organic P (83–100%) with prominent peaks assigned to myo- and scyllo-inositol hexakisphosphate (IP6), although trace amounts of the neo- and d-chiro-IP6 stereoisomers were also present. Phosphonates were identified in the soils from the coldest and wettest locations; α- and β-glycerophosphate and mononucleotides were minor components of organic P in all soils. The composition of organic P in these soils contrasts with that reported previously for Australian soils from warm, dry environments where inositol phosphate (IP6) peaks were less dominant or absent and humic-P and α- and β-glycerophosphate were proportionally larger components of organic P. Instead, the soil organic P composition exhibited similarities to soils from other cold, wet environments. This provides preliminary evidence that climate is a key driver in the variation of organic P speciation in soils.

Solution 31P nuclear magnetic resonance (NMR) spectroscopy is the most common technique for the detailed characterisation of soil organic P, but is yet to be applied widely to Australian soils. We investigated the composition of soil P in 18 diverse Australian soils using this technique. Soils were treated with a mixture of sodium hydroxide–ethylenediaminetetra-acetic acid (NaOH-EDTA), which resulted in the extraction of up to 89% of total soil P. It was possible to identify up to 15 well-resolved resonances and one broad signal in each 31P NMR spectrum. The well-resolved resonances included those of orthophosphate, α- and β-glycerophosphate, phytate, adenosine-5′-monosphosphate, and scyllo-inositol phosphate, as well as five unassigned resonances in the monoester region and two unassigned resonances downfield (higher ppm values) of orthophosphate. The majority of 31P NMR signal in the NaOH-EDTA extracts was assigned to orthophosphate, representing 37–90% of extractable P. Orthophosphate monoesters comprised the next largest pool of extractable P (7–55%). The most prominent resonances were due to phytate, which comprised up to 9% of total NaOH-EDTA extractable P, and α- and β-glycerophosphate, which comprised 1–5% of total NaOH-EDTA extractable P. A substantially greater portion of organic P (2–39% of total NaOH-EDTA extractable P) appeared as a broad peak in the monoester P region; we propose that this is due to P found in large, ‘humic’ molecules. Orthophosphate diesters (1–5% of total NaOH-EDTA extractable P) and pyrophosphate (1–5% of total NaOH-EDTA extractable P) were minor components of P in all soil extracts. These results suggest that organic P in large humic molecules represents the second most abundant form of NaOH-EDTA extractable soil P (behind orthophosphate). Furthermore, small P-containing compounds, such as phytate, represent a much smaller proportion of soil P than is commonly assumed.

Abstract. Inositol phosphates (IPs) are a major pool of identifiable organic phosphorus (P) in soil. However, insight into their distribution and cycling in soil remains limited, particularly of lower-order IP (IP5 and IP4). This is because the quantification of lower-order IP typically requires a series of chemical extractions, including hypobromite oxidation to isolate IP, followed by chromatographic separation. Here, for the first time, we identify the chemical nature of organic P in four soil extracts following hypobromite oxidation using solution 31P NMR spectroscopy and transverse relaxation (T2) experiments. Soil samples analysed include A horizons from a Ferralsol (Colombia), a Cambisol and a Gleysol from Switzerland, and a Cambisol from Germany. Solution 31P nuclear magnetic resonance (NMR) spectra of the phosphomonoester region in soil extracts following hypobromite oxidation revealed an increase in the number of sharp signals (up to 70) and an on average 2-fold decrease in the concentration of the broad signal compared to the untreated soil extracts. We identified the presence of four stereoisomers of IP6, four stereoisomers of IP5, and scyllo-IP4. We also identified for the first time two isomers of myo-IP5 in soil extracts: myo-(1,2,4,5,6)-IP5 and myo-(1,3,4,5,6)-IP5. Concentrations of total IP ranged from 1.4 to 159.3 mg P per kg soil across all soils, of which between 9 % and 50 % were comprised of lower-order IP. Furthermore, we found that the T2 times, which are considered to be inversely related to the tumbling of a molecule in solution and hence its molecular size, were significantly shorter for the underlying broad signal compared to for the sharp signals (IP6) in soil extracts following hypobromite oxidation. In summary, we demonstrate the presence of a plethora of organic P compounds in soil extracts, largely attributed to IPs of various orders, and provide new insight into the chemical stability of complex forms of organic P associated with soil organic matter.

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.

Organic phosphorus is potentially an important source of phosphorus (P) for agriculture, although it is not directly available for plant or microbial uptake. However, organic P can be converted into available inorganic P though hydrolysis or mineralisation. The rate of P release from organic P forms depends partly on the specific organic P compounds present in the soil. Until recently characterising soil organic P has been limited by the lack of appropriate analytic techniques. Consequently, organic P dynamics remains poorly understood. In this thesis, the focus was on improving techniques for the characterisation of soil organic P using solution ³¹P nuclear magnetic resonance (NMR) spectroscopy, applying these techniques to characterise a range of Australian soils and developing a better understanding of the cycling and potential bioavailability of soil organic P. The characterisation of soil organic P relies on the correct identification of resonances. Orthophosphate monoester peaks were identified by spiking model organic P compounds into NaOH- EDTA soil extracts. In this way, seven major resonances that were common to most of the NMR spectra were assigned to adenosine-monophosphate (AMP), scyllo-inositol hexakisphosphate, α- and β-glycerophosphate and myo-inositol hexakisphosphate (phytate). More importantly, spiking highlighted the similarly in appearance and chemical shift of some of the orthophosphate monoester resonances, particularly those of phytate and α- and β-glycerophosphate. This may have resulted in the misidentification and over-estimation of the concentrations of these species in previous studies. To provide a detailed quantitative assessment of soil organic P using ³¹P NMR spectroscopy, a modified method of spectral deconvolution, which included using an internal standard (methylenediphosphonic acid; MDP), was developed. The method of deconvolution implemented in this thesis considered P contained in larger humic molecules. A broad signal, in addition to the routinely fitted sharp peaks, was fitted to the orthophosphate monoester region of the NMR spectrum. A large proportion of monoester P (32–78%) could be assigned to this signal. When the broad signal was not taken into account phytate concentrations were over-estimated by 54%. It is likely that the concentrations of other specific orthophosphate monoester compounds were also over-estimated. The potential over-estimation of phytate concentrations has implication for the understanding of phytate stability in soils. High phytate concentrations in soils are usually explained by the stability of phytate in soils or the limited presence or activity of specialised enzymes (phytase). Lower phytate concentrations suggest phytate maybe less stable in soils than previously supposed. Therefore, the rate of phytate degradation in a calcareous soil was investigated. Phytate was applied to a calcareous soil at four different concentrations (ranging from 58–730 mg kg⁻¹) and the effect of wheat straw as an additional source of carbon was also examined. Regardless of treatment, phytate concentrations decreased over the 13-week incubation period and were adequately fitted to a first order decay model. There was no clear trend in the rate of phytate loss with treatment and the half life of phytate ranged from 4 to 8 weeks. The loss of phytate coincided with an increase in orthophosphate concentration, that in some cases more than doubled the native soil P concentrations, and there was very little variation in extraction efficiency. This result provided evidence for the microbial degradation of phytate. It demonstrated that in the calcareous soil examined, phytate was not highly stable, but a bioavailable source of organic P. The composition of soil P in 18 diverse Australian soils was also examined. Across all NaOH-EDTA soil extracts analysed, phytate comprised up to 9%, but averaged only 3% of total extractable P. Two other resonances that were also prominent in all the ³¹P NMR spectra and comprised a similar proportion of total organic P were due to α- and β-glycerophosphate. By examining the alkaline hydrolysis of a phospholipid (phosphatidlycholine), the potential source of α- and β-glycerophosphate was identified. Although α- and β-glycerophosphate and phyate gave rise to the most intense peaks, the broad signal, which was attributed to humic P, represented the most abundant form of soil organic P (27–72% of total extractable organic P). Therefore, it was suggested that the development of methods that aim to increase the availability of stabilised forms of organic P should give preference to increasing the availability of P contained in humic P complexes. Understanding P cycling not only relies on analytical methods that enable the accurate identification and quantification of soil organic P but also requires methods that can gauge the susceptibility of different organic P species to enzymatic hydrolysis. Therefore, enzymatic hydrolysis was combined with ³¹P NMR spectroscopy to identify and compare the specific organic P species in the enzyme labile and non-enzyme labile fractions of a range of NaOH-EDTA soil extracts. Phosphorus-31 NMR analysis of NaOH-EDTA soil extracts treated with active and inactivated phytase enzyme preparations showed that phytase hydrolysed the majority of the small, orthophosphate monoester compounds (α- and β-glycerophosphate, phytate, scyllo-inositol hexakisphosphate) and pyrophosphate, but orthophosphate diesters (DNA) and humic P were generally unaffected. The ³¹P NMR spectra revealed that not only was organic P hydrolysed but new orthophosphate monoester species were formed, possibly as a result of enzymatic phosphorylation. Although combining enzymatic hydrolysis and ³¹P NMR spectroscopy enabled the identification of individual organic P species that were susceptible or resistant to enzyme hydrolysis, there is still a need for further improvement and refinement of the technique in order to provide an accurate estimate of the potentially available fraction of soil organic P.
Thesis (Ph.D.) -- University of Adelaide, School of Agriculture, Food and Wine, 2010