The chemical nature of soil organic phosphorus: A critical review and global compilation of quantitative data

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The chemical nature of soil organic phosphorus: A critical review and global compilation of quantitative data Timothy I. McLarena,∗, Ronald J. Smernikb, Michael J. McLaughlinb, Ashlea L. Dooletteb, Alan E. Richardsonc, Emmanuel Frossarda a

Group of Plant Nutrition, Department of Environmental Systems Science, Swiss Federal Institute of Technology (ETH) Zurich, Lindau, Switzerland b Soils Group, School of Agriculture, Food and Wine and Waite Research Institute, The University of Adelaide, Urrbrae, SA, Australia c CSIRO Agriculture & Food, Canberra, ACT, Australia ∗ Corresponding author: e-mail address: [email protected]

Contents 1. Introduction 1.1 Background 1.2 Approach of historical review 2. Era 1: Mid-1800s to 1930s—The existence of soil organic phosphorus and reliance on chemical extraction and isolation techniques 2.1 Main findings 2.2 Discussion 3. Era 2: 1940s—Supporting evidence on the existence of inositol phosphates in soil 3.1 Main findings 3.2 Discussion 4. Era 3: 1950s to 1970s—Chromatographic techniques with a focus on inositol phosphates and molecular weight distribution of soil organic phosphorus 4.1 Main findings 4.2 Discussion 5. Era 4: 1980s to early 2000s—Simultaneous detection of broad classes of soil organic phosphorus using solution phosphorus-31 nuclear magnetic resonance spectroscopy 5.1 Main findings 5.2 Discussion 6. Era 5: Early 2000s to current—Spectral deconvolution fitting for characterizing soil phosphomonoesters 6.1 Main findings 6.2 Discussion

Advances in Agronomy ISSN 0065-2113 https://doi.org/10.1016/bs.agron.2019.10.001

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2019 Elsevier Inc. All rights reserved.

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7. Evolving view on the structure, formation and cycling of soil organic phosphorus Acknowledgments References

56 61 61

Abstract Historically, the chemical nature of organic phosphorus (P) in soil has largely been considered to comprise of recognizable biomolecules that predominantly include inositol phosphates, nucleic acids and phospholipids. However, these forms alone do not explain the existence of, or account for the processes responsible for, a larger pool of “unresolved” organic P that exists in soils. We critically reviewed the historic literature and carried out a global compilation of quantitative data to understand the chemical nature of soil organic P, including insight on what might constitute unresolved forms. We identified five key eras spanning the mid-1800s to current. Understanding of the chemical nature of organic P has largely reflected the predominant analytical technique in use, which generally involved focusing on a particular class of organic P. While inositol phosphates have been a focus throughout most eras, quantitative data reveal that the composition of the majority of organic P (typically >50%) in soil remains unresolved. Insight on its chemical nature has revealed that it is largely comprised of phosphomonoesters (P–O–C) and is associated with large molecular weight fractions, including the soil humic acid fraction. Furthermore, there is strong evidence that this is concomitant with the existence of a broad spectral feature that appears along with sharp peaks attributable to specific compounds in the phosphomonoester region of solution 31P nuclear magnetic resonance spectra. Here, we highlight the need to improve our understanding of the chemical nature and cycling of diverse forms of organic P in soil, including that of “unresolved” pools. This will necessitate the use of multiple techniques and approaches in soil biogeochemistry that require a holistic approach to understanding soil organic matter dynamics, and the association of organic P with soil organic carbon.

1. Introduction 1.1 Background Phosphorus (P) is an essential element for living organisms because it is vital to cellular function and it cannot be replaced with another element. It is an innate constituent of the phospholipids that comprise cell membranes, the molecules that carry genetic code (deoxyribonucleic acid—DNA, and ribonucleic acid—RNA), and the “molecular unit currency” of intracellular energy transfer (adenosine triphosphate) (Westheimer, 1987). Ultimately all living organisms source their P either directly or indirectly via food sources from soil. Consequently, an insufficient supply of P from soil to biological systems can constrain growth and productivity (Frossard et al., 2000).

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Worldwide, concentrations of total P in surface soils generally range from 200 to 1000 mg P kg
1 (Yang and Post, 2011). Soil P can be identified as occurring in two broad chemical forms: (1) inorganic P, which includes orthophosphate anions in solution, orthophosphate minerals, and orthophosphate sorbed to mineral surfaces and organic matter; and (2) organic P, in which P atoms are covalently bonded to carbon (C) either directly (P–C) or via a phosphoester linkage (phosphomonoester (P–O–C) or phosphodiester (C–O–P–O–C)). Condensed forms of P (i.e., pyrophosphate and polyphosphates), which are also often found in living organisms and soils, although technically inorganic, are often grouped with organic P as they are not routinely detected by the colorimetric techniques used to distinguish inorganic from organic P through direct quantification of orthophosphate (Turner et al., 2005a). The relative amounts of inorganic and organic P that comprise total soil P vary greatly between soil types and land-use (Cross and Schlesinger, 1995; Harrison, 1987; Negassa and Leinweber, 2009), with organic forms generally comprising between 20% and 80% of the total P in soil (Anderson, 1980; Harrison, 1987). Higher percentages of organic P tend to occur in the upper horizons of soils under grassland or forest as compared to that of intensively managed arable systems (Stutter et al., 2015), and also in soils of temperate regions compared to tropical regions (Yang et al., 2013). The proportion of total P present as organic P changes with pedogenesis (Walker and Syers, 1976; Yang and Post, 2011). Walker and Syers (1976) developed a model that described changes in the amount and composition of soil P with time (Fig. 1). It describes a decrease in the concentration of total P in soil with increasing time, but with a concomitant increase in the organic P fraction. Consequently, the turnover rate of soil organic P (SOP) becomes increasingly important for biological uptake with pedogenesis (Turner et al., 2013). Soil organic P is important in terrestrial ecosystems for several reasons, including: (1) P is an innate constituent of soil organic matter (McGill and Cole, 1981) and total organic P is correlated with total (organic) C in soil (Kirkby et al., 2011); (2) it can accumulate in fertilized soil which contributes to an agricultural inefficiency of fertilizer P (McLaren et al., 2015a; Schefe et al., 2015); (3) through mineralization, it is an important source of orthophosphate for living organisms, which also has implications for “organically” managed systems where fertilizer P is either not commercially available or where its application is limited by choice or environmental concern (B€ unemann, 2015; Dodd and Sharpley, 2015; Richardson et al., 2005); and (4) it can be a source of P to aquatic and marine ecosystems where eutrophication may eventuate (Dodd and Sharpley, 2015; Turner, 2005).

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Fig. 1 The relative distribution of inorganic and organic forms of phosphorus (P) in soils with increasing time of soil development. Reprinted from Fig. 1 of Walker, T.W., Syers, J.K., 1976. The fate of phosphorus during pedogenesis. Geoderma 15, 1–19 with minimal alteration. Copyright (1976), with permission from Elsevier.

It is therefore important that we understand the chemical nature of organic P in soils and the mechanisms that govern its flux through the soil-plant system. Knowledge on how P is cycled through soil organic matter has been hindered to a large extent by uncertainty on the chemical nature of SOP (Doolette and Smernik, 2015; George et al., 2018; Haygarth et al., 2018). In particular, there is a need to further understand the molecular chemistry of organic P in soil and how that translates within a broader context (CadeMenun, 2017; Haygarth et al., 2018; Leinweber et al., 2018). There is also a need to address issues on the consistency of methodological techniques used to identify the chemical nature of SOP, particularly the reliance on solution 31P nuclear magnetic resonance (NMR) spectroscopy and spectral deconvolution fitting procedures (Doolette and Smernik, 2015; George et al., 2018; Haygarth et al., 2018). In this paper we critically review the collective scientific literature on the chemical nature of SOP. In particular, our aims were to: (1) understand the evolution of knowledge on the chemical nature of SOP; (2) critically review the literature on the identification and quantification of SOP; (3) carry out a global compilation of quantitative data on the chemical nature of SOP; and (4) gain new insight on what might comprise pools of “unresolved” organic P in soil.

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1.2 Approach of historical review Our approach was to review the collective scientific literature related to the chemical nature of SOP, which included reviewing over 500 published journal papers from the mid-1800s to current. Papers related to the dynamics of SOP were only considered if they provided novel insight on the chemical nature of SOP. Our review focused on papers published in English, but in some instances included non-English papers. The review is structured in a historical format (divided into five major eras) that summarizes the main findings of each era that furthered our understanding of the chemical nature of SOP. The findings from each era are then followed by a discussion that critically evaluates the research and its contribution to our understanding of SOP. Key eras of research that were partitioned into five eras were based on a number of factors, such as: (1) a significant change in methodology; (2) a highly influential paper that resulted in new insight on the chemical nature of SOP; or (3) a noticeable shift in the nomenclature or terminology regarding SOP. However, not all of the papers that were reviewed are cited here, but rather those that we considered representative of the broader literature within each era. For example, papers that applied an existing method to a different soil environment or set of treatments, which did not reveal any new insight on the chemical nature of SOP within that era were omitted. Furthermore, we note that due to the nature of research and publication, there was usually an overlap between what we designate as eras and so this designation should be taken as a guide only. A global compilation of quantitative data on the chemical nature of SOP was carried out, which involved summarizing the quantitative data reported in the majority of studies using the predominant technique at the time. No quantitative data on the chemical nature of SOP was reported in Era 1 and only one study during Era 2 (Bower, 1945), which was combined with quantitative data reported in Era 3. Quantitative data reported in Era 3 was largely based on chromatographic techniques on soil extracts. Quantitative data reported in Era 4 was largely based on solution 31P NMR spectroscopy on soil extracts and spectral integration of NMR spectra, where broad classes of organic P were reported. Quantitative data reported in Era 5 (up to 2017) was largely based on solution 31P NMR spectroscopy followed by spectral deconvolution fitting of the orthophosphate and phosphomonoester region based on the methods of Turner et al. (2003e) and B€ unemann et al. (2008b) (or modifications thereof ). Lastly, studies within each era were not included in the global compilation of quantitative

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data if there was incomplete information so that concentrations of total SOP or its chemical nature could not be reliably determined on a soil basis (mg P kg
1). Furthermore, studies were also excluded if there were major issues with spectral quality in which pools of SOP could not be reliably determined (e.g., Adams, 1990).

2. Era 1: Mid-1800s to 1930s—The existence of soil organic phosphorus and reliance on chemical extraction and isolation techniques 2.1 Main findings Evidence that a proportion of the total P in soil could exist in an organic form can be traced back to the mid-19th century (Mulder (1844) as cited in Stewart (1910)). This was largely based on the observation that P was not easily separated from humus material (i.e., the “matie`re noire,” which translated into English means the “black matter”), and that P occurred in stoichiometric ratios with C and nitrogen (N) (Table 1) (Stewart, 1910). The existence of SOP, however, remained in some doubt until specific methods were developed that could show P was covalently bonded to C (i.e., SOP), rather than being present as inorganic P sorbed within an organic matrix (Stewart, 1910). Potter and Benton (1916) first provided evidence for the existence organic P in soil with the development of an alkaline extraction method in which inorganic P could be differentiated from organic P through its precipitation on addition of excess Mg2+. Even then, some researchers remained unconvinced that this so-called “organic P” was

Table 1 Concentrations of the “matière noire” (black matter) and its carbon (C), nitrogen (N) and phosphorus (P) content in two soils. matière noire Ratio Soil

matière noire

C

N

P

P:N

N:C

P:C

A

60,840

25,860

2805

524

1:5

1:9

1:49

B

61,660

25,790

2885

508

1:6

1:9

1:51

Average

61,250

25,825

2845

516

1:6

1:9

1:50

Soil was extracted with acid prior to ammonia extraction. Values are expressed in pounds per 2 million pounds of dry soil, as reported by the author. The table was taken and adapted from Stewart, R., 1910. Quantitative Relationships of Carbon, Phosphorus, and Nitrogen in Soils. Bulletins of the Agricultural Experiment Station, vol. 145. University of Illinois, pp. 91–128.

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not sorbed colloidal inorganic P (Gortner and Shaw, 1917). This was later dismissed by Potter and Snyder (1918) with the presentation of a modified procedure for the extraction of organic P. Even as the existence of organic P in soil was being established, speculation began as to its chemical composition (Auten, 1923; Potter and Benton, 1916; Stewart, 1910). The obvious candidates were the major forms of organic P known to be present in living organisms, specifically nucleic acids, phospholipids and phytate (Auten, 1923; Schreiner, 1923; Shorey, 1913). Shorey (1913) wrote “It is evident, then, that organic P compounds are added to soils in every addition of animal or vegetable material, and it is reasonable to expect that some of them would persist for a time at least and make up a portion of the organic matter of the soil.” In order to understand what forms of organic P could be present in soil there were two main approaches used by researchers of this era. The first was to try and isolate recognizable biomolecules from soil using chemical extraction procedures, and the second was to investigate the fate of these biomolecules (i.e., the rate of their conversion to inorganic P) when added to soil (Auten, 1923; Potter and Snyder, 1918). For the former, researchers relied on their understanding of the chemical properties of recognizable biomolecules (i.e., differences in pH and temperature stability, kinetic reactivity, solubility in different solvents, and color reactivity), which were used to fractionate the organic matrix (Potter and Benton, 1916). However, this was problematic due to the inherently complex nature of soil organic matter. For the latter, researchers hypothesized that if a particular form of organic P was only sparingly hydrolyzed after its addition to soil (i.e., it resisted degradation), then this would infer an ability to form a stable pool of SOP (Auten, 1923; Potter and Snyder, 1918). Some of the earliest studies on the chemical nature of SOP claimed to have revealed the presence of nucleic acids (Aso, 1904; Shorey, 1912) and phospholipids (Aso, 1904). Aso (1904) considered phospholipids to be a minor constituent of the SOP, which was consistent with other researchers who found phospholipids were readily hydrolyzed upon addition to soil (Auten, 1923). Shorey (1913) used an innovative but complex extraction procedure to isolate nucleic acids from soils through identification of the decomposition products of nucleic acids (i.e., phosphoric acid, pentose sugars, cytosine and xanthine bases). Using this approach, Shorey (1913) considered nucleic acids to be a major component of the organic P in soil, stating that “in many cases they form a very appreciable portion of the organic matter of soils.” While there does not appear to be any

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attempt to isolate phytate from soil during this era, it was still considered a likely candidate for a stable pool of SOP due to its presence in living organisms (Schreiner, 1923). Inconclusive results on the chemical nature of SOP were obtained when recognizable biomolecules were added to soil (Auten, 1923; Potter and Snyder, 1918). Based on a series of experiments, Potter and Snyder (1918) presented supporting evidence that nucleic acids appear to be evident in some soils, whereas phytate appeared to be present in other soils. In the first part of their study, soil that contained negligible concentrations of organic matter was collected in order to investigate whether phytate could be extracted from soil with acid. They found <4% of the P in added phytate was extracted with the acid solution; this small fraction of added P was assumed to be orthophosphate present as a minor impurity in the phytate material. In the second part of the study, acid hydrolysis curves of nucleic acid and phytate were determined over time. The purpose was to compare their acid hydrolysis curves with that of SOP, in order to gain some insight on their presence and potential bioavailability. In this study, they concluded that about 60% of the nucleic acids and about 13% of phytate were hydrolyzed. The authors then assessed the rate of acid hydrolysis (at 100 °C) of organic P from three loam soils and compared that with nucleotides and an impure phytate material (Fig. 2). Between 20% and 31% of the extractable

Fig. 2 The acid hydrolysis (at 100 °C) curves (inorganic P release) of three loam soils, and those of nucleotides and an impure phytate material over time. The hydrolysis curve of Soil 1 is different in shape to those of Soils 2 and 3. The latter two are similar in shape to the hydrolysis curves of the phytate material and nucleotides. The figure was taken and adapted from Potter, R.S., Snyder, R.S., 1918. The organic phosphorus of soil. Soil Sci. 6, 321–332. Copyright (1918), with permission from Wolters Kluwer Health.

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organic P was hydrolyzed at the end of the experiment, with two of the three soils exhibiting similar hydrolysis curves to those of nucleotides and the phytate material. The authors concluded that the bioavailability of organic P differed between soils and that phytate most likely resisted degradation due to its resistance to acid hydrolysis, its high sorption affinity to soil compared to orthophosphate, and consequently the preference of microorganisms to utilize orthophosphate. In a review of early literature, Auten (1923) surmised that of the organic P added to soil, much would be comprised of nucleic acids and phytate (Shorey, 1913; Suzuki and Yoshimura, 1907), whereas only a small portion would be phospholipids (Aso, 1904). However, based on theoretical calculations of the total P content of living organisms in soil-plant systems, which included generalized assumptions on the amount of living biomass and their P concentrations, it was suggested that these sources could not reasonably account for the amount of total organic P observed in soils. Auten (1923) concluded therefore, that there must be a pool of organic P in soils which is not directly or immediately attributable to living organisms. The author carried out an incubation experiment with a sterilized silica sand that subsequently had been inoculated with a “soil infusion,” presumably of soil microorganisms. The soils received additions of nucleic acids, phytate and lecithin (a phospholipid), and rates of hydrolysis were assessed over time. It was found that after 3 months of incubation, 85%, 67% and 66% of the added organic P substrates had been hydrolyzed, respectively. From these results, it was concluded that nucleic acids, phytate and phospholipids were unlikely to comprise the stable pool of SOP because they were largely degraded in a relatively short period of time. Interestingly, Auten (1923) then considered the formation of a condensed “humus-like” substance that could be synthesized in the laboratory from amino acids and carbohydrates as proposed by Maillard (1912). Auten (1923) hypothesized that if inorganic P was added to the procedure of Maillard (1912), then P could combine and be found within the resultant product, which was tested and confirmed in the study of Auten (1923). Furthermore, it was found that the humus-like substance became more complex over time, from which it was suggested that nucleic acids, phospholipids and inositol phosphates (IP) were only a minor component of SOP.

2.2 Discussion Understanding the chemical nature of SOP during this era was generally limited due to the complexity of soils and the availability of appropriate

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techniques for its characterization. For the most part, much of the understanding on what constitutes SOP was speculative and where experimental data were obtained the results were sometimes contradictory and often inconclusive (Auten, 1923; Potter and Snyder, 1918). Phytate was generally considered to be the most likely candidate for a stable pool of SOP during Era 1, largely because studies indicated that nucleic acids and phospholipids did not persist when added to soil (Aso, 1904; Potter and Benton, 1916), whereas, at least in some soils, phytate appeared to persist (Potter and Snyder, 1918). A mechanism was proposed that might explain how phytate could accumulate in soil based on its high sorption affinity by soil constituents (Potter and Snyder, 1918). While research during this era may not have greatly advanced our understanding of the chemical nature of SOP, it had major influence on future work through the observation concerning the complex composition of SOP. The chemical nature of organic P in soil was partly confirmed as being that of organic P found in living organisms (i.e., nucleic acids, phospholipids and phytate) (Schreiner, 1923) that, after death of the organism, becomes part of SOP along with greater knowledge on the chemical properties of these organic compounds (Dyer et al., 1940; Potter and Snyder, 1918; Shorey, 1913). An alternative hypothesis, albeit less widely held at the time, was that the organic P in soil is likely to also be part that of “humus-like” substances, formed via abiotic Maillard-type reactions (Maillard (1912) as cited in Auten (1923)). While the formation of organic P via abiotic processes has not been established, knowledge that a portion of the organic P in soil cannot be directly attributed to recognizable biomolecules has been a recurring theme in SOP research since this time.

3. Era 2: 1940s—Supporting evidence on the existence of inositol phosphates in soil 3.1 Main findings Dyer et al. (1940) were the first to claim direct isolation of IP from soil in a seminal paper published in Science that was followed up with a series of related subsequent papers (Dyer and Wrenshall, 1941a,b; Wrenshall and Dyer, 1941). The method for isolating the “phytin material” relied on a series of chemical extractions that essentially remained in use for decades thereafter (until the 1980s). The main steps were: (1) an acid pre-treatment to remove calcium that would otherwise cause problems with dissolution and/or precipitation of organic material in subsequent steps; (2) hot alkaline

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extraction to solubilize IP and other organics; (3) hot hypobromite treatment to oxidize the majority of organic matter (IP being resistant to this treatment); and (4) addition of FeCl3 to selectively precipitate the IP. Further purification steps yielded a white crystalline solid with an appearance and stoichiometry (particularly with regards to the ratio of iron to P) consistent with that of an authentic sample of iron phytate. Wrenshall and Dyer (1941) also investigated the stability of a range of organic P compounds toward enzymatic degradation. Phosphatases were extracted with water from the mucous membrane of pig intestine, which was considered to contain monoesterases and diesterases but negligible amounts of phytases (Levene and Dillon, 1932; Plimmer, 1913), and phytase was extracted with water from wheat bran (Plimmer, 1913). These enzymes were added to a variety of organic compounds (sodium phytate, and a crude extract of phytate obtained from soil, sodium phytate after bromination treatment, iron phytate, aluminium phytate, calcium phytate, and nucleic acid). After 10 days, the phosphatases degraded a large proportion (75%) of the nucleic acids, but only a small proportion (6%) of sodium phytate. Conversely, after 7 days, the phytase degraded a large proportion (>72%) of all added compounds of organic P, except iron- and aluminium-phytate. The authors suggested that a possible mechanism for phytate accumulation in soil was through interactions with sesquioxides that render it insoluble and resistant to enzymatic (microbial) degradation. In 1940, a further paper verified the existence of IP in soils from Hawaii (Yoshida, 1940). However, in this case only isolation of free inositol was achieved (i.e., inositol without any attached phosphate groups). The ratio of inositol to phosphate in the soil fraction from which the inositol was eventually purified led the author to suggest that inositol monophosphate was the predominant form of organic P present in the soil. While this contradicted the findings of Dyer et al. (1940), it provided further evidence for the presence of IP which, in different soils and under different laboratories, accounted for either a small percentage or as much as half of the total soil P (Bower, 1945).

3.2 Discussion The method of Dyer et al. (1940) provided an opportunity to isolate and possibly quantify what was considered a potentially important fraction of SOP (Bower, 1945; Dyer et al., 1940; Yoshida, 1940). However, information on the efficacy of the procedure remains lacking and the results on

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the chemical nature of the isolated phytin material were generally contradictory (Bower, 1945; Dyer et al., 1940). Nevertheless, there were three main outcomes of this era: (1) the development of a technique that could estimate soil IP (Dyer et al., 1940); (2) confirmation of a mechanism for the stabilization of phytate in soil via its interaction with sesquioxides (Wrenshall and Dyer, 1941); and (3) data that suggested IP could constitute up to half of the organic P in soil (Bower, 1945). However, again, up until the end of Era 2, very little was definitively known on the chemical nature of the wider pool of SOP, for which information either remained speculative or was derived from indirect measures only.

4. Era 3: 1950s to 1970s—Chromatographic techniques with a focus on inositol phosphates and molecular weight distribution of soil organic phosphorus 4.1 Main findings The next major advance in the characterization of SOP involved the application of chromatographic separation to soil extracts (Smith and Clark, 1951). Anion-exchange chromatography was applied to “phytin material” extracted from soil, which had up until that time been assumed to consist entirely of IP (predominantly phytate). Smith and Clark (1951) showed, however, that “phytin” isolated from soil behaved differently to phytate isolated from plants. While some IP (especially phytate) appeared to be present in the supposed phytin material, they estimated that two-thirds or more of the organic P was in fact not IP. They also reported that the soil extracts contained a compound that eluted from the anion-exchange column after that of phytate, but its identity was unknown at that time (Fig. 3). In a paper published in Nature, Anderson (1955) described an alternate method to that of Smith and Clark (1951) for characterizing IP in soils based on 2D paper chromatography (also see Anderson (1956) for more detail). The first dimension involved elution of P compounds with a solution of methanol and ammonia, whereas the second dimension involved elution of P compounds with a solution of acetone and acetic acid. Using this method on “phytin material” extracted from three soils and without the hypobromite treatment, Anderson (1956) reported that IP comprised between 27% and 38% of the extractable organic P (based on a hot HCl and cold NaOH extraction technique). The majority of the total organic P was attributed to inositol hexaphosphate, whereas lower order IP (i.e., inositol tetra-, tri-, di-, and mono-phosphates) were either not detected or found in trace amounts.

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Fig. 3 Chromatographic elution curves of organic P from a loam soil of slightly acid pH (A) before and (B) after the addition of “sodium phytate and derivatives,” and also in a silt loam soil of slightly alkaline pH (C) before and (D) after the addition of “sodium phytate and derivatives.” The “†” symbols identify the elution peaks of the “sodium phytate and derivatives” material in the spiked samples (B and D), and also indicate at the same location in the original samples (A and C). The elution peak at fractions 70–90 was thought to be due to inositol tetra(quadri)phosphates, peaks at fractions 140 and 152 were thought to be due to inositol pentaphosphates, whereas a peak at fraction 178 was thought to be that of the inositol hexaphosphates. The “‡” symbols identify the peak of an unknown compound that eluted after that of phytate. The figure was taken and adapted from Smith, D.H., Clark, F.E., 1951. Anion-exchange chromatography of inositol phosphates from soil. Soil Sci. 72, 353–360. Copyright (1951), with permission from Wolters Kluwer Health.

Improvements of methodologies for isolating and characterizing IP in soil was further reported in a series of papers by Caldwell and Black (1958a,b,c). Caldwell and Black (1958a) described the phytin material as containing meso-inositol hexaphosphate and a supposed isomer of

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meso-inositol hexaphosphate. The former appears to correspond with phytate (i.e., myo-inositol hexaphosphate) and the latter is analogous with the compound that eluted after phytate from the anion-exchange column, as previously reported by Smith and Clark (1951). The term “meso” refers to molecules with multiple chiral centers, but which are not optically active because those chiral centers are chemically identical through symmetry; this is true of several inositol isomers, including myo- and scyllo- and so is not useful as a unique descriptor for an inositol isomer. While the conformational structure of the compound that eluted after “phytate” was not known, the authors suggested it to be an isomer of inositol hexaphosphate. This was based on it eluting after “phytate” on an anion exchange column. It had been established that elution of IP generally followed the order of increasing number of phosphate groups, hence a compound eluting after phytate should have (at least) six phosphate groups (Caldwell and Black, 1958b; Smith and Clark, 1952). Caldwell and Black (1958c) applied their methods to quantify inositol hexaphosphates across 49 soils of varying pH (acid to slightly alkaline) and soil type (loamy sand to clay texture). They found that the proportion of total organic P comprised of inositol hexaphosphates ranged from 3% to 52% (on average 17%). Furthermore, concentrations of inositol hexaphosphates were correlated with concentrations of total SOP (after subtraction of the inositol hexaphosphates). Neither pH nor the concentrations of free iron oxide (determined by extraction with sodium citrate with dithionite) was significantly correlated with concentrations of inositol hexaphosphates, which suggested stabilization via strong interactions with sesquioxide minerals is not the only stabilization mechanism for these compounds. These authors also argued for the microbial synthesis of inositol hexaphosphates based on increased concentrations of inositol hexaphosphates in an artificial soil mixture identified using their chromatographic method after several months incubation (Caldwell and Black, 1958b). Chromatographic characterization of soil IP was most comprehensively researched throughout the 1960s to early 1980s by Dennis Cosgrove and co-workers. This included an improved nomenclature of the identified compounds of organic P. First, Cosgrove (1962) published a paper in Nature that reported the “supposed isomer of inositol hexaphosphate” of Caldwell and Black (1958a) was scyllo-inositol hexaphosphate (Cosgrove, 1962). In this paper, a third stereoisomer of inositol hexaphosphate was also identified which was initially reported as “DL-inositol hexaphosphate” (Cosgrove, 1962), but later found to be optically active and specifically

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Fig. 4 The conformational structures of (A) myo-inositol, (B) neo-inositol, (C) D-inositol, and (D) scyllo-inositol. The figure was taken and adapted from Cosgrove, D.J., 1966. Synthesis of the hexaphosphates of myo-, scyllo-, neo- and D-inositol. J. Sci. Food Agric. 17, 550–554. Copyright (1966), with permission from Wiley.

the D-enantiomer of chiro-inositol hexaphosphate (Cosgrove, 1969). A fourth stereoisomer of inositol hexaphosphate was detected and identified as neo-inositol hexaphosphate (Fig. 4) (Cosgrove and Tate, 1963). Cosgrove (1962, 1963a) describe in detail the exhaustive and complex procedures used to determine the chemical nature of IP in soils. In particular the difficulties of using anion-exchange chromatography where chiro and neo stereoisomers may elute with that of the myo stereoisomer of inositol hexaphosphate. It was not until later that the chiro, myo, neo and scyllo stereoisomers of the combined inositol penta- and hexa-phosphates could be more easily distinguished with the use of gas-liquid chromatography (Irving and Cosgrove, 1982). On this basis, the myo stereoisomer and to a lesser extent the scyllo stereoisomer were found to be the most abundant forms in soil. Moreover, there was little variation (<12% relative standard deviation) between the relative proportions of stereoisomers when assessed across four contrasting soils, whereby on average 64% of myo, 23% of scyllo, 7% of chiro, and 7% of neo for the combined inositol penta- and hexa-phosphates was observed (Irving and Cosgrove, 1982). Lastly, Irving and Cosgrove (1981) appear to be the first to describe inositol hexaphosphates (and pentaphosphates), which was common in previous studies, as inositol hexakisphosphates (IP6) (and pentakisphosphates). The reason for this was not provided, but since the term “kis” is a Greek prefix referring to “times,” it is likely that the addition of “kis” reflects a more appropriate nomenclature for the same compound (Murthy, 2007; Sarma, 2004).

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While the main focus of Cosgrove’s research was identifying the diversity of IP in soil, rather than their quantification (a point made by Cosgrove (1963a) himself ), attempts were also made to identify the sources and/or processes responsible for the presence of stereoisomers other than the myo form which was assumed to be added to soils via seeds (Cosgrove, 1964, 1969). While he was largely unsuccessful in this, he did show an absence of stereoisomers, other than the myo form in fungi, yeast and acorns (Cosgrove, 1964), and that the chiro stereoisomer of inositol hexaphosphate was optically active, which provided evidence against the chemical epimerization of the myo stereoisomer as a likely source (Cosgrove, 1969). Importantly, knowledge on the origin of different stereoisomers of inositol hexaphosphate in soil remains largely unknown to this day (Anderson and Malcolm, 1974; Cosgrove, 1963a). The work of Cosgrove and co-workers was successful in identifying possible sources and/or processes that could be responsible for the presence of lower order IP in soil. Both biological (i.e., via enzymatic means) and non-biological (i.e., via chemical means) hydrolysis or dephosphorylation of pure inositol hexaphosphate to lower order IP was demonstrated via differing pathways in the order of phosphate removal (Cosgrove, 1963b, 1966, 1970). Pathways for the chemical synthesis of lower order forms of myo-IP, by reacting myo-inositol with sodium trimetaphosphate were identified, as was a non-biological epimerization of myo-inositol pentaphosphate to scyllo-inositol pentaphosphate (Cosgrove, 1972). A similar process for the conversion of DL-myo-inositol pentaphosphate to DL-chiro-inositol pentaphosphate was postulated, although this would likely require a biological process to be converted to the D-enantiomer. Concomitant work by George Anderson and co-workers focused on quantification of inositol penta- and hexa-phosphates in soils. Several other studies used Anderson’s techniques, or modifications thereof, to estimate concentrations of IP in soils (Table 2). Collectively these studies generated a view on the dominance of IP as a major pool of SOP. In a landmark review on SOP by Anderson (1980), it was stated that IP in soil “are quickly stabilized and have accumulated to such an extent in some soils that they constitute more than half of the organic P.” However, it was later acknowledged in Anderson (1980) that IP more generally comprise <20% of the total organic P in soils from Australia, Canada, the United Kingdom, and the United States. In addition, Anderson (1980) stated that “in some soils only a small percentage of the organic P has been characterized, and at best, nearly 40% remains to be identified. Most of it occurs in material of very large molecular weight (MW), but otherwise little is known about it.”

ARTICLE IN PRESS 17

Chemical nature of soil organic phosphorus

Table 2 A global compilation of all quantitative data on inositol phosphates in soils from across 13 countries and 25 published studies (total of 358 samples) up to the end of Era 3 (1950s–70s). “Other” forms of Inositol organic P (% of phosphates Total organic organic P) (% of organic P) P (mg kg21) Number of a Range Average Range Average Range Average Location samples

Australia

130

6–2088

214

0–38

16

62–100 84

Bangladesh

40

39–560

196

9–84

54

16–91

46

Canada

49

70–710

293

1–23

10

77–99

90

Chile

9

654–1492 1173

42–67 58

33–58

42

Denmark

1

354–354

354

46–46 46

54–54

54

England

17

88–1750

364

11–45 28

55–89

72

Georgia

4

115–167

144

30–40 34

60–70

66

Ghana

10

75–275

168

8–23

15

77–92

85

New Zealand

5

440–1360

732

8–43

25

57–92

75

Nigeria

4

170–600

350

23–29 26

71–77

74

Scotland

30

127–938

529

1–58

41

42–99

59

Sri Lanka

3

270–288

281

1–1

1

99–99

99

United States

56

40–446

247

3–52

19

48–97

81

Overall average

28

188–848

388

14–42 29

58–86

71

a Citations for each location include: Australia (Cosgrove, 1963a; Irving and Cosgrove, 1981; Steward and Tate, 1971; Williams and Anderson, 1968), Bangladesh (Islam and Ahmed, 1973; Islam and Mandal, 1977; Mandal and Islam, 1979), Canada (Appiah and Thomas, 1982; Dormaar, 1967; Halstead and Anderson, 1970; McKercher and Anderson, 1968a,b; Thomas and Lynch, 1960), Chile (Borie et al., 1989), Denmark (Pedersen, 1953), England (Anderson, 1964; Omotoso and Wild, 1970; Oniani et al., 1973), Georgia (Oniani et al., 1973), Ghana (Appiah and Thomas, 1982), New Zealand (Martin and Wicken, 1966), Nigeria (Omotoso and Wild, 1970), Scotland (Anderson, 1956, 1964; Anderson et al., 1974; Anderson and Malcolm, 1974; Halstead and Anderson, 1970; McKercher and Anderson, 1968a), Sri Lanka (Oniani et al., 1973), United States (Bower, 1945; Caldwell and Black, 1958c; Smith and Clark, 1951). Pools of “other” forms of organic P are taken as the difference between total organic P and that measured as inositol phosphates.

We carried out a global compilation of all quantitative data derived up to the end of Era 3 in order to assess what would have been known on the abundance of soil IP (Table 2). This included a dataset of 358 samples across 13 countries. Average concentrations of SOP ranged from

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Timothy I. McLaren et al.

188 to 848 mg kg
1 (on average 388 mg kg
1) across all soils. Pools of IP and “other” forms of organic P (i.e., non-IP) accounted for on average 29% and 71% of the total organic P across all soils, respectively. Unfortunately, no consistent methodology was used across the studies to quantify the IP content of soil as presented in Table 2. Some authors suggested that the measured concentrations of IP may have been underestimated due to incomplete recovery of organic P through the resin columns (Martin, 1970), or possibly due to incomplete precipitation of the IP (Anderson, 1963; Mandal and Islam, 1979). On the other hand, Irving and Cosgrove (1981) reported that soil IP would be overestimated in some studies due to the formation of material that co-eluted with that of IP. In order to prevent this, Irving and Cosgrove (1981) recommended the inclusion of a hypobromite oxidation step to remove any non-IP material prior to chromatographic analysis. The authors demonstrated that concentrations of IP could be overestimated without hypobromite oxidation in some soils (Table 3). Research continued to improve our understanding of the processes related to the accumulation of IP in soil. This largely involved understanding the sorption dynamics of IP to soil constituents (Anderson et al., 1974; Anderson and Arlidge, 1962), the stability of phytate with pH ( Jackman and Black, 1951b), the enzymatic release of orthophosphate from phytate Table 3 Concentrations of total organic P for six Australian soils that were determined on material prepared for estimating inositol phosphates with a hypobromite oxidation pre-treatment, compared to that without on the same soils (Williams and Anderson, 1968). Total organic P (mg kg21 soil) Soil

Hypobromite

Williams and Anderson (1968)

187

46.3

46.5

236

27.8

42.9

246

5.1

8.2

432

0.3

1.0

607

25.3

23.6

639

25.8

41.9

The table was taken and adapted from Irving, G.C.J., Cosgrove, D.J., 1981. The use of hypobromite oxidation to evaluate two current methods for the estimation of inositol polyphosphates in alkaline extracts of soils. Commun. Soil Sci. Plant Anal. 12, 495–509. Copyright (1981), with permission from Taylor & Francis.

ARTICLE IN PRESS Chemical nature of soil organic phosphorus

19

( Jackman and Black, 1951a, 1952), and the degradation or assimilation of phytate by plants and microorganisms (Greaves and Webley, 1969; Martin, 1973). These studies generally supported previous work on processes that would lead to the stabilization of IP in soil. An important component of research during Era 3 was understanding the “unresolved” pools of organic P, which occurred concurrently with that of IP. The term is analogous with “unidentified” or “unknown” forms of SOP and largely refers to the SOP that could not be elucidated or identified as recognizable biomolecules, which was generally thought to account for more than 50% of the total organic P in soil (Martin, 1964). A preliminary view of this organic P was that it was P associated with humic substances, phospholipids or nucleic acids (Dormaar, 1963, 1968; Wrenshall and McKibbin, 1937). The problem with this was that humic substances were difficult to extract and identify (Dormaar, 1963, 1967), whereas phospholipids and nucleic acids were generally found to be minor fractions of the total organic P in soil (Adams et al., 1954; Anderson, 1958, 1961, 1970; Dormaar, 1970; Kowalenko and McKercher, 1970). Nevertheless, new insight was made with the application of physical fractionation using size exclusion chromatography to soil extracts according to MW. There was evidence to suggest much of the organic P in soil was contained in large MW fractions and comprised of macromolecules derived from “humification” processes (Moyer and Thomas, 1970; Swift and Posner, 1972). Thomas and Bowman (1966) first reported the distribution of organic P in soil extracts according to MW. This was carried out on a soil of pH 7.2 and 8.6% organic matter, which was extracted using Dowex A-l chelating resin with a HF-HCl acid pre-treatment, and then passed through a Sephadex® G-75 column. A major portion of the organic P in these soil extracts was contained in MW material >50 kDa. Moyer and Thomas (1970) later repeated the experiment on the same soil, but then qualitatively determined the presence of IP in various MW fractions. These authors reported that 36% of the organic P was in MW fractions >50 kDa, 31% of the organic P was in MW fractions between 1 and 50 kDa, and 8% of the organic P was in MW fractions <1 kDa, with IP being detected only in the latter two lower MW fractions. Further differentiation of the organic P in various MW fractions (Fig. 5) and differing metal contents on the same soil was later reported by Veinot and Thomas (1972). A more comprehensive analysis on the MW distribution of organic P was then carried out by Steward and Tate (1971) on eight soils collected across Australia.

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Timothy I. McLaren et al.

Fig. 5 Chromatographic elution curve on a resin extract from a loam soil of near neutral pH that was fractionated with Sephadex gels. The approximate molecular weight range of each fraction were designated as: G-75 I fraction >50 kDa, G-75 II fraction between 1 kDa and 50 kDa, and G-75 III fraction <1 kDa. The figure was taken and adapted from Veinot, R.L., Thomas, R.L., 1972. High molecular weight organic phosphorus complexes in soil organic matter: inositol and metal content of various fractions. Soil Sci. Soc. Am. J. 36, 71–73. Copyright (1972), with permission from the Soil Science Society of America Journal.

These authors found that, on average, 79% and 21% of the organic P was detected in MW fractions >30 kDa and <30 kDa, respectively. Similarly, Goh and Williams (1982) determined the MW distribution of P across 10 soils collected from three chronosequences in New Zealand. On average, 64% of the total P in soil extracts was detected in MW fractions >50 kDa. Condron and Goh (1989) reported further that, on average, 28% of the extractable organic P was detected in MW fractions >100 kDa for six soils under irrigated pasture in New Zealand. Overall, these studies indicate the presence of a significant portion of the SOP that could be described as occurring in large MW fractions, and suggest the presence of organic P in addition to that of IP and other identifiable forms in soils.

4.2 Discussion Understanding the chemical nature of SOP was greatly improved with the combination of extracting “phytin material” and chromatographic

ARTICLE IN PRESS Chemical nature of soil organic phosphorus

21

techniques, which provided a plethora of information on the diversity of soil IP (Cosgrove, 1962; Smith and Clark, 1951). Several new forms of SOP were identified using this approach, including inositol hexa-, penta-, tetra-, tri-, di-, and mono-phosphates (Anderson, 1955, 1956; Cosgrove, 1963a; Smith and Clark, 1951). The most commonly detected and most abundant forms identified in soil were myo-inositol hexaphosphate (Cosgrove, 1963a; Smith and Clark, 1951), scyllo-inositol hexaphosphate (Caldwell and Black, 1958a; Cosgrove, 1962), D-chiro-inositol hexaphosphate (Cosgrove, 1962, 1969), and neo-inositol hexaphosphate (Cosgrove and Tate, 1963). There was also supporting evidence for the presence of multiple stereoisomers (myo, scyllo, D-chiro, and neo) for the lower order inositol penta-, tetra-, tri-, and di-phosphates (Anderson and Malcolm, 1974; Halstead and Anderson, 1970). However, only the inositol hexaphosphates have continued to be identified beyond Era 3, with their presence confirmed using an independent technique (i.e., solution 31P NMR spectroscopy). Clearly, further research is needed to confirm the diversity and abundance of inositol penta-, tetra-, tri-, and di-phosphates, and their multiple stereoisomers (myo, scyllo, D-chiro, and neo) in soils. Quantifying the abundance of IP in soils was problematic through much of Era 3. The general consensus was that IP were quantitatively an important pool of SOP (Anderson, 1980; Cosgrove, 1977; Dalal, 1977). However, their relative contribution to the total pool of organic P varied greatly between soils with widespread recognition that the composition of a large proportion of organic P remained “unresolved” (Anderson, 1980; Cosgrove, 1977; Dalal, 1977). This view is consistent with a global compilation of quantitative data on IP that revealed they comprised about 30% of the total organic P across all soils (Table 2). Nevertheless, there appears to be an over emphasis on the abundance of IP in soils with several key reviews suggesting that IP comprised more than half of the total organic P in some soils (Anderson, 1980; Dalal, 1977), whereas studies by Cosgrove and co-workers were more conservative. Thus it is likely that concentrations of IP reported during this era were generally an overestimate and as such should be treated with caution (Irving and Cosgrove, 1981). Concentrations of SOP that could not be attributed to IP comprised on average about 70% of the total organic P (Table 2). The chemical nature of this “unresolved” organic P was unlikely to be that of phospholipids and nucleic acids due to their low concentrations in soil (Adams et al., 1954; Anderson, 1970; Kowalenko and McKercher, 1970), while physical fractionation methods revealed that much of it could be described as

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Timothy I. McLaren et al.

occurring in large MW fractions (Goh and Williams, 1982; Moyer and Thomas, 1970; Steward and Tate, 1971). No consistent definition on what was considered as “large” MW was used in the literature, but the term was often associated with the “unresolved” pool of SOP (Harrison, 1987; Tate, 1984). Furthermore, it was suggested that the large MW fractions generally contained little or no IP (Moyer and Thomas, 1970; Veinot and Thomas, 1972), and concentrations of P were correlated to those of organic N with increasing MW (Swift and Posner, 1972). This is interesting as soil organic matter was known at the time to have relatively constant proportions of C, N and P (Walker and Adams, 1958; Williams and Donald, 1957), whereas IP6 are C poor and lack N. This provides further evidence that the “unresolved” portion of SOP is associated with organic P in large MW fractions (Anderson, 1980; Dalal, 1977; Harrison, 1987). Very little was known on the dynamics of this unresolved SOP. A study by Goh and Williams (1982) indicated that the concentration of P in large MW fractions (>50 kDa) of surface soil layers at the Manawatu chronosequence increased with soil age. In addition, Condron and Goh (1989) found that organic P in large MW fractions (>100 kDa) approximately doubled after 25 years in unfertilized soils under irrigated pasture, and also increased with the addition of inorganic forms of fertilizer P. Interestingly, there appears to be indirect evidence that the increase in organic P in large MW fractions is closely associated with increases in the other main elemental constituents of soil organic matter (i.e., C and N) (Condron and Goh, 1989). While information on the mechanisms responsible for the accumulation of organic P in large MW fractions is limited, these studies suggest the mechanisms are associated with factors affecting soil development (e.g., time and climate) and biological activity (microbial and plant growth). At the end of Era 3, there were several main conclusions that could be obtained on the chemical nature of organic P and its abundance in soil: (1) there was a diversity of IP in soil, with the myo and scyllo stereoisomers of inositol hexaphosphate being dominant within this fraction; (2) pools of IP were an important component of SOP, which comprised on average up to 30% of the total organic P across all soils; (3) phospholipids and nucleic acids generally comprised <10% of the total organic P in soil; and (4) the chemical nature of the majority of organic P in soil remained “unresolved” but was tightly associated with soil organic C and N, and contained in large MW fractions.

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23

5. Era 4: 1980s to early 2000s—Simultaneous detection of broad classes of soil organic phosphorus using solution phosphorus-31 nuclear magnetic resonance spectroscopy 5.1 Main findings A new era of understanding the chemical nature of SOP began with the application of solution 31P NMR spectroscopy to soil extracts, which enabled the simultaneous spectroscopic detection of all forms of organic P that could be brought into solution. Newman and Tate (1980) were the first to report on the use of the new approach with the publication of 31 P NMR spectra of extracts from New Zealand soils under grasslands (Fig. 6). The majority of signal intensity in the NMR spectra of soil extracts was found in the orthophosphate and phosphomonoester region (between δ 7.0 and 3.0 ppm), whereas signals of lower intensity (or absent in some soils) were found in the phosphodiester (δ
0.8 ppm), pyrophosphate (δ
5.5 ppm), polyphosphate (δ
21.4 ppm) and phosphonate (δ 19.8 ppm and 18.3 ppm) regions. Within the orthophosphate and phosphomonoester region, spiking (addition of known organic and inorganic P species to soil extracts) was used to assign a peak at δ 5.30 ppm to orthophosphate, and peaks at δ 5.26 ppm, 4.37 ppm, 4.02 ppm and 3.85 ppm to myo-IP6. The assignment of a peak at δ 3.56 ppm to choline phosphate by

Fig. 6 The first published solution 31P nuclear magnetic resonance (NMR) spectrum on a soil extract. The figure was taken and adapted from Newman, R.H., Tate, K.R., 1980. Soil phosphorus characterisation by 31P nuclear magnetic resonance. Commun. Soil Sci. Plant Anal. 11, 835–842. Copyright (1980), with permission from Taylor & Francis.

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Timothy I. McLaren et al.

Fig. 7 Solution 31P nuclear magnetic resonance (NMR) spectra of the orthophosphate and phosphomonoester region on (A) a soil extract from a native grassland and (B) on a solution containing myo-inositol hexaphosphate. The “†” symbols identify the peaks arising from myo-inositol hexaphosphate and the “‡” symbol identifies the peak the authors attributed to choline phosphate. The figure was taken and adapted from Newman, R.H., Tate, K.R., 1980. Soil phosphorus characterisation by 31P nuclear magnetic resonance. Commun. Soil Sci. Plant Anal. 11, 835–842. Copyright (1980), with permission from Taylor & Francis.

Newman and Tate (1980) has subsequently been questioned (Fig. 7) (Turner and Richardson, 2004). Extracts were also spiked with 2-aminoethylphosphonate (C2H8NO3P) to provide evidence that a peak at δ 19.8 ppm was due to a phosphonate, with sodium pyrophosphate to confirm a peak at δ
5.5 ppm was due to pyrophosphate, and with potassium metaphosphate to provide evidence that a peak at δ
21.4 ppm was due to polyphosphate. Based on previously reported chemical shifts of organic compounds in alkaline solution, they also suggested other peaks in the phosphomonoester region were due to sugar phosphates and mononucleotides (Mandel and Westley, 1964; Moon and Richards, 1973), and peaks in the phosphodiester region were due to phospholipids and DNA (Hanlon et al., 1976; Henderson et al., 1974). The work of Newman and Tate (1980) instigated a conceptual change in the approach to characterizing SOP. Compared to previous eras, analysis of SOP now involved a simple and rapid extraction that sought to solubilize as much of the SOP as possible, which could then be subsequently characterized using solution 31P NMR spectroscopy (Condron et al., 1985; Newman and Tate, 1980). This was markedly different to the sample preparation for chromatography, which used a series of complex extraction and purification steps, which were primarily designed to isolate IP material (Anderson, 1956), or the use of non-polar solvents

ARTICLE IN PRESS Chemical nature of soil organic phosphorus

25

(e.g., ethanol, chloroform, and methanol) designed to extract phospholipids (Hance and Anderson, 1963; Kowalenko and McKercher, 1970; Stott and Tabatabai, 1985). A further advantage of this procedure was that the extractable organic P from the “humic” component in soil did not have to be removed prior to analysis, as recommended for chromatographic approaches (Irving and Cosgrove, 1981). Adoption of solution 31P NMR spectroscopy for organic P determination brought about a change in nomenclature describing the dominant classes of SOP (Newman and Tate, 1980). Soil organic P was described on the basis of the association of P to C via a direct bond (phosphonates) or an ester linkage (i.e., phosphomonoesters and phosphodiesters) (Condron et al., 1985; Newman and Tate, 1980). Moreover, identifiable peaks present in NMR spectra, could then be interpreted as those relating to recognizable biomolecules (e.g., myo-IP6) (Tate and Newman, 1982; Zech et al., 1987). However, precise quantification of these individual peaks usually was not reported, rather broad classes of organic P were determined based on integrating the NMR signal into defined ranges (Condron et al., 1985). The orthophosphate and phosphomonoester region, which contained the majority of the NMR signal for most soil extracts, was often poorly resolved and contained several overlapping peaks, which made it difficult to specifically identify and quantify individual P compounds (Bedrock et al., 1994; Condron et al., 1985; Gil-Sotres et al., 1990; Zech et al., 1985). Even small changes in sample conditions or analysis setup resulted in changes in spectral quality and peak position (Cade-Menun and Preston, 1996; Crouse et al., 2000). Indeed, the first NMR spectrum of the phosphomonoester region reported in Newman and Tate (1980) and reproduced here in Fig. 7, remained as one of the most well-resolved peaks within this era (Condron et al., 1990; Guggenberger et al., 1996b; Hawkes et al., 1984). Two main approaches were used to identify the forms of organic P within the phosphomonoester region: (1) spiking the soil extract with a reference material in order to enhance the peak(s) under investigation; or (2) comparing the chemical shifts of known forms of organic P, including those in different chemical matrices, with the observable NMR signal from soil extracts (Adams and Byrne, 1989; Condron et al., 1985; Newman and Tate, 1980; Pant et al., 1999). Several studies claimed to have identified organic P compounds beyond those initially identified by Newman and Tate (1980) in the phosphomonoester region, such as aromatic phosphate esters (Bedrock et al., 1994), dihydroxyacetone phosphate

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Timothy I. McLaren et al.

(Pant et al., 1999), glycerophosphate (Adams and Byrne, 1989; Pant et al., 1999), ribose-5-phosphate and glucose-6-phosphate (Adams and Byrne, 1989). Adams and Byrne (1989) discounted glucose-1-phosphate as a likely constituent of soil phosphomonoesters because it had a distinctive chemical shift of δ 2.7 ppm, which was free of signal in soil extracts, whereas ribose-5-phosphate (δ 5.0 ppm) and glucose-6-phosphate (δ 5.3 ppm) were considered potential constituents because their chemical shifts fell within the range where signals were detected in soil extracts. The phosphodiester signal observed in the NMR spectra of soil extracts was generally assumed to be due to phospholipids and nucleic acids, based on initial assignments by Newman and Tate (1980) (Bedrock et al., 1994; Condron et al., 1985, 1990, 1996; Guggenberger et al., 1996a; Hawkes et al., 1984; Sumann et al., 1998; Zech et al., 1985, 1987). Phosphodiesters were often described as containing nucleic acids and phospholipids as one peak. However, this could also be with a peak maximum at δ
0.8 ppm (Condron et al., 1985), as a “band” of peaks with a peak maximum at δ
0.8 (Newman and Tate, 1980), as a broad peak between δ 0.5 and
1.5 ppm (Hinedi et al., 1989), or as separate peaks next to each other with peak maxima at δ 0.3 and
0.9 ppm (Zech et al., 1985). Condron et al. (1990) also suggested that two peaks within the phosphodiester region were those of teichoic acids (between δ 0.95 and 0.36 ppm), and nucleic acids and phospholipids (between δ
1.00 and
1.04 ppm) (Fig. 8). The assignment of teichoic acids was partly based on a comparison of chemical shifts within the NMR signal of soil extracts with that of the chemical shifts of teichoic acid in non-soil extracts, along with knowledge that teichoic acid was identified in some living organisms (Ezra et al., 1983; Ward, 1981). Several subsequent studies used this assignment to claim the presence of teichoic acids in their extracts (Guggenberger et al., 1996b, 2000; Rubaek et al., 1999). In general, limited information was given on the types of phospholipids and nucleic acids that would make up the phosphodiester region (Makarov et al., 2002). Most studies considered DNA to be the primary constituent of nucleic acids (Guggenberger et al., 1996b; Newman and Tate, 1980). There was recognition, however, that some phosphodiesters originally present in the soil should undergo alkaline hydrolysis and thus be present in the phosphomonoester region of a NMR spectrum (Tate and Newman, 1982). Tate and Newman (1982) also recognized that RNA would rapidly hydrolyze under alkaline conditions and would thus not be expected to be present in the phosphodiester region.

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27

Fig. 8 A solution 31P nuclear magnetic resonance (NMR) spectrum of the orthophosphate, phosphomonoester and phosphodiester region from a Luvisol soil under native boreal forest. The values are chemical shifts and were attributed by the authors to orthophosphate (δ 5.32–5.38 ppm), phosphomonoesters (δ 3.61–4.66 ppm), teichoic acids (δ 0.95–0.36 ppm), and phosphodiesters (nucleic acids and phospholipids) (δ
1.00 to
1.04 ppm). The figure was taken and adapted from Condron, L.M., Frossard, E., Tiessen, H., Newman, R.H., Stewart, J.W.B., 1990. Chemical nature of organic phosphorus in cultivated and uncultivated soils under different environmental conditions. J. Soil Sci. 41, 41–50. Copyright (1990), with permission from Wiley.

Makarov et al. (2002) later used solution 31P NMR spectroscopy to identify the chemical nature of phosphodiesters in 0.1 M NaOH extracts from the Oa (organic) horizon of a Cambisol in Austria and an Ah (mineral) horizon of a Chernozem in Russia, which included duplicate samples that were treated with a non-polar solvent (methanol-chloroform) in order to remove phospholipids from the soil. The authors noted that the NMR spectrum of the phosphodiester region for the untreated soil was similar to that of the treated soil. Furthermore, no organic P was detected in the methanolchloroform extracts of the mineral soil, whereas there was 40 mg kg
1 of organic P (3% of extractable organic P) in the methanol-chloroform extracts of the organic soil. In the latter, there were two prominent peaks present in the phosphodiester region at δ 1.5 and 0.6 ppm. The authors concluded that the dominate NMR signal in the phosphodiester region at around δ 0 ppm was exclusively that of DNA, whereas signals slightly downfield from DNA were likely to be that of phospholipids and teichoic acids, again based on indirect measures (Condron et al., 1990). Quantitative evaluation of the chemical nature of SOP was generally limited to broad classes of organic P, rather than individual compounds. A global compilation of all quantitative data on the chemical nature of organic P in soil extracts by the end of Era 4 is presented in Table 4.

Table 4 A global compilation of all quantitative data on soil extracts using solution 31P NMR spectroscopy during Era 4 (1980s to early 2000s), which covered soils from across 16 countries and 26 published studies (total of 204 samples).

Locationa

Australia

Number of samples

Total organic P (mg kg21)

Phosphomonoesters (% of organic P)

Range

Range

Average

Range

Average

Range

Average

Average

Phosphodiesters (% of organic P)

Phosphonates (% of organic P)

16–55

34

66–82

72

18–32

26

0–4

2

Brazil

12

19–90

55

90–100

95

0–10

5

0–0

0

Canada

18

90–568

224

46–100

85

0–54

15

0–1

0

Denmark

3

76–137

102

72–78

74

16–24

20

4–7

6

England

25

92–782

395

54–96

79

3–47

17

0–23

5

Germany

11

69–457

252

50–85

73

12–44

21

0–11

5

Mexico

5

75–98

81

76–85

81

15–24

19

0–0

0

Netherlands

9

122–279

177

87–100

98

0–13

2

0–0

0

20

88–566

267

74–100

90

0–24

9

0–12

1

6

23–52

34

71–85

77

15–29

23

0–0

0

New Zealand Republic of Liberia Russia

18

244–1726

638

56–95

81

3–36

16

0–11

3

4

111–395

280

60–98

76

2–40

24

0–0

0

Spain

5

51–277

161

47–87

71

13–53

29

0–0

0

Tanzania

5

44–75

60

80–88

84

12–20

16

0–0

0

Thailand

24

10–347

144

21–94

69

6–79

30

0–9

1

United States

33

21–669

175

33–100

81

0–67

19

0–5

0

Overall average

13

72–411

193

61–92

80

7–37

18

0–5

2

Scotland b

a

Citations for each location include: Australia (Adams and Byrne, 1989), Brazil (Cardoso et al., 2003), Canada (Cade-Menun and Preston, 1996; Condron et al., 1990; Zhang et al., 1999), Denmark (Guggenberger et al., 1996a), England (Hawkes et al., 1984; McDowell, 2003; Turner et al., 2003b), Germany (Guggenberger et al., 1996a; Zech et al., 1987), Mexico (Zech et al., 1985), Netherlands (Koopmans et al., 2003), New Zealand (Condron et al., 1985; Condron et al., 1996; Newman and Tate, 1980; Tate and Newman, 1982), Republic of Liberia (Forster and Zech, 1993), Russia (Makarov, 1998; Makarov et al., 1995), Scotland (Bedrock et al., 1994), Spain (Gil-Sotres et al., 1990), Tanzania (Solomon and Lehmann, 2000), Thailand (M€ oller et al., 2000), United States (Dai et al., 1996; Robinson et al., 1998; Turner et al., 2003a). b In the study of Gil-Sotres et al. (1990), values for phosphonates could not be obtained as they were reported along with polyphosphates and unidentified compounds as “Others.”

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29

This included a dataset of 204 samples across 16 countries. Concentrations of total pools of SOP ranged from 72 to 411 mg kg
1 (on average 193 mg kg
1) across all sites. The contribution of broad classes of organic P was found to decrease in the order phosphomonoesters > phosphodiesters > phosphonates, which across all sites comprised on average 80%, 18% and 2% of the total organic P in soil, respectively. During this era, several studies investigated the chemical nature of organic P in the humic acid fraction extracted from soil (i.e., organic P soluble in alkali but insoluble in acid) (Bedrock et al., 1995; Makarov, 1997; Makarov et al., 1996). Ogner (1983) collected samples from four soils that were defined as “humus” soils, and prepared their associated humic acids. The humic acid fractions contained between 660 and 2390 mg total P kg
1, and were of large MW (>12,000 Da) due to exclusion of small MW molecules via membrane dialysis. The author reported the solution 31 P NMR spectra on their humic acid extracts was visually similar to those of soil extracts as reported by Newman and Tate (1980). In particular, the NMR spectra of humic acids generally contained phosphonates, a diversity of phosphomonoesters, phosphodiesters, and a small amount of orthophosphate (13–16% of total P). Bedrock et al. (1994) also collected samples from three soils that were defined as “peat” soils and one sample that was defined as a “mineral” soil, from which humic acids were then prepared. The humic acid fraction contained between 1250 and 13,300 mg total P kg
1. Solution 31 P NMR spectra revealed the presence of phosphonates, a diversity of phosphomonoesters (37–67% of total P), phosphodiesters, pyrophosphate, and some orthophosphate (16–30% of total P). While phosphomonoesters were the dominant class of organic P, the presence of myo-IP6 could not be confirmed. Guggenberger et al. (1996b) investigated the humic and fulvic acid fractions (i.e., organic P soluble in alkali and acid, respectively) from the 0 to 10 cm soil layer (A horizon) of four Oxisols under tropical pasture. Concentrations of total P in the humic acids ranged from 23 to 70 mg P kg
1, and in the fulvic acids from 4 to 31 mg P kg
1. Solution 31P NMR spectra of the humic acid fraction revealed the presence of phosphonates, phosphomonoesters (44–59% of total P), phosphodiesters, pyrophosphate, and some orthophosphate (3–6% of total P); spectra of the fulvic acids also contained these forms of P, except phosphodiesters. Overall, the chemical nature of organic P in the humic acid fraction appeared to be similar to that in extracts of whole soils (Fig. 9). Two main approaches were used during Era 4 to investigate the processes relating to “unresolved” pools of organic P. The first approach was based on

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Timothy I. McLaren et al.

Fig. 9 A solution 31P nuclear magnetic resonance (NMR) spectra on a (A) whole extract and on a (B) humic acid fraction of a soil sample from what was defined as a “mineral” soil. Reprinted from Figs. 2D and 3D of Bedrock, C.N., Cheshire, M.V., Chudek, J.A., Goodman, B.A., Shand, C.A., 1994. Use of 31P-NMR to study the forms of phosphorus in peat soils. Sci. Total Environ. 152, 1–8 with minimal alteration. Copyright (1994), with permission from Elsevier.

abiotic pathway reactions similar to that originally proposed by Auten (1923) (Brannon and Sommers, 1985a,b). In summary, Brannon and Sommers (1985a) proposed a pathway for the existence of P in humic polymers via the phosphorylation of organic compounds with a free amine (–NH2) group, which could covalently bond with aromatic rings within the humic polymer via a nucleophilic addition reaction. The authors synthesized model humic polymers, which were characterized, and then compared with humic acids extracted from a silt loam soil. Concentrations of P were 2540 and 9420 mg kg
1 in the model humic polymers that were synthesized with phosphoserine and phosphoethanolamine, respectively. Gel chromatography revealed the absorbance pattern and distribution of P was slightly different between the humic acids from the model humic polymer with phosphoethanolamine and that of the soil. However, the infrared absorption spectra of the model humic polymer with phosphoethanolamine was similar to that reported for soil humic substances in the literature. The second approach, conceptually proposed by Borie and Zunino (1983), involved various abiotic and biotic reactions that produced “humic P” associated with organic matter. This included: (1) reaction of allophanic sites with orthophosphate and “humic acids,” which are primarily via the microbial biomass; (2) the production of organic substances from the microbial biomass that undergo degradation and directly contribute to humic P to soil; and (3) P that becomes associated with humus via complexation with metals.

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31

Throughout Era 4, many studies used solution 31P NMR spectroscopy to investigate the chemical nature of SOP across different ecosystems, soil types, land use and agricultural management systems. In particular, the chemical nature of SOP was compared: (1) along soil chronosequence gradients, including along climosequences and toposequences (Makarov et al., 1996; Tate and Newman, 1982); (2) between fertilized treatments of long-term field experiments (Condron et al., 1985; Hawkes et al., 1984); (3) between land-uses (Condron et al., 1990); and (4) across climatic zones (Sumann et al., 1998). Overall, these studies led to an improved understanding of the role of broad classes of SOP (i.e., phosphomonoesters, phosphodiesters and phosphonates) within the organic P cycle. Similarly to Era 3, many studies continued to further our understanding of the abiotic processes responsible for stabilizing IP in soil (Celi et al., 1999, 2000; Ognalaga et al., 1994; Shang et al., 1990, 1992), although a point of difference was that toward the end of Era 4, there was also increasing interest on how phytate could undergo biological utilization (Richardson, 2001). This primarily involved understanding the enzymatic action of microbes and plants (through root exudates) to hydrolyze soil phytate (Adams and Pate, 1992; Findenegg and Nelemans, 1993). Lastly, different extraction techniques were being investigated to improve the spectral quality of solution 31P NMR spectra on soil extracts. Cade-Menun and Preston (1996) investigated four different chemical extraction techniques (1:20 soil to solution ratio) using: (1) Chelex (1:6 soil to chelex ratio) and 0.25 M NaOH; (2) Chelex (1:6 soil to chelex ratio) and H2O; (3) 0.25 M NaOH and 0.05 M EDTA; and (4) 0.25 M NaOH. The NaOH-EDTA extraction technique, as modified from the method of Bowman and Moir (1993), was found to have the best extraction efficiency for soil P, while not compromising resolution of extracted organic P signals in subsequent 31P NMR spectra (Fig. 10). Most importantly, these results showed that the choice of chemical extractant could influence the distribution of P as determined by NMR spectroscopy. The NaOHEDTA extraction technique and subsequent preparation for NMR analysis has since been modified by various researchers, but essentially remains the predominant approach for extracting SOP prior to its characterization using solution 31P NMR spectroscopy (Cade-Menun and Liu, 2014; Doolette and Smernik, 2011).

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Fig. 10 Solution 31P NMR spectra of soil extracts from a forest floor that had been prepared with four different extraction techniques (1:20 soil to solution ratio) using: (1) Chelex (1:6 soil to chelex ratio) and 0.25 M NaOH; (2) Chelex (1:6 soil to chelex ratio) and H2O; (3) 0.25 M NaOH and 0.05 M EDTA, and; (4) 0.25 M NaOH. The figure was taken and adapted from Cade-Menun, B.J., Preston, C.M., 1996. A comparison of soil extraction procedures for 31P NMR spectroscopy. Soil Sci. 161, 770–785. Copyright (1996), with permission from Wolters Kluwer Health.

5.2 Discussion The advent of solution 31P NMR spectroscopy to soil extracts resulted in several fundamental changes to the understanding of the chemical nature of SOP (Newman and Tate, 1980). Instead of approaching the task of SOP characterization by isolating specific organic P compounds (e.g., IP) as in previous eras, the whole pool of extractable organic P could be characterized using a relatively simple and rapid extraction and simultaneous analysis. The chemical nature of organic P was subsequently described on

ARTICLE IN PRESS Chemical nature of soil organic phosphorus

33

the basis of its linkage to C, i.e., phosphomonoesters, phosphodiesters and phosphonates (Newman and Tate, 1980) because this is what 31P chemical shift is most sensitive to. Phosphomonoesters were found to be the predominant form of organic P detected, comprising on average 80% of the total organic P in soil (Table 4). Low spectral quality remained a significant problem in many studies which prevented accurate identification and quantification of specific organic P compounds. The NMR spectra of soil extracts commonly comprised a broad peak and a few poorly resolved sharp peaks. Therefore, little information was provided on the presence (and dynamics) of specific organic P compounds during this era, including IP, for which so much detailed information had already been obtained. While soil type, choice of chemical extractant, pH of the solution, and NMR experimental setup was shown to have a major influence on this (Cade-Menun and Preston, 1996; Crouse et al., 2000), spectral quality was often also compromised by the use of low field strength (and low-resolution) NMR spectrometers (Preston, 2015; Schulthess, 2011). There were two main approaches to peak identification used in this era to gain insight on the possible forms of P present in the phosphomonoester region: (1) the “spiking” approach and (2) using reported values of chemical shifts of organic P obtained in non-soil chemical matrices. Both of these techniques have limitations that can lead to erroneous results. The original study of Newman and Tate (1980) showed clear evidence for the presence of myo-inositol hexaphosphate, based on the presence of four NMR signals that increased with the addition of the authentic compound (Newman and Tate, 1980). However, these authors also erroneously identified a peak as choline phosphate using the same approach, which was later shown to be scyllo-inositol hexaphosphate (Doolette et al., 2009; Turner and Richardson, 2004). It should be noted though that myo-inositol hexaphosphate is well suited to identification through spiking among P species commonly detected in soil extracts because its 31P NMR spectrum consists of four peaks, due to the presence of four chemically distinct P atoms in each molecule. Most other common P species contain P in only one unique molecular environment, hence their NMR spectra comprises a single peak and therefore are more prone to mis-identification. Most studies in this era interpreted the phosphomonoester regions as containing myo-inositol hexaphosphate, sugar phosphates, mononucleotides and choline phosphate (Adams and Byrne, 1989; Condron et al., 1985; Dai et al., 1996; Guggenberger et al., 1996a,b; Makarov, 1998; Zech et al., 1985, 1987).

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This also included the alkaline hydrolysis products of some phosphodiesters (e.g., RNA and phospholipids), which would subsequently be present in the phosphomonoester region (Gil-Sotres et al., 1990; Tate and Newman, 1982). However, there was limited evidence to exclusively support the presence of any particular form of organic P due to poor spectral quality, and a lack of studies incorporating validation of these compounds using other techniques, especially the chromatographic techniques developed during the previous era. A similar approach was used for the phosphodiester region, also with issues of spectral quality and peak assignment. The assignments of phospholipid and teichoic acid remain inconclusive and should be treated with caution, whereas that of DNA appears to be more substantiated because of its known presence and relative abundance in all living organisms. Nevertheless, quantitative assessments of these compounds using NMR have not been compared with alternative techniques. While the study of Makarov et al. (2002) was limited, the use of organic solvents indicate that alkaline solvents are unlikely to extract phospholipids at concentrations typically considered adequate for NMR analysis. Pools of phospholipids in soil would in any case still represent a small fraction of the total pool of organic P, which is consistent with previous eras (Dormaar, 1968; Kowalenko and McKercher, 1970; Makarov et al., 2002). Furthermore, Makarov et al. (2002) also showed that NMR signals downfield from DNA could contain the intermediary products of RNA as it undergoes alkaline hydrolysis. However, there is still insufficient information to reliably assign signals in the phosphodiester region, apart from DNA at around δ 0 to
1.5 ppm. Understanding the chemical nature of “unresolved” pools of SOP also remained limited in this era, which primarily focused only on the application of solution 31P NMR spectroscopy to humic acid extracts prepared from soil. In general, the results revealed that the organic P in the humic acid fraction was: (i) of large MW; (ii) dominated by a broad signal in the phosphomonoester region; and (iii) visually similar to spectra of “whole” soil extracts (Bedrock et al., 1994; Ogner, 1983). These results would appear to be consistent with the identification of large pools of non-IP in large MW organic matter in Era 3. While caution is needed when interpreting these results due to the inherently low spectral quality at the time, they are somewhat consistent with results obtained using higher resolution NMR spectrometers in Era 5 (early 2000s to current) (He et al., 2006). Advances in the understanding of mechanisms associated with the “unresolved” pool of SOP were also limited during Era 4. Mechanisms were

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35

proposed for the synthesis of “complex” forms of organic P based on abiotic and biotic pathways (Borie and Zunino, 1983; Brannon and Sommers, 1985a), which appear to be an extension of the ideas originally proposed by Auten (1923). However, at this stage, there was insufficient evidence to support either pathway. Knowledge on the chemical nature of organic P was advanced during Era 4 compared to that of previous eras. This included: (1) a large proportion of organic P exists as phosphomonoesters (on average 80% of total organic P); (2) a large proportion of organic P could be found in humic acid fractions; and (3) that the phosphomonoester region in particular was composed of both a series of identifiable sharp peaks and a broad but poorly characterized signal. In addition, these findings are consistent with that of Era 3 in that a large proportion of the organic P was found in large MW fractions and was not specifically identified as being IP.

6. Era 5: Early 2000s to current—Spectral deconvolution fitting for characterizing soil phosphomonoesters 6.1 Main findings A comprehensive review by Turner et al. (2002) on IP in the environment demonstrated the importance of understanding its biogeochemistry in terrestrial ecosystems. A key conclusion of the review was that IP accumulated in soils to become the predominant class of identifiable organic P, but its cycling and mobilization remained poorly understood. While the review focused on the biogeochemistry of IP, it expanded on previous reviews on the chemical nature of organic P in soil (Anderson, 1975, 1980; Dalal, 1977; Harrison, 1987), and provided a renewed basis for investigating the chemical nature of SOP. Since 2003, considerable improvements in spectral resolution and sensitivity of NMR spectra have occurred through a combination of improved methodologies for extraction and sample preparation, higher field strength NMR spectrometers and techniques for improving the resolution of NMR spectra (Cade-Menun, 2005a; Cade-Menun and Liu, 2014; McLaren et al., 2015c; Schulthess, 2011). Near the beginning of this era, Turner et al. (2003d) reported detailed information on a wide range of P compounds identified using solution 31P NMR spectroscopy, including IP stereoisomers (Turner et al., 2012; Turner and Richardson, 2004), which could be used to help identify specific 31P NMR peaks in soil extracts. This included information on the degradation of P compounds under conditions used

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Timothy I. McLaren et al.

for NMR spectroscopy, which complimented previous work (CadeMenun et al., 2002; Crouse et al., 2000; Makarov et al., 2002). The “library” of NMR data on known organic P compounds provided a foundation for more detailed interpretation of spectra during Era 5. In a landmark paper, Turner et al. (2003e) published a solution 31P NMR spectrum of an extract from a soil surface under grassland in the United Kingdom, which exhibited greater spectral resolution than that observed through most of the previous era. A key difference between the earlier NMR studies and the study of Turner et al. (2003e) was the use of NaOH-EDTA, as proposed by Cade-Menun and Preston (1996), combined with a higher field strength (600 MHz) NMR spectrometer; compared to a 80 MHz NMR reported in Newman and Tate (1980) or 250 MHz NMR reported in Cade-Menun and Preston (1996). The increased spectral resolution as presented in Turner et al. (2003e) enabled improved detection of P species and their quantification, particularly for myo- and scyllo-IP6. Based on improved spectral resolution, Turner and co-workers (e.g., Turner et al., 2003e) applied procedures for spectral deconvolution fitting to report concentrations of myo-IP6 in 29 soils from the 0 to 10 cm layer under pastures in the United Kingdom. Based on identified peaks, Turner et al. (2003e) used a Lorentzian function fitting procedure (Bruker WinNMR program) to quantify organic P compounds in NMR spectra (Fig. 11). After identifying the chemical shifts of the peaks, peak areas were calculated from the peak maxima to the baseline of the spectra. The net peak areas of myo-IP6 were then summed, expressed as a proportion of the total net peak area in the NMR spectra, and multiplied by the total concentration of P in the soil extract (based on digestion and subsequent determination of orthophosphate in the digested samples). Supporting evidence for the efficacy of the spectral deconvolution fitting procedure was provided using known concentrations of authentic organic P compounds in pure 1 M NaOH solution. This involved adding a known concentration of myo-IP6 (and other known organic P compounds) in NaOH solution and calculating their recovery, which ranged from 93% to 114% of the added P. Based on this approach, concentrations of myo-IP6 ranging from 26 to 189 mg P kg
1 were calculated across all soils, corresponding to between 11% and 35% of total organic P in the soil extracts. This study provided a simpler and more rapid procedure for identifying and quantifying myo-IP6 and other organic P compounds in soil extracts than the previous eras. The deconvolution fitting procedure proposed by Turner et al. (2003e)

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Fig. 11 Solution 31P nuclear magnetic resonance (NMR) spectrum of the orthophosphate and phosphomonoester region on a soil extract from a pasture soil in the United Kingdom. The shaded regions indicate the peaks associated with the six phosphate (P1–P6) groups of myo-inositol hexakisphosphate, and how their net peak area was quantified using spectral deconvolution fitting procedures. The figure was taken and adapted from Turner, B.L., Mahieu, N., Condron, L.M., 2003e. Quantification of myo-inositol hexakisphosphate in alkaline soil extracts by solution 31P NMR spectroscopy and spectral deconvolution. Soil Sci. 168, 469–478. Copyright (2003), with permission from Wolters Kluwer Health.

was extensively used thereafter in a range of other NMR studies to report the chemical nature of SOP in various ecosystems and under different management. Turner et al. (2003c) reported a more detailed study on the identity and diversity of organic P in soil extracts based on a series of spiking experiments and assignments. The presence of up to 24 species of P across 29 soils under pasture was suggested, although some peaks remained “unidentified” or attributed to “mononucleotides” (Table 5). Furthermore, Turner et al. (2003e) introduced nomenclature on the structural configurations of IP6 in soil extracts using solution 31P NMR spectroscopy, which was later expanded on by Turner and Richardson (2004) and Turner et al. (2012) (Table 6). The authors hypothesized that the relative dominance of structural configurations in soil extracts may be influenced by factors other than solution pH, such as metal counter ions. This study also reported that the extracting solution may influence the configuration of the identified compound.

ARTICLE IN PRESS Table 5 Peak assignments of various P compounds in NaOH-EDTA extracts across 29 soils under pasture collected from the United Kingdom using solution 31P NMR spectroscopy. Chemical shift (ppm) Mean

Range

Assignment

20.72

20.56–21.78

2-Aminoethyl phosphonic acid

19.15

18.99–19.27

Phosphonolipid

7.43

7.19–7.58

Unknown, but possibly aromatic diesters

6.78

6.72–6.88

R-(
)-1,10 -binaphthyl-2,20 -

6.55

6.48–6.63

diyl hydrogen phosphate

6.24

6.18–6.34

Inorganic orthophosphate

5.90

5.84–6.00

Inositol hexakisphosphatea

5.68

5.61–5.77

Unidentified

5.39

5.30–5.47

Glucose-6-phosphate

5.24

5.18–5.29

Unidentified

5.12

5.04–5.16

Phosphatidic acid

4.95

4.90–5.00

Inositol hexakisphosphatea

4.85

4.80–4.89

β-Glycerophosphate

4.72

4.67–4.78

Mononucleotides/ethanolamine phosphate

4.57

4.53–4.65

Inositol hexakisphosphatea

4.45

4.38–4.49

Inositol hexakisphosphatea

4.26

4.20–4.35

Mononucleotide

4.10

4.06–4.14

Choline phosphate

3.64

3.52–3.82

Unidentified

3.27

3.13–3.43

Glucose-1-phosphate

1.75

Phosphatidyl ethanolamine

1.57

Phosphatidyl serine

0.78

Phosphatidyl choline


0.26


0.15 to
0.36

DNA


4.05


3.92 to
4.15

Pyrophosphate


3.63,
9.61

Adenosine diphosphate


20.18

Inorganic polyphosphate

a

myo-Inositol hexakisphosphate gives four signals in the ratio 1:2:2:1, corresponding to the positions of the P groups on the inositol ring (Turner et al., 2003d). Reprinted from Turner, B.L., Mahieu, N., Condron, L.M., 2003c. The phosphorus composition of temperate pasture soils determined by NaOH–EDTA extraction and solution 31P NMR spectroscopy. Org. Geochem. 34, 1199–1210 with minimal alteration: table 3. Copyright (2003), with permission from Elsevier.

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Chemical nature of soil organic phosphorus

Table 6 Chemical shifts (ppm) and structural configurations of four stereoisomers of inositol hexakisphosphate (IP6) in soil extracts (pH > 13). Phosphate Chemical shift Stereoisomer Configuration positions Orientation (δ ppm)

scyllo-IP6

6 ax

C1, C2, C3, C4, ax C5, C6

4.03

myo-IP6

1-eq/5-ax

C5

ax

4.38

C1, C3

ax

4.53

C4, C6

ax

4.89

C2

eq

5.79

C2, C5

ax

4.58

C1, C3, C4, C6 eq

6.67

C1, C3, C4, C6 ax

4.93

C2, C5

eq

5.17

C1, C6

ax

4.33

C2, C5

eq

5.66

C3, C4

eq

6.90

C3, C4

ax

4.66

C2, C5

ax

5.08

C1, C6

eq

6.48

neo-IP6

4-eq/2-ax

2-eq/4-ax

D-chiro-IP6

4-eq/2-ax

2-eq/4-ax

The terms “ax” refers to axial and “eq” refers to equatorial. Adapted with permission from Turner, B.L., Cheesman, A.W., Godage, H.Y., Riley, A.M., Potter, B.V.L., 2012. Determination of neo- and D-chiro-inositol hexakisphosphate in soils by solution 31 P NMR spectroscopy. Environ. Sci. Technol. 46, 4994–5002: table 2: https://pubs.acs.org/doi/abs/ 10.1021/es204446z. Copyright (2012) American Chemical Society: Further permissions related to the material excerpted should be directed to the American Chemical Society.

A further development by Turner and co-workers was to use hypobromite oxidation, based on earlier approaches (Irving and Cosgrove, 1981; Suzumura and Kamatani, 1993), of soil extracts prior to NMR analysis to remove P associated with organic matter and spiking to confirm the presence of three isomers of IP6, other than phytate: scyllo-IP6 (Turner and Richardson, 2004), and D-chiro- and neo-IP6 (Turner et al., 2012). The presence of unidentified signals (downfield from orthophosphate) after bromination, suggested the presence of IP instead of a previous assignment of aromatic phosphodiesters (Bedrock et al., 1994); these signals were later confirmed to be those of D-chiro- and neo-IP6 (Turner et al., 2012).

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In addition, the peak initially assigned as choline phosphate by Newman and Tate (1980) was re-assigned to scyllo-IP6. Overall, the relative abundance of the identified IP6 stereoisomers were generally consistent with the previous findings of Irving and Cosgrove (1982), and on average comprised 56% myo, 33% scyllo, 6% neo, and 5% D-chiro forms. Lastly, while lower order IPs have not been identified in soil extracts with NMR, Turner et al. (2012) suggested their presence based on the premise that the remaining NMR signal not attributable to IP6 in soil extracts after treatment with bromination would be due to IP that resist hypobromite oxidation. The authors suggested concentrations of lower order IPs comprised on average 20% of the total organic P across three soils. Cade-Menun and co-workers used an alternative approach to that of Turner et al. (2003e) to partition signal in NMR spectra of soil extracts. In addition to initially fitting peaks for the myo and scyllo stereoisomers of IP6, Hill and Cade-Menun (2009) further partitioned the phosphomonoester and phosphodiester regions (Fig. 12). Since then, Cade-Menun and

Fig. 12 Solution 31P nuclear magnetic resonance (NMR) spectrum of the orthophosphate, phosphomonoester, phosphodiester and pyrophosphate regions on an extract from a cropped soil in the United States. The dark shaded regions indicate the peaks associated with myo-inositol hexakisphosphate, and the light shaded peak is that of scyllo-inositol hexakisphosphate. The authors also partition their spectra into three regions of phosphomonoesters, four regions of phosphodiesters (teichoic acid (TA), phospholipids (Lipids), DNA, and “other” phosphodiesters), orthophosphate and pyrophosphate. The figure was taken and adapted from Hill, J.E., Cade-Menun, B.J., 2009. Phosphorus-31 nuclear magnetic resonance spectroscopy transect study of poultry operations on the Delmarva Peninsula. J. Environ. Qual. 38, 130–138. Copyright (2009), with permission from the Journal of Environmental Quality.

ARTICLE IN PRESS Chemical nature of soil organic phosphorus

41

co-workers have reported the presence of a number of specific compounds and a variety of “unknown” peaks, see Table 7 (CadeMenun et al., 2017; Jiang et al., 2017; Liu et al., 2015; Schneider et al., 2016). Understanding the chemical nature of organic P in soil extracts using solution 31P NMR spectroscopy requires appropriate preparation procedures in order to obtain “high quality” NMR spectra (CadeMenun, 2005a; Turner et al., 2005a). The work of Cade-Menun and co-workers has highlighted the importance of the chemical matrix used to extract organic P from soil, and its subsequent preparation for NMR analysis. Several review articles on the procedures for measurement of SOP using solution 31P NMR spectroscopy have been published (Cade-Menun, 2005a,b; Cade-Menun and Liu, 2014; Condron et al., 2005; Turner et al., 2005a). The use of NMR spectrometers with greater resolution, combined with other techniques, has also provided some advances in characterizing P associated with the soil humic acid fraction. For example, He et al. (2006) analyzed humic and fulvic acids obtained from the Elliot “standard soil” from the International Humic Substances Society (IHSS, 2013). The authors showed that the humic acid fraction contained orthophosphate (21%) and phosphomonoesters (79%), while the fulvic acid fraction contained the same species but in different proportions: orthophosphate (40%) and phosphomonoesters (60%). While the NMR spectra appeared to lack any sharp peaks, the organic P was primarily of large MW (i.e., >3 kDa) and a significant proportion (>50%) was potentially bioavailable based on the release of P as effected by ultraviolet radiation and enzymatic hydrolysis. Later, He et al. (2011) determined the amount and chemical nature of organic P in two humic acid fractions that were considered to differ in their stability (Olk et al., 1995): “mobile” humic acid (MHA) and “recalcitrant” calcium humate (CaHA) fractions, which were obtained from three fertilized surface soils (0–15 cm, silt loam) under continuous corn (Zea mays L.). Most notably, the spectra of both fractions contained a variety of sharp peaks, including those due to scyllo-IP6 and the hydrolysis products of phospholipids. An important development in the characterization of organic P speciation through Era 5 was the introduction of two-dimensional (2D) NMR techniques. Of particular value have been heteronuclear correlation experiments, including 1H–31P heteronuclear single quantum coherence (HSQC). In this approach, the two dimensions represent the 31P and the 1 H spectra, and “cross peaks” arise for pairs of “coupled” nuclei, for which there is transfer of magnetization from one nucleus (1H) to another (31P)

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Timothy I. McLaren et al.

Table 7 Peak assignments of various P compounds in NaOH-EDTA extracts of soils from a long-term field experiment in Canada using solution 31P NMR spectroscopy. Phosphorus form or Category compound class Chemical shift (δ ppm)

Inorganic P Orthophosphate

Organic P

6.00

Pyrophosphate


4.27

Polyphosphates


4.06,
4.63,
8.68,
10.33,
11.41,
15.87,
22.89,
26.89,
29.91

Phosphonates

24.41, 20.29, 17.50, 13.61, 12.40, 10.66, 9.55, 8.76

Phosphomonoesters myo-IHP

5.58, 4.67, 4.15, 4.09

scyllo-IHP

3.76

neo-IHP

6.39, 4.25

chiro-IHP 4e/2a

6.56, 5.23, 3.94

chiro-IHP 2e/4a

6.17, 4.75, 4.34

Glucose 6-phosphate 5.15 α-Glycerophosphate 4.83 β-Glycerophosphate 4.62 Mononucleotides

4.57, 4.53, 4.46, 4.38, 4.24

Choline phosphate

3.84

Unknown

5.02

Monoester 1

6.90, 6.26

Monoester 2

5.86, 5.76, 5.44, 5.36, 4.94, 4.02

Monoester 3

3.65, 3.53, 3.35, 3.05

Phosphodiesters Diester 1

1.61, 1.00, 0.80, 0.03,
0.28

DNA


0.70,
0.83

Diester 2


1.73,
2.43

The term ‘IHP’ refers to ‘inositol hexakisphosphate’. Adapted with permission from Liu, J., Hu, Y., Yang, J., Abdi, D., Cade-Menun, B.J., 2015. Investigation of soil legacy phosphorus transformation in long-term agricultural fields using sequential fractionation, P K-edge XANES and solution P NMR spectroscopy. Environ. Sci. Technol. 49, 168–176: table S5 in the Supporting Information. Copyright (2015) American Chemical Society: Further permissions related to the material excerpted should be directed to the American Chemical Society.

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43

(Keeler, 2010). In practice, this usually requires the nuclei be within three covalent bonds, e.g., between the 1H nuclei on one C and the 31P nuclei of a phosphate bound to the same C. A key advantage of 2D NMR in analysis of SOP in soil extracts is that it allows compounds with very similar 31P chemical shifts to be resolved based on the chemical shift of their coupled 1 H nuclei. Vestergren et al. (2012) first applied 1H–31P HSQC NMR spectroscopy to soil extracts to characterize organic P in organic soil horizons from boreal forests (Fig. 13) and this approach was extended to the analysis of more soils in subsequent papers (Vincent et al., 2012, 2013). The solution 31P NMR spectra obtained on these samples gave broad NMR signals. However, 2D NMR revealed new information on the chemical nature of organic P,

Fig. 13 A solution 1H–31P heteronuclear single quantum coherence (HSQC) nuclear magnetic resonance (NMR) spectrum displaying the δ 6.0 to 4.0 region (31P NMR spectra) and δ 4.9 to 3.6 region (1H NMR spectra) on a Na2S treated NaOH-EDTA extract of the “organic” horizon of a humus soil in boreal forest. Peak assignments are: A ¼ β-glycerophosphate, B ¼ α-glycerophosphate, combined cross peaks of C, C0 , C00 and C000 ¼ myo-inositol hexakisphosphate, D ¼ guanosine 20 monophosphate, G ¼ uridine 20 monophosphate, H ¼ uridine 30 monophosphate, T ¼ scyllo-inositol hexakisphosphate, and cross peaks X2, X3 and X4 ¼ “unidentified” compounds. Where there is signal overlap in the 31P dimension, dashed lines provide a link to signals that have been resolved in the 1H dimension. Adapted with permission from Fig. 2B of Vestergren, J., Vincent, A.G., Jansson, M., Persson, P., Ilstedt, U., Gro€bner, G., Giesler, R., Schleucher, J., 2012. High-resolution characterization of organic phosphorus in soil extracts using 2D 1H–31P NMR correlation spectroscopy. Environ. Sci. Technol. 46, 3950–3956. Copyright (2012) American Chemical Society.

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Timothy I. McLaren et al.

particularly in the phosphomonoester region. Using this approach, the authors showed that an NMR signal attributed to the P at the axial C1/C3 position (i.e., at δ 4.65 ppm) and C5 position (i.e., at δ 4.55 ppm) of myo-IP6, also contained signal due to uridine 20 monophosphate and guanosine 20 monophosphate (and possibly an “unknown” phosphomonoester). It was revealed that the NMR signal of β-glycerophosphate overlaps with that of uridine 30 monophosphate. Nucleotides were not only detected but could be identified as isomers with phosphate at the 20 and 30 positions (i.e., guanosine 20 monophosphate, and uridine 20 and 30 monophosphate), suggesting they are due to the alkaline hydrolysis of RNA, since the vast majority of mononucleotides present in living cells are present as the 50 phosphates (Vestergren et al., 2012). In addition, Vincent et al. (2012) suggested the phosphomonoester region did not contain a broad signal due to a transverse relaxation (T1ρ) experiment of an organic horizon extract, which revealed peak linewidths ranging between 9 and 24 Hz. Vincent et al. (2013) published the peak assignments of an extensive array of compounds found in an organic horizon based on these NMR approaches (Table 8). Despite improvements in spectral resolution in this era, Smernik and Dougherty (2007) demonstrated the need to be cautious when assigning peaks, as even myo-IP6 (with its distinctive set of peaks) could potentially be mis-identified. The authors demonstrated via phytate spiking that the presence of two prominent peaks approximately 0.3 ppm apart in the phosphomonoester region (around δ 4.9 and 4.6 ppm) could not always be assumed to be the P peaks of the axial C4/C6 and C1/C3 phosphates of myo-IP6, as it was later shown that these peaks were often those due to α- and β-glycerophosphate (Doolette et al., 2009). Smernik and co-workers also suggested the quantification of myo-IP6 in soil extracts using NMR and the spectral deconvolution fitting procedure of Turner et al. (2003e) may need to be revised to better account for the underlying presence of a broad signal in this spectral region. Smernik and Dougherty, 2007 proposed an alternative means to quantify myo-IP6 in soil extracts based on a spiking and spectral subtraction approach. They reported that concentrations of myo-IP6 were small (<5% of SOP) across four Australian soils, and that the dominant signal for organic P appeared to be a broad underlying feature in the phosphomonoester region. Dougherty et al. (2007) provided further evidence of the underlying broad signal by using a hydrofluoric acid pre-treatment of the soil, which solubilizes most clay minerals, including the iron and aluminium oxides that phytate has a

ARTICLE IN PRESS 45

Chemical nature of soil organic phosphorus

Table 8 Peak assignments of various P compounds in NaOH-EDTA extracts based on solution 31P NMR spectroscopy and solution 31P-1H (correlation) NMR spectroscopy. Chemical shift (δ ppm—31P) (δ ppm—1H) Class

Label Compound

A

β-Glycerophosphate

4.95

4.07

Monoester

B

α-Glycerophosphate

5.27

3.68, 3.75

Monoester

C

myo-inositol hexakisphosphate

4.65 (C4, C6)

4.52

Monoester

4.55 (C5)

4.27

5.03 (C1, C3)

4.48

5.97 (C2)

4.48

C0 00

C

C000 0

D

Guanosine 2 monophosphate

4.33

4.89

Monoester

E

Guanosine 30 monophosphate

4.90

4.58

Monoester

F

2-Aminoethyl phosphonic acid

18.98

1.59, 3.28

Phosphonate

G

Uridine 20 monophosphate

4.62

4.54

Monoester

0

H

Uridine 3 monophosphate

4.85

4.38

Monoester

I

Choline phosphate

4.25

4.08

Monoester

J

Ethanolamine phosphate

4.91

3.69

Monoester

K

α-D-glucose-1-phosphate

3.39

5.34

Monoester

L

Guanosine 50 monophosphate

4.78

3.84, 3.89

Monoester

M

DNA


0.5, to 0.0 3.80–4.20

Diester

4.62–4.98 0

N

Adenosine 5 monophosphate

4.77

3.85, 3.90

Monoester

O

Phosphatidyl choline

0.69

4.23, 3.79

Diester

P

Phosphatidyl ethanolamine

1.79

3.81

Diester

4.93

Monoester

0

0

0

Q

Adenosine 2 , 5 diphosphate 4.54 (2 )

R

4.81 (50 )

3.88, 3.94

17.39

1.80

S

MDPA (reference)

Phosphonate Continued

ARTICLE IN PRESS 46

Timothy I. McLaren et al.

Table 8 Peak assignments of various P compounds in NaOH-EDTA extracts based on solution 31P NMR spectroscopy and solution 31P-1H (correlation) NMR spectroscopy.—Cont’d Chemical shift Label Compound

(δ ppm—31P) (δ ppm—1H) Class

T

scyllo-inositol hexakisphosphate

4.14

4.47

Monoester

U

MPA (reference)

21.30

1.00

Phosphonate

A1

Orthophosphate

6.29

Orthophosphate

A4

Polyphosphate (end-chain)


3.70 to
4.27

Polyphosphate

A5

Pyrophosphate

A6

Polyphosphate (mid-chain groups)


18.5 to
21.0

X

Unknown

5.75

3.39

Monoester

X2

Unknown

4.46

4.93

Monoester

X3

Unknown

4.84

4.37

Monoester

X4

Unknown

4.84

4.34

Monoester

Y1

Unknown

4.00

6.65

Aromatic

Y2

Unknown

3.32

2.33

Monoester

Y3

Unknown

0.88

2.68–2.88

Diester

Y4

Unknown

4.94

4.53

Monoester

Pyrophosphate Polyphosphate

This includes P compounds as reference materials and those found in soils collected from the “organic” horizon along the V€asterbotten chronosequence (90 to 7800 years old) of boreal forests in northern Sweden. The table was taken and adapted from Vincent, A.G., Vestergren, J., Gr€ obner, G., Persson, P., Schleucher, J., Giesler, R., 2013. Soil organic phosphorus transformations in a boreal forest chronosequence. Plant Soil 367, 149–162. Copyright (2013), with permission from Springer.

strong affinity for. Alkaline (NaOH-EDTA) extracts of soils that had undergone hydrofluoric acid pre-treatment contained no myo-IP6 peaks but retained a broad signal in the monoester region, including through the chemical shift region where the peaks for myo-IP6 had been (Fig. 14). Consequently, B€ unemann et al. (2008b) revised the deconvolution fitting procedure of Turner et al. (2003e) to include a broad underlying feature (Fig. 15). The accuracy of quantification of myo-IP6 using this method was later tested by Doolette et al. (2010) using two approaches: (1) the aforementioned spiking and subtraction method of Smernik and Dougherty, 2007;

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47

Fig. 14 Solution 31P nuclear magnetic resonance (NMR) spectra of the orthophosphate and phosphomonoester region on a soil extract without hydrofluoric acid pre-treatment (A) and after hydrofluoric acid pre-treatment (B). The “†” symbols identify the peak locations arising from myo-inositol hexaphosphate. The soil was collected from the surface horizon of a permanent pasture in southeastern Australia. The figure was taken and adapted from Dougherty, W.J., Smernik, R.J., B€ unemann, E.K., Chittleborough, D.J., 2007. On the use of hydrofluoric acid pretreatment of soils for phosphorus-31 nuclear magnetic resonance analyses. Soil Sci. Soc. Am. J. 71, 1111–1118. Copyright (2007), with permission from the Soil Science Society of America Journal.

Fig. 15 A simplified view of the spectral deconvolution fitting procedure used to partition the NMR signal within the orthophosphate and phosphomonoester region (δ 6.2–3.0 ppm) based on the method of B€ unemann et al. (2008b). In the first step, a broad signal (shaded area) is fitted that lies underneath the sharp signal, and then after it is taken into account and removed, in the second step, the sharp signals are fitted. In this example, only the sharp signals of myo-inositol hexakisphosphate (myo-IP6) in the second step have been highlighted (shaded area) for the purposes of a model.

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Timothy I. McLaren et al.

and (2) mathematically by adding a known amount of myo-IP6 to soil extracts and calculating its concentration using the deconvolution method of Turner et al. (2003e) (i.e., without a broad peak underneath the sharp peaks) and also by the deconvolution method of B€ unemann et al. (2008b) (i.e., with a broad peak present in addition to the sharp peaks). The authors found that concentrations of the added myo-IP6 were more accurately determined by the subtraction method or when a broad peak was included in the deconvolution fitting procedure, compared to when a broad peak was not accounted for. Doolette et al. (2011a) was the first to describe in detail the deconvolution fitting procedure that includes a broad peak and termed the organic P responsible for this broad NMR signal as “humic P.” McLaren et al. (2015b) provided further and more direct evidence for the importance of phosphomonoesters that contribute to a broad signal within NMR spectra. The chemical nature of organic P was investigated across five different soils, which had been extracted with NaOH-EDTA and separated into “small” (<10 kDa) and “large” (>10 kDa) MW fractions by ultrafiltration. Solution 31P NMR spectroscopy was carried out on the “unfractionated” soil extracts and then on the 10 kDa filtrates and 10 kDa retentates after passage through a 10 kDa filtration membrane. Organic P speciation was markedly different in the large MW (>10 kDa) fraction (Fig. 16) which was dominated by a

Fig. 16 Solution 31P NMR spectra of the orthophosphate and phosphomonoester region on (A) the original (unfractionated) soil extract, (B) 10 kDa retentate, and (C) 10 kDa filtrate of Ap horizon (0–20 cm layer) under cultivation collected in France. The vertical scale of each spectrum for the 10 kDa retentates was increased by a factor of 4 to highlight its spectral features. Adapted with permission from Fig. 2 (France) of McLaren, T.I., Smernik, R.J., McLaughlin, M.J., McBeath, T.M., Kirby, J.K., Simpson, R.J., Guppy, C.N., Doolette, A.L., Richardson, A.E., 2015b. Complex forms of soil organic phosphorus—a major component of soil phosphorus. Environ. Sci. Technol. 49, 13238–13245. Copyright (2015) American Chemical Society.

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broad signal within the phosphomonoester region, despite some “distinct” peaks also being visible. The results confirmed that deconvolution of the NMR signal from the peak maxima of sharp peaks should not proceed to the baseline of the spectra, as this would include NMR signal from other forms of organic P (i.e., the broad signal). On average, 33% of the extractable organic P was detected in MW fractions >10 kDa for these five soils (McLaren et al., 2015b). Evidence for the existence of phosphomonoesters within a broad peak using solution 31P NMR spectroscopy was further supported through enzymatic characterization. Using 10 diverse soils, Jarosch et al. (2015) used phosphatase enzymes in combination with gel filtration chromatography on NaOH-EDTA extracts to determine the MW distribution (nominal cut-off of 5 kDa) of the organic P in soil extracts. They found that the chemical nature of recognizable biomolecules as determined using solution 31P NMR spectroscopy was in general agreement with their corresponding forms as indicated using enzymatic addition assays (e.g., the amount of P soluble by nuclease and acid phosphatase was similar to that detected by 31 P NMR as phosphodiesters—DNA). Ultimately, the majority of organic P in these soils was as “enzyme-stable” (i.e., organic P remaining in solution after enzyme action), and the amount of this “enzyme-stable” SOP was consistent with the amount of P comprising the broad signal in the phosphomonoester region as determined using solution 31P NMR spectroscopy, and amount of organic P detected in large MW material (>5 kDa) using gel filtration chromatography (Fig. 17). Mechanisms that might contribute to the association of organic P with the broad signal have been further investigated by Smernik and co-workers. This included understanding the organic P composition of some of the direct sources of P-containing organic matter added to soils, i.e., plants and microorganisms. Solution 31P NMR spectra of the samples, however, only revealed the presence of sharp peaks and no broad feature (Doolette and Smernik, 2016; Noack et al., 2012; Smernik et al., 2015). Similar findings were reported by B€ unemann et al. (2008b) for soil incubation experiments using four soils (two collected from the field and two “model” soils). The authors found that the phosphomonoester region of the field soils contained two characteristic features: (1) a series of sharp peaks (e.g., δ 4.6, 4.7, 5.1 ppm); and (2) a broad feature spanning from approximately δ 3.5ppm to δ 6.5 ppm. However, the two model soils only contained the former after microbial incubation and the addition of a C source (i.e., peaks at δ 4.6, 4.7, 5.1 ppm). Consequently, B€ unemann et al. (2008b) speculated that the difference between the model

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Timothy I. McLaren et al.

Fig. 17 The MW distribution of organic P in NaOH-EDTA extracts using gel filtration chromatography for a soil under grassland in Switzerland (A), and a correlation analysis between pools of organic P in large molecular weight material (i.e., >5 kDa) and pools of enzyme stable organic P for 10 diverse soils collected from across the world under different management (B). Molybdate reactive P (MRP) and molybdate unreactive P (MUP) is generally attributed to pools of inorganic and organic P, respectively. Reprinted from Fig. 5 of Jarosch, K.A., Doolette, A.L., Smernik, R.J., Tamburini, F., Frossard, E., B€ unemann, E.K., 2015. Characterisation of soil organic phosphorus in NaOH-EDTA extracts: a comparison of 31P NMR spectroscopy and enzyme addition assays. Soil Biol. Biochem. 91, 298–309 with minimal alteration. Copyright (2015), with permission from Elsevier.

and field soils, i.e., the presence of the broad peak, is due to the synthesis of complex forms of organic P in the latter as a result of slow “humification” processes. Information on the mechanisms controlling the flux of organic P and its composition within the broad signal in soil remains limited. A global compilation of all quantitative data on the chemical nature of organic P in soil extracts by the end of Era 5 is shown in Table 9, which included a dataset of 411 samples across 18 countries. In addition to the range of identifiable compounds (Table 9), these studies highlight the significance of the “unidentified” forms of organic P in the phosphomonoester region, including that present as a broad signal, which constituted on average 45% of total organic P. Of the identifiable organic P, myo-IP6 and scyllo-IP6 constituted on average 20% and 10%, respectively, of the total organic P, α- and β-glycerophosphate (often attributed to the alkaline hydrolysis products of phospholipids) comprised on average 7% of the total SOP, whereas nucleotides (often attributed to the alkaline hydrolysis of RNA) comprised on average 4% of the total organic P in soil.

Table 9 A global compilation of all quantitative data on soil extracts using solution 31P NMR spectroscopy collected from across 18 countries and 41 published studies (total of 411 samples) up to the end of Era 5 (Early 2000s to 2017). Total organic P (mg kg-1) Phosphomonoesters (% of organic P) Number of a Range Average Unidentified myo-IP6 scyllo-IP6 neo-IP6 D-chiro-IP6 Unidentified IPb α-Glyc β-Glyc ChoPd G6Pe Nucleotides samples Location

76

27–533

119

67.1

5.0

2.7

0.0

0.0

0.2

4.7

4.5

0.0

0.0

3.2

Brazil

10

81–561

262

1.9

30.7

12.1

0.0

0.0

0.0

19.3

21.3

0.0

1.7

6.9

Canada

20

48–295

182

29.2

30.1

6.5

0.6

2.9

0.0

2.7

5.7

0.9

0.9

10.3

China

5

74–98

87

22.9

26.1

6.1

0.0

0.0

0.0

6.6

7.3

2.5

3.8

7.7

Colombia

1

135–135 135

45.2

17.8

3.7

0.0

0.0

0.0

5.9

5.9

0.0

0.0

5.2

England

41

129–1083 442

54.1

12.3

10.8

0.0

0.0

0.4

0.0

0.0

0.0

0.0

0.0

Falkland Islands

3

543–773 665

37.0

27.3

16.2

3.0

2.2

0.0

0.0

0.0

0.0

0.0

0.0

France

1

392–392 392

70.2

11.0

6.9

0.0

0.0

0.0

2.2

2.2

0.0

0.0

4.1

Germany

6

74–370

145

34.4

21.5

11.6

0.4

2.0

0.0

5.4

3.3

0.0

0.0

1.5

Ireland

4

188–592 414

49.6

33.7

14.5

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

Madagascar 7

43–393

135

55.6

12.1

7.9

0.0

0.0

12.4

0.7

0.7

0.0

0.0

1.7

New Zealand

103

5–991

317

55.3

21.2

4.2

0.3

0.1

0.0

0.0

0.0

0.0

0.0

0.0

Northern Ireland

13

406–486 443

28.0

16.3

10.0

3.7

6.7

0.0

3.2

6.4

2.3

0.0

11.8

Scotland

14

37–446

79.7

0.0

18.6

0.0

0.0

1.1

0.0

0.0

0.0

0.0

0.0

247

ARTICLE IN PRESS

Australia

Continued

Table 9 A global compilation of all quantitative data on soil extracts using solution 31P NMR spectroscopy collected from across 18 countries and 41 published studies (total of 411 samples) up to the end of Era 5 (Early 2000s to 2017).—Cont’d Total organic P (mg kg-1) Phosphomonoesters (% of organic P) Number of a samples Location Range Average Unidentified myo-IP6 scyllo-IP6 neo-IP6 D-chiro-IP6 Unidentified IPb α-Glyc β-Glyc ChoPd G6Pe Nucleotides

Sweden

95–651

220

44.1

23.7

7.1

0.0

0.0

0.2

3.9

0.0

0.0

0.0

9.0

Switzerland 10

4–1012

236

41.0

13.6

6.2

0.0

0.0

0.0

1.9

1.9

0.0

0.0

4.8

United States

36

18–1899 231

48.3

23.3

8.3

0.7

2.9

0.0

0.8

1.5

0.9

0.8

2.7

Wales

14

344–658 483

37.2

28.4

18.9

0.0

0.0

0.6

1.1

2.2

0.0

0.0

2.0

Overall average

23

147–632 286

44.5

19.7

9.6

0.5

0.9

0.8

3.2

3.5

0.4

0.4

3.9

a

Citations for each location include: Australia (Doolette et al., 2017, 2010, 2011a,b; Jarosch et al., 2015; McLaren et al., 2015b,c, 2017; Moata et al., 2016), Brazil (Deiss et al., 2016; Gatiboni et al., 2013), Canada (Cade-Menun et al., 2010; Liu et al., 2015; Schneider et al., 2016), China (Liu et al., 2013), Colombia ( Jarosch et al., 2015), England (Turner et al. (2003e), including Turner et al. (2003c) and Turner et al. (2005b), and Stutter et al. (2015)), Falkland Islands (Turner et al., 2012), France (McLaren et al., 2015b), Germany ( Jarosch et al., 2015; McLaren et al., 2015b; Missong et al., 2016), Ireland (Murphy et al., 2009), Madagascar ( Jarosch et al., 2015; Turner, 2006), New Zealand (Chen et al., 2004; McDowell et al., 2005, 2007; McDowell and Stewart, 2006; Turner et al., 2007a, 2014), Northern Island (Cade-Menun et al., 2017), Scotland (Stutter et al., 2015), Sweden (Ahlgren et al., 2013; McLaren et al., 2015b; Vincent et al., 2012; Vincent et al., 2013), Switzerland (Annaheim et al., 2015; Jarosch et al., 2015), United States (Cheesman et al., 2014; Dou et al., 2009; Giles et al., 2015; He et al., 2008; Hill and Cade-Menun, 2009; McLaren et al., 2015b; Weyers et al., 2016), and Wales (Turner et al. (2003e), including Turner et al. (2003c) and Turner et al. (2005b), Stutter et al. (2015), and Ebuele et al. (2016)). b In some studies, pools of inositol hexakisphosphates were reported together (e.g., neo-IP6 and D-chiro-IP6). In these cases, concentrations were reported as “Unidentified IP.” c In some studies, the net peak of area α- and β-Glycerophosphate was reported together. Therefore, in these situations, we divided the value equally between the two glycerophosphate isomers as an approximation. d The term ‘ChoP’ refers to choline phosphate. e The term ‘G6P’ refers to glucose-6-phosphate.

ARTICLE IN PRESS

47

ARTICLE IN PRESS Chemical nature of soil organic phosphorus

53

6.2 Discussion The application of solution 31P NMR spectroscopy to soil extracts remained the dominant approach used in Era 5 to understand the chemical nature of organic P in soil. Improvements in spectral quality and processing meant more information could be gained on the chemical composition of organic P in soil extracts. The most significant advance was the use of spectral deconvolution, especially when applied to the phosphomonoester region for the identification of organic P compounds as sharp peaks (Turner et al., 2003e) as well as “unresolved” or complex forms of organic P as a broad signal (B€ unemann et al., 2008b). During Era 5, a major development was that more information could be gained on pools of “unresolved” SOP as identified as a broad signal in the phosphomonoester region of 31P NMR spectra of NaOH-EDTA extracts. This interpretation was based on (1) visual-based measures of NMR spectra from a range of soil types, in particular those of naturally occurring soils with low or absent concentrations of IP (McLaren et al., 2014; Smernik and Dougherty, 2007) and on the humic acid fraction (He et al., 2006); (2) preferential removal of myo-IP6 on HF-treatment from soils that still reveals NMR signal in the phosphomonoester region (Dougherty et al., 2007); (3) mathematical based approaches that require inclusion of a broad signal for complete recovery of the added P spike (Doolette et al., 2010, 2011a); (4) enzyme-based measures that revealed a large proportion of organic P is non-enzyme labile and which showed the concentration of this non-enzyme labile organic P correlated with the concentration of organic P attributable to the broad signal in 31P NMR spectra ( Jarosch et al., 2015); and (5) MW measures that reveal 31P NMR spectra of large MW organic P were dominated by a broad signal ( Jarosch et al., 2015; McLaren et al., 2015b). The significance of this was that the identification and properties of organic P as the broad signal was consistent with nonNMR measures in previous eras, which were considered to be that of organic P not attributed to IP, phospholipids and nucleic acids (Eras 2 and 3), was contained in large MW material (Era 3), comprised of phosphomonoester linkages (Era 4), and containing similar spectral features to humic acids (Bedrock et al., 1994). Recognition of the broad signal in NMR spectra and differences in opinion of its relative quantitative contribution to SOP remain (Doolette and Smernik, 2015). Nonetheless, based on a global compilation of all NMR data for Era 5, the proportion of SOP that is not attributed to identifiable

ARTICLE IN PRESS 54

Timothy I. McLaren et al.

forms of organic P when averaged across studies ranged from 2% to 80% with an average of 45%. Studies at the lower end of this range tended to apply deconvolution fitting procedures that do not include a broad signal, and consequently a greater contribution of the SOP was attributed to myo-IP6 (e.g., see Deiss et al. (2016) and Cade-Menun et al. (2010)). Consequently, a limitation of this approach is that the reported pool of SOP characterized by NMR does not include a large pool of “unresolved” organic P, which does not reconcile with other studies using non-NMR techniques that indicate the substantial presence of large MW organic P (Dalal, 1977; K€ ogel-Knabner and Rumpel, 2018). Another major development was the detection of clearly observable sharp peaks within the phosphomonoester region, which could be attributed to specific compounds of organic P (Doolette et al., 2009; Turner et al., 2012; Vestergren et al., 2012), and then quantified using spectral deconvolution fitting (B€ unemann et al., 2008b; Hill and Cade-Menun, 2009; Turner et al., 2003e). Furthermore, the application of (2D) 1H–31P HSQC NMR spectroscopy to soil extracts enabled the detection of multiple P compounds despite the appearance of a singular peak in the solution (1D) 31P NMR spectra (Vestergren et al., 2012). Era 5 resulted in major advance in the identification and quantification of a range of specific compounds of organic P compared to that obtained in Era 4. The most consistently detected (and identifiable) forms of organic P in soil extracts using NMR included: (1) myo-, scyllo-, D-chiro- and neo-IP6 (Turner and Richardson, 2004; Turner et al., 2012; Vestergren et al., 2012); (2) α- and β-glycerophosphate as the alkaline hydrolysis products of phospholipids (Doolette et al., 2009; Vestergren et al., 2012); and (3) some RNA mononucleotides as the alkaline hydrolysis products of RNA (Doolette et al., 2009; Smernik et al., 2015; Turner et al., 2003d; Vestergren et al., 2012). Some studies also claim the presence of sugar phosphates and choline phosphate (e.g., see Cade-Menun et al. (2015) and Giles et al. (2015)), but evidence for their detection by NMR in soil extracts remains limited (Vestergren et al., 2012; Vincent et al., 2012). Despite this, non-NMR techniques have identified them at very low concentrations (Waithaisong et al., 2015). Lastly, improvements in spectral quality have also revealed the presence of several sharp peaks within the phosphomonoester region whose identity remains unknown (B€ unemann et al., 2008b; Cade-Menun et al., 2017; Vestergren et al., 2012). Importantly, the number of “identified” organic P compounds identified in spectra and their concentrations are influenced to a large extent by the chosen method of deconvolution fitting; either the method of Turner et al. (2003e) or modifications thereof (Hill and Cade-Menun, 2009;

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Vincent et al., 2012), which involve fitting a series of sharp peaks from the peak maxima to the baseline of the spectra; or the method of B€ unemann et al. (2008b) or modifications thereof (McLaren et al., 2015c), which involves fitting a broad signal and then a series of sharp peaks that coincide with the spectral range covered by the broad signal. Work that has focused on assessing the efficacy of deconvolution fitting procedures in soil extracts has demonstrated that NMR signals exhibiting sharp peaks in the phosphomonoester region (e.g., myo-IP6) are likely to be overestimated if the presence of an underlying broad signal is not considered (Doolette and Smernik, 2015; Doolette et al., 2010, 2011a). A major difference between Eras 3 and 5 was the level of detail provided on the diversity of IP. This is because the chromatographic methods used in studies from Era 3 were better able to distinguish lower order IP from IP6. Similarly, studies that claim to identify RNA mononucleotides in the phosphomonoester region of their NMR spectra often report the presence of up to 5 peaks (Turner et al., 2006; Vincent et al., 2010). Under alkaline conditions, RNA hydrolyzes to produce a mixture of 20 - and 30 -mononucleotides, i.e., a total of eight compounds. However, the peaks for these species fall in a crowded region of the 31P NMR spectrum and so there is a degree of overlap, both within the RNA monomers and with β-glycerophosphate (Smernik et al., 2015), and also likely with other monoester compounds, including IP. It is thus difficult to accurately and reliably assign signal to RNA-derived organic P by NMR. Clearly, more work is needed on identifying and quantifying IP and RNA mononucleotides in soil extracts. The use of selective extractants (e.g., hypobromite oxidation) and 2D NMR techniques are likely to provide further insight. Limited progress has occurred in our understanding of the composition of phosphonates and phosphodiesters in soils using NMR spectroscopy between Eras 4 and 5. The most consistently identified forms include: (1) DNA (McLaren et al., 2015b; Wang et al., 2017); (2) intact phospholipid (Makarov et al., 2002; Vestergren et al., 2012); and (3) 2-aminoethyl phosphonate (Newman and Tate, 1980; Vestergren et al., 2012). Again, more information is needed on their composition and abundance in soil. The most common pre-treatment prior to solution 31P NMR spectroscopy remains extraction with NaOH-EDTA (Cade-Menun and Liu, 2014). Two potential limitations of this are: (1) incomplete extraction of SOP; and (2) creation of “artifacts” through alkaline hydrolysis that might lead to an erroneous interpretation of a subsequent NMR spectrum. In terms of the first consideration, quantitative data collected across Era 5 revealed the

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technique recovered on average 66% of the total P in soil, although much lower recoveries were recorded for some soils, particularly alkaline soils (e.g., McLaren et al. (2014)). Unfortunately, there is no reliable method for specifically assessing the recovery of organic P (as opposed to total P) using this technique (Turner et al., 2005a). Therefore, it is not possible to ascertain whether the non-extracted P is exclusively inorganic P, or whether particular forms of organic P are not being extracted. Importantly, it remains unclear whether the variable proportion that is not extracted is similar in composition to the portion that is extracted, or represents a further and almost completely uncharacterized pool of complex organic P in large MW material. In terms of the second consideration (the potential for alkaline hydrolysis), it is well established that IPs are stable in NaOH-EDTA solution, while both phospholipids and RNA are partially hydrolyzed in NaOH-EDTA (Doolette et al., 2009; Smernik et al., 2015), but in these cases, the reaction products (α- and β-glycerophosphate, and 20 - and 30 mononucleotides, respectively) are still organic P compounds and are specific to the class of organic P compound from which they were formed. However, more broadly, there is evidence that alkaline solutions can influence the composition of soil organic C (Hayes et al., 1975; Wilson and Goh, 1983; Worobey and Webster, 1981). Therefore, we suggest that techniques used to understand the effect of alkaline solutions on the chemical nature of soil organic matter should also be incorporated for that of SOP (Drosos et al., 2017b; Piccolo, 2002; Preston, 2015; Simpson et al., 2011). It is important to note that some attempt has been made on the non-destructive characterization of SOP (Kizewski et al., 2011; Kruse et al., 2015; Vogel et al., 2016). Some of the more prominent methods include: P K-edge X-ray absorption near-edge structure (XANES) spectroscopy (e.g., Kruse and Leinweber, 2008; Andersson et al., 2016), solid-state 31 P NMR spectroscopy (e.g., Shand et al., 1999; Dougherty et al., 2005), and deep ultraviolet (DUV) Raman microspectroscopy (e.g., Vogel et al., 2017). However, to date none of these studies have been demonstrated to be a reliable method for identifying the chemical nature of SOP. Nevertheless, their future development and potential use as additional tools to understand the chemical nature of SOP is encouraged.

7. Evolving view on the structure, formation and cycling of soil organic phosphorus The existence of organic P in soil has now been established for over 100 years (Potter and Benton, 1916; Potter and Snyder, 1918), with its

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chemical nature largely being assumed to reflect that of organic P found in living organisms (Condron et al., 2005; Darch et al., 2014). This suggests that the synthesis of organic P from inorganic P only occurs within living organisms (including soil microorganisms and various order of eukaryotes; plants and animals), and organic P is thus released to the soil upon cell death, where it may be stabilized by the soil constituents and accumulate over time (Fig. 18) (Anderson, 1980; Condron et al., 2005; Dalal, 1977; Nash et al., 2014; Turner et al., 2002). The four main classes of identifiable organic P in soils are: (1) IP (Cosgrove, 1963a; Turner et al., 2012); (2) phospholipids (Doolette et al., 2009; Dormaar, 1970; Hance and Anderson, 1963); (3) nucleic acids (DNA and RNA) (Adams et al., 1954; Makarov et al., 2002; Wrenshall and Dyer, 1941); and (4) phosphonates which have generally been detected in trace amounts (Hawkes et al., 1984; Newman and Tate, 1980; Tate and Newman, 1982). Inositol phosphates have a key role in P storage and are dominant in some plant parts, most notably seeds, but also appear to be present in a wide range of other plant parts where P status is high

Fig. 18 A simplified model indicating the pathways (black arrows) in a soil-plant system that are known to transfer P between inorganic and organic forms, and alternative pathways (gray arrows) that may contribute to the formation of “unknown” forms of organic P that occur in soil. “Unknown” process 1 is related to degradation of recognizable biomolecules and then their polymerization, whereas “unknown” process 2 to the phosphorylation of carbon molecules that become or have already been polymerized.

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(Doolette and Smernik, 2016; Noack et al., 2012). Conversely, nucleic acids and phospholipids are ubiquitous to every living cell and are the dominant form of organic P compounds in most living cells (B€ unemann et al., 2008a,b; Noack et al., 2012), but undergo rapid microbial turnover in soil and, in general, seem to comprise <10% of the total pool of SOP. Inositol phosphates are widely considered the predominant constituent of a stable pool of SOP (Condron et al., 2005; Darch et al., 2014; Turner et al., 2002); the most prominent IP compound being the myo stereoisomer of IP6 (phytate). Indeed, myo-IP6 is one of the most well-studied forms of SOP across all eras (Cosgrove, 1977; Giles et al., 2011; Harrison, 1987; Turner et al., 2002, 2007b). Concentrations of IPs in soil vary considerably, but some studies suggest that they may comprise over 80% of the total organic P in specific soils (Ahlgren et al., 2013; Cheesman et al., 2014; McDowell and Stewart, 2006). More generally, across most soils, IP account for less than one-third of the total organic P when assessed across multiple techniques (Doolette et al., 2011b; Jarosch et al., 2015; McKercher and Anderson, 1968b; Omotoso and Wild, 1970; Steward and Tate, 1971) and may also either be absent or found at negligible concentrations (Cosgrove, 1963a; Doolette et al., 2011b; Fisher et al., 2014; McLaren et al., 2014; Turner et al., 2006; Turner and Engelbrecht, 2011; Vincent et al., 2010). Clearly, IPs are not able to explain the existence of, or account for the processes responsible for, a stable pool of organic P in all soils. The view that IPs are an important component of SOP was empirically established during Eras 2 and 3, with the isolation and analysis of IP using chromatographic techniques (Anderson, 1964; Bower, 1945; Cosgrove, 1963a; Dyer et al., 1940) and then in later eras through solution 31P NMR spectroscopy. The initial focus on IP is unsurprising given the targeted nature of the methodologies at the time being directed at their characterization, and other methods for nucleic acids and phospholipids revealing that they were minor fractions of the total pool of SOP (Adams et al., 1954; Hance and Anderson, 1963; Kowalenko and McKercher, 1970). However, even in these early eras, it was recognized that the composition of a large proportion of the organic P remained “unresolved” (Anderson, 1980; Dalal, 1977). Widespread focus on IP as being the predominant component of the phosphomonoesters in soil remained through Era 4, with phosphomonoesters being shown to represent the majority of SOP. Most notably, during Era 5, IP6 received much attention and as such were viewed as the most important “identifiable” component of SOP (Condron et al., 2005; Darch et al., 2014; Giles et al., 2011; Nash et al., 2014;

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Turner et al., 2002). Again, this is somewhat unsurprising given the nature of the analytical and deconvolution fitting procedures being focused on identifiable peaks with IP6 being the most predominant (Doolette and Smernik, 2015). While our understanding of soil IP has advanced considerably in recent decades, especially with regard to IP6, an over emphasis on them has perhaps contributed to a lesser understanding on the diversity and abundance of “other” forms of SOP. Quantitative evidence collected across all eras shows that for most soils, the structure and composition of the majority of organic P remains “unresolved.” The complex and elusive nature of this pool of organic P hinders current research progress and future effort is required to better understand its chemical composition, and to identify the mechanisms controlling its presence and turnover in soil. Knowledge on the chemical nature of “unresolved” SOP remains limited. However, some aspects of its chemical nature have been established: (1) it cannot be directly attributed to recognizable biomolecules, i.e., the organic P in this pool does not directly reflect that found in living organisms (B€ unemann et al., 2008a,b; Noack et al., 2012); (2) it is contained in large MW fractions, which suggests it is present in large compounds or polymers that are innately complex ( Jarosch et al., 2015; McLaren et al., 2015b; Steward and Tate, 1971); (3) it is intimately associated with soil organic matter due to correlations between SOP, C and N (Kirkby et al., 2011; Moata et al., 2016; Swift and Posner, 1972); (4) it is largely associated with the humic acid fraction of soil (Bedrock et al., 1994; He et al., 2006); and (5) it appears to be predominantly comprised of phosphomonoester linkages, based on its 31P NMR chemical shift (He et al., 2006; McLaren et al., 2015b). Similarly, there is a paucity of information on the mechanisms that contribute to the formation, stabilization and turnover of “unresolved” SOP. Long-term studies suggest that concentrations of phosphomonoesters as the broad signal remained unchanged after 62 years under a variety of P fertilization and cropping strategies (Annaheim et al., 2015). Significant increase in the magnitude of the broad peak, but without change in its percentage contribution to total SOP, was, however, observed in a permanent grassland after 13 years and up to 100 years of P fertilization compared to an unfertilized control (McLaren et al., 2017; Schefe et al., 2015). Lastly, there is some recent evidence to suggest that the proportion of phosphomonoesters as the broad signal relative to total pools of SOP are higher in warm and dry environments compared to that in cold and wet environments (Doolette et al., 2017).

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The presence of both small and specific organic P molecules and complex forms of organic P in soil and the apparent absence of the latter in living organisms (B€ unemann et al., 2008a; Noack et al., 2012), suggests that current models on the dynamics of SOP in terrestrial ecosystems require further revision. As shown in Fig. 18, we propose a model that incorporates both a direct pathway and two additional processes that might regulate the flux of organic P soil systems. Several alternative processes have been hypothesized for the formation of “unknown” forms of SOP, which include: (1) the phosphorylation of organic compounds via abiotic processes (Auten, 1923; Brannon and Sommers, 1985a), a possibility supported by evidence in non-soil systems that phosphomonoesters can be formed via abiotic processes through reaction of phosphate with either a –OH or –NH group (Kamerlin et al., 2013); (2) the association of orthophosphate and organic compounds via metal bridges (Borie and Zunino, 1983; Gerke, 2010); and (3) the polymerization of an array of organic P degradation products derived from microbial and plant residue turnover, including their interaction with soil minerals. Understanding the chemical nature of “unresolved” pools of SOP also needs to be integrated with knowledge on the chemical nature and cycling of C, N and sulfur within soil organic matter (K€ ogel-Knabner and Rumpel, 2018). A common view is that soil organic matter is largely derived from microbial detritus, rather than via direct deposition of recalcitrant plant materials (Liang and Balser, 2010; Miltner et al., 2012), and that it is comprised of a relatively small but heterogeneous array of biomolecules that are either intact or at various stages of degradation (Kelleher and Simpson, 2006; Lehmann et al., 2008). A further view is that soil organic matter is largely comprised of humic substances, which are themselves a heterogeneous array of biomolecules that become part of large supramolecular structures via selfassembly (Nuzzo et al., 2017; Piccolo and Spiteller, 2003). The mechanisms involved appear to involve the stabilization of microbial and plant biopolymers in soil via organo-mineral associations, hydrophobic, van der Waals and electrostatic interactions, metal bridges and ligand exchange (Drosos et al., 2017a,b; L€ utzow et al., 2006; Mitchell et al., 2018). Historically, the chemical composition of soil organic matter has often focused on organic C, oxygen, hydrogen and N (K€ ogel-Knabner and Rumpel, 2018; Piccolo, 2002; Preston, 2015; Simpson et al., 2011), whereas the P component has received less attention. It is likely that similar techniques used to understand the complex nature of soil organic matter may also help elucidate the chemical nature of the “unresolved” pools of SOP.

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Acknowledgments The authors would like to thank Dr. Christopher Guppy, Dr. Astrid Oberson, Dr. Federica Tamburini, and Dr. Rene Verel for helpful discussions relating to various aspects covered in this review.

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