Analysis and speciation of organic phosphorus in

environmental matrices: Development of methods to improve 31P NMR analysis

Johan Vestergren

Doctoral Thesis, Department of Chemistry

Umeå University, 2014

Responsible publisher under swedish law: the Dean of the Faculty of Science and

Technology

This work is protected by the Swedish Copyright Legislation (Act 1960:729)

ISBN: 978-91-7601-132-4

Electronic version available at http://umu.diva-portal.org/

Tryck/Printed by: Service Center KBC

Umeå, Sweden, 2014

“I have wished to see chemistry applied to domestic objects, to malting, for

instance, brewing, making cider, to fermentation and distillation generally,

to the making of bread, butter, cheese, soap……..”

- Thomas Jefferson

i

Table of Contents

Table of Contents i Abstract iii List of Abbreviations iv List of Publications v Introduction 1

Phosphorus in nature 1 Phosphorus limitation of ecosystems’ plant productivity 4 Phosphorus: Its way to bioavailability 6 31P NMR Spectroscopy: an innovative tool for studying the speciation of organic

phosphorus and the need for advanced techniques to study P-species. 8 Liquid NMR in soil science 10 Solid state 31P MAS NMR on intact soils 13

Aim of thesis 15 Materials and Methods 16

NMR spectroscopy 16 The NMR signal 16 The chemical shift 18 Indirect spin-spin couplings 18 Line broadening contributing factors 19 The 2D 1H-31P HSQC NMR experiment 21 Inductively coupled plasma optical emission spectrometry (ICP-OES) 22 X-ray fluorescence spectroscopy (XRF) 23 Other analytical methods used in this thesis 24 Site descriptions 24 Sampling and general preparations 25 Preparations for solid state NMR 26 Sample preparations for liquid NMR by conventional extraction 26 Sulfide treatment 27 NMR experiments 28 SIMCA analysis 29

Summary of Papers 30 Paper I; High Resolution Characterization of Organic Phosphorus in Soil Extracts

using 2D 1H-31P NMR Correlation Spectroscopy 30 Paper II; Soil organic phosphorus transformations in a boreal forest

chronosequence 32 Paper III; Effect of Trees on Forms of Soil Phosphorus in an Agroforestry

Parkland in Burkina Faso (manuscript) 33 Paper IV; Changes in organic P-composition in boreal forest humus soils: the

role of iron and aluminium 34 Conclusions and future work 36

ii

Acknowledgements 38 References 42

iii

Abstract

Phosphorus (P) is an essential element for life on our planet. It is central in

numerous biochemical processes in terrestrial and aqueous ecosystems

including food production; and it is the primary growth-limiting nutrient in

some of the world’s biomes. The main source of P for use as agricultural

fertilizer is mining of non-renewable mineral phosphate. In terrestrial

ecosystems the main source is soil P, where the largest fraction is organic P,

composed of many species with widely differing properties. This fraction

controls the utilization of P by plants and microorganisms and influences

ecosystem development and productivity. However, there is only scarce

knowledge about the molecular composition of the organic P pool, about the

processes controlling its bioavailability, and about its changes as soils

develop. Therefore, the aim of this thesis was to develop robust solution- and

solid-state 31P nuclear magnetic resonance spectroscopy (NMR) methods to

provide molecular information about speciation of the organic P pool, and to

study its dynamics in boreal and tropical soils. By studying humus soils of a

groundwater recharge/discharge productivity gradient in a Fennoscandian

boreal forest by solution- and solid-state NMR, it was found that P speciation

changed with productivity. In particular, the level of orthophosphate diesters

decreased with increasing productivity while mono-esters such as inositol

phosphates increased. Because the use of solution NMR on conventional

NaOH/EDTA extracts of soils was limited due to severe line broadening

caused by the presence of paramagnetic metal ions, a new extraction method

was developed and validated. Based on the removal of these paramagnetic

impurities by sulfide precipitation, a dramatic decrease in NMR linewidths

was obtained, allowing for the first time to apply modern multi-dimensional

solution NMR techniques to soil extracts. Identification of individual soil P-

species, and tracking changes in the organic P pools during soil development

provided information for connecting P-speciation to bioavailability and

ecosystem properties. Using this NMR approach we studied the

transformation of organic P in humus soils along a chronosequence (7800

years) in Northern Sweden. While total P varied little, the composition of the

soil P pool changed particularly among young sites, where also the largest

shift in the composition of the plant community and of soil microorganisms

was observed. Very old soils, such as found Africa, are thought to strongly

adsorb P, limiting plant productivity. I used NMR to study the effect of

scattered agroforestry trees on P speciation in two semi-arid tropical

woodlands with different soil mineralogy (Burkina Faso). While the total P

concentration was low, under the tree canopies higher amounts of P and

higher diversity of P-species were found, presumably reflecting higher

microbial activity.

iv

List of Abbreviations

ATP Adenosine Triphosphate

CSA Chemical Shift Anisotropy

D2O Deuterium oxide or “heavy water”

DNA Deoxyribonucleic Acid

EA-IRMS Automated Elemental Analyzer Isotope Ratio Mass Spec.

EDTA Ethylenediaminetetraacetic Acid

FID Free Induction Decay

HSQC Heteronuclear Single Quantum Coherence Spectroscopy

HR High Resolution

I Magnetic Spin

ICP-OES Inductively Coupled Plasma-Optical Emission Spectroscopy

LRC Land Rising Coast (translation from Swedish)

MAS Magic Angle Spinning

MDPA Methylene Diphosphonic Acid

MPA Methyl Phosphonic Acid

N Nitrogen

NaOH Sodium Hydroxide

Na2S Sodium Sulfide

NMR Nuclear Magnetic Resonance Spectroscopy

P or 31P Phosphorus

PC Phosphatidylcholine

ppb Parts per billion (µg/L)

ppm Parts per million

RF Radio Frequency

RNA Ribonucleic Acid

T1 Longitudinal Relaxation or Spin-Lattice Relaxation

T2 Transversal Relaxation or Spin-Spin Relaxation

XRF X-Ray Fluorescence Spectroscopy

v

List of Publications

This thesis is based on the following papers:

I: Vestergren J., Vincent AG., Ilstedt U., Jansson M., Persson P., Gröbner G.,

Giesler R., Schleucher J. “High Resolution Characterization of Organic

Phosphorus in Soil Extracts using 2D 1H, 31P NMR Correlation”. Environ. Sci.

Technol, 46, 3950−3956, (2012)

II: Vincent AG., Vestergren J., Gröbner G., Persson P., Schleucher J., Giesler R.

“Soil Organic Phosphorus Transformations in a Boreal Forest

Chronosequence” Plant Soil 367:149–162 (2013)

III: Vestergren J., Vincent A.G., Ouattara K., Persson P., Gröbner G., Giesler R.,

Schleucher J., Ilstedt U. “Agroforest Parkland Influence on Phosphorus

Speciation – A 31P NMR Study”, Manuscript

IV: Vincent AG., Schleucher J., Gröbner G., Vestergren J., Persson P., Jansson

M., Giesler R. “Changes in Organic Phosphorus Composition in Boreal Forest

Humus Soils: the Role of Iron and Aluminium”, Biogeochemistry, 108, 1-3, 485-499 (2011)

Additional papers which are not included in the thesis:

Vestergren J., Sundman A., Persson P., Giesler R., Schleucher J., Jonsson

A., Jansson M., Gröbner G. ”Absorption Dynamics of Phosphate Species on

Ion Exchange Resin – A 31P NMR study”. Manuscript

Kruse J., Abraham M., Amelung W., Baum C., Bol R., Kühn O., Lewandowski

H., Niederberger J., Oelmann Y., Rüger C., Santner J., Siebers M., Siebers N.,

Spohn M., Vestergren J., Vogts A., Leinweber P. “: Advanced Methods in

Soil Phosphorus Research: A Review”. submitted to Journal of Plant

Nutrition and Soil Science

Vincent A., Ilstedt U., Vestergren J., Giesler R., Persson P., Gröbner G,

Schleucher J. “Phosphorus in Forest Soils - Disentangling the Chemistry of an

Essential Nutrient” Fakta Skog (Forest Facts) 4 (2013)

1

Introduction

Phosphorus in nature

Life on this planet is in all its aspects in need of phosphorus (P). Life would

not persist without this unique chemical element. P is well known for its

essential role in biochemical processes, its use as fertilizer in agricultural

production, and its notorious role as pollutant in aqueous environments [1,

2]. In general, organic P is the dominant form of P found in soils, but also in

stream, lake and sea water. Organic P is present as numerous P-species and

controls primary production in many terrestrial and aquatic ecosystems, as

found in tropical areas as well as in boreal locations [3]. In these systems

restricted availability of P occurs not only through low P-levels but in many

cases through P-fixation; a process which includes physical adsorption of P

to organic and mineral material and precipitation into chemically bound

complexes of P [4-8].

2

Figure 1: Change of phosphorus use over time. Demand for rock phosphate

has increased dramatically while importance of manure has declined.

Adapted from [9]; based on concept from [10])

Therefore, in many parts of the world, food and forestry production is

severely restricted by the limited availability of P for the plants. Due to this

fact large-scale mining and manufacturing of P-fertilizer from rock

phosphate has increased dramatically in the last century (s. Figure 1) to

improve productivity, especially for food crops. Since human demand for P

will grow due to an increase in population and climate warming [11] a

dramatic increase in demand for P-fertilizer is expected in the future.

However, due to the current non-sustainable P-management and the

expected decline in P-mining, the reduced availability of P-fertilizer is

predicted to become an imminent problem in the next 50 years (Figure

2)[9].

3

Figure 2: The request for Phosphorus in the future (picture by Magnus

Bard; published in “Dagens Nyheter” 2011-12-31). Translation of text in

picture: “Yes, there is one more thing. Phosphorus - we are starting to run

out of that, too.”

P-availability will therefore impose fundamental constraints on production

capacity affecting resource availability, and even social and economic

development. Politics and science therefore have to provide solutions for

avoiding P-scarcity but also P-pollution, to achieve a sustainable P-economy

for ensuring food security as well as a stable society and economy in the

future. Establishing a sustainable P-management and sustainable

agriculture, demands profound knowledge of the ability of plants and

microorganisms to utilize P; a process which is an important part of the

global P-dynamics and with wide ranging implications for ecosystems [7].

However, at present, there is a severe lack in knowledge about the molecular

processes controlling the speciation and reactivity of the organic P-pool and

its resulting P-bioavailability in ecosystems [12]. Therefore, identification of

the different organic P-species is crucial in tracking their individual origin

and understanding the species-dependent dynamics and bioavailability of P

in soils, and ultimately, to ascertain their role in ecosystems and their effects

on soil fertility. Related questions like these need to be answered, in

particular because little is known about the response of P biogeochemistry to

global environmental changes, and the possibility that P fertilizer for

4

agriculture may soon become scarce [13, 14]. Therefore, the general aim of

this thesis was to carry out a multidisciplinary study to provide not only new

basic knowledge about the speciation and dynamics of organic P-species in

boreal and tropical ecosystems but also to generate the necessary

methodology and knowledge for the sustainable management.

Figure 3: Sustainability and phosphorus dynamics: Main factors are

environment, society and economy (adapted from [9]).

Phosphorus limitation of ecosystems’ plant productivity

In tropical parts of the world with their many millions year old soils, P is

often the limiting nutrient since primary P-containing minerals are depleted

with soil age [15, 16]. In these old tropical soils, as typical found in Africa (s.

Figure 4), the remaining P is often bound strongly to secondary iron and

aluminum minerals and is considered not to be bioavailable. Furthermore

atmospheric input of P is quite low [17]. As a consequence, P limitation

limits agricultural and forest productivity and is - besides social, political and

economic imbalances - a major cause of famine in this part of the world [18].

However, P limitation is not only associated with highly weathered soils in

the tropics but can also occur in boreal systems (s. Figure 4), as recent

5

studies have demonstrated [19-21]. As the second largest biome, the boreal

forest accounts for a third of the global woodland cover and is crucial for

biodiversity, economy and as an important carbon sink [22]. A large body of

research has studied the factors behind growth of this boreal ecosystem and

ca. 25 elements of soil nutrients were identified with N and P often the most

important ones. Fertilization with these elements is expected to increase tree

growth.

Figure 4: Example of tropical environment (left: Burkina Faso) and boreal

environment (right: forest in northern Sweden); (photos: Burkina Faso:

Andrea Vincent and boreal forest: Johan Vestergren)

For a long time, N was seen as being the main limiting nutrient in boreal

forests. Therefore most research focused on understanding the role and

mechanism of N for forest growth [23]. For P much less research was carried

out, partially due to the appearence of soil P in many different molecular

species. Only recently it was found out that P can also act as an important

limiting factor for plant growth in boreal ecosystems [15, 20, 24] . The likely

explanation for this locally limiting role of P is that a large fraction of soil P is

chemically bound in stable complexes, in particular on soil surfaces. This

holds also for the organic P-pool, which mostly consists of mono- and

diesters of phosphoric acid. This is a direct consequence of the unusually

large reactivity of phosphate groups towards environmental particles in

comparison with other nutrients [8, 25, 26]. These mineral and organic soil

particles are, together with amorphous aluminium and iron compounds, the

main factors determining soil sorption capacity for organic P [27, 28]. Soil

adsorption stabilizes the organic P-compounds against microbial

degradation; a process which prevents biological utilization of this P-pool by

plants [12]. However, there is only sparse knowledge about the mechanisms

of these surface reactions and how they affect the organic P-speciation,

enzymatic degradation and bioavailability. Adsorption of P-species probably

6

also affects losses of P from terrestrial P into aquatic systems, because soils

with surface reactive properties causing P-limitation are also found at the

transition zone between terrestrial and aquatic ecosystems. In freshwater

ecosystems the organic P-availability also affect primary production and the

heterotrophic energy mobilization rate [29]. A likely climate impact in the

northernmost ecosystems will result in an increased leaching loss of

dissolved organic P into these aquatic systems.

Phosphorus: Its way to bioavailability

All the mineral P located in the surface layer of our planet is found in its

oxidized phosphate form in primary minerals such as the minable apatite

family [16]. In contrast N compounds can be biologically created from

atmospheric N2 by different N-fixing organisms. Weathering and microbial

breakdown of P-containing minerals releases the P to the soil. Here, plants

and organisms in the soil microbial community consume P. By converting

inorganic P such as phosphate, pyro- and polyphosphate (s. Figure 5) e.g.

into organic P, they generate the P-containing molecular building blocks

needed for their growth. Most input of organic P to the soils originates from

P-diesters [12]. Even so, P monoesters are the prevailing form of organic P in

soil. Organic P is continuously degraded into plant accessible phosphate by

enzymes and acids, produced by roots and microbes, so that over time P

cycles between being fixed in organisms and being released into the soil as P-

monoesters. (s. Figure 5).

7

Figure 5: Examples of inorganic and organic P-compounds as abundant in

soils, plants and microorganisms. The inorganic forms are basically

phosphate polymers ranging from one phosphate group, orthophosphate, to

several in polyphosphate.

In general, P in solution is the main source for plants, but P concentrations

in solution are not sufficient for continuous growth. Therefore, most P

needed, has to originate from solid soil particles. Transformation into a

soluble form occurs via abiotic and biotic processes. There

adsorption/desorption from soil surfaces and precipitation/dissolution

control the release of P into solution and the biological conversion of P from

inorganic to organic P-species and vice-versa [7].

D-glucose-6-phosphate

guanosine 5´monophosphate

myo- inositol hexakisphosphate

pyrophosphate

2-aminoethyl phosphonate

Scyllo-Inositol hexakisphosphate

phosphatidylcholine

α-glycero phosphate

β-glycero phosphate

R

R R

R R

R R=PO3H

2

8

Boreal forest soils contain a range of organic P compounds, often as a major

fraction of total soil P [30]. Much of this organic P occurs in small organic

molecules with high energy content and high nutrient values. These

molecules include numerous molecular species, often monoesters such as

inositol-phosphate or nucleotides, diesters such as DNA or RNA,

phospholipids from cell membranes of organisms and phosphonates. All of

them, as well as inorganic polyphosphate and pyrophosphate (s. Figure 5)

require hydrolysis before biological utilization. Organic P is continuously

degraded into plant accessible phosphate by enzymes and acids, which are

produced by roots and microbes. After some time of repeating death and

degradation cycles of plants and microbes, the P is released once more into

the soil as P-monoesters.

However, the release of phosphate and thereby its bioavailability for plants is

controlled by the degradation rates of the organic P-compounds and their

release by desorption from mineral surfaces in the soil matrix and presence

of multivalent cations (Fe, Al, Mn…) in the surfaces. However, the strength

of adsorption, which can prevent release completely in very mineral rich soil

as found in the tropics [31], depends not only on the soil matrix but also

crucially on the type of organic P- species.

The tendency for adsorption of a P-species increases with the number of

phosphate groups and therefore its negative charge at a given pH. Therefore

phosphate diesters have a lower propensity for adsorption than monoesters

and are easier accessible for microbes, making them more bioavailable than

e.g. monoesters [32]. Monoesters like inositol phosphates but also inorganic

phosphates can due to their high charge bind much stronger to mineral

surfaces and even form very stable complexes with multivalent metal ions

[28].

31P NMR Spectroscopy: an innovative tool for studying the speciation of organic phosphorus and the need for advanced techniques to study P-species.

While the role of N in boreal forests and other ecosystems has been studied

in large detail [23], much less is known about the role of P as a nutrient and

a limiting factor in these systems [20, 21, 24, 33]. The main reason for this

limited knowledge about soil P is the sheer complexity and heterogeneity of

soils. As a Science editorial stated “ The processes occurring in the top few

centimeters of the earth’s surface are the basis of all life on dry land, but the

opacity of soil has severely limited our understanding of how it functions”

[34]. The problem with P stems from the fact that P is abundant in many

different molecular species, which can in addition also be bound physically

9

to soil surfaces and form complexes with the metal ions present in these soils

[35-37]. Therefore, identification of these numerous organic P-species,

elucidating their origin and understanding their chemical and biological

dynamics in soils (and in aquatic environments which is not the main scope

here) is crucial in ascertaining their role in ecosystems and their effects on

soil fertility. In addition, comprehensive information about soil P is essential

in understanding the response of P-biochemistry to global environmental

changes and in sustainable P-fertilizer management in agriculture [11, 13,

14]. Therefore, it is quite obvious that advanced experimental techniques

with the capabilities of analyzing the speciation and dynamics of organic P in

these complex systems at the molecular level are greatly needed.

NMR has emerged in recent years as the most powerful and versatile

technique to obtain molecular information on the nature of soil components

and their interactions [38]. NMR can now also be used to elucidate soil

processes at various scales. While liquid-state NMR on soil extracts can be

applied to extract structural information of individual components, diffusion

NMR and related techniques can study the formation of colloidal soil

aggregates from organic matter. Solid-state NMR techniques such as magic

angle spinning (MAS) NMR and high resolution (HR) MAS NMR add

another layer of information by being able to explore a soil matrix directly

and to extract details about larger complexes and their internal arrangement,

in addition, the adsorption behavior of smaller molecules (organic

molecules, contaminants) to soil interfaces and aggregates can be studied.

Finally, also magnetic resonance imaging as known from its medical

applications, is now applied in exploring distributions of specific

components across soil layers, as is localized spectroscopy of specific spots in

soil profiles [38].

Phosphorus consists of just one isotope, 31P, which is NMR-visible with a

high sensitivity. Already basic liquid 31P NMR on soil extracts can be used to

provide identification of various organic P-species of unknown origin [39].

As a result, liquid NMR studies on extracts have been widely employed not

only to identify P-containing molecules in complex mixtures (metabolomics

approach [38]) but also to determine their structure (molecular

identification approach [38]). More recently, also solid state NMR

techniques (31P MAS NMR) gained an increased interest in soil science, since

these approaches can directly study unperturbed solid soil matrices, as well

as complexes of small organic P-species adsorbed to mineral- or biological

surfaces, or colloidal particles [40-44].

10

Liquid NMR in soil science

NMR on liquid samples containing the molecules of interest in solution, is

the most commonly used type of NMR experiments in chemistry, structural

biology and even soil science. In solution, molecules tumble fast which leads

to well resolved and easy-to-analyze isotropic NMR signals. Thus, liquid

NMR provides molecular details that allow structure determination of

molecules, or their identification, because the positions values of these

signals are then indicative for specific P-compounds [45].

NMR as an approach for studying soil organic P has been employed since the

80’s to identify different species of organic P in soil matrices [46]. Because

the sample has to be liquid, soil specimen have to be treated in a way which

releases their P-species into the solution to be used in the NMR experiments.

For this purpose various extraction procedures have been developed, based

on strong acids or bases to extract organic P from soils in a quantitative way

[47, 48]; or step-wise using different extraction conditions to extract

different portions of P in each step [49]. A single-step extraction using

sodium hydroxide/ethylenediaminetetraacetic acid (NaOH/EDTA)

treatment was developed by Cade-Menun, and colleagues [47, 48, 50, 51],

and has become the most widely used extraction protocol, suitable for most

soils and even other matrices such as marine particles and sediments [52,

53]. Extracts produced this way presumably contain nearly all bio-available

P, and are routinely studied by liquid NMR to identify different organic P-

species and to compare them quantitatively [47, 48, 50]. However, the

conditions used in most extraction protocols alter the chemical structures of

various P-species [54-56]. A typical 1D 31P NMR spectrum of a boreal soil

extract is shown in figure 6.

11

Figure 6: Typical 1D liquid 31P NMR spectrum of a boreal forest soil extract:

Different peaks indicate individual P-species which can be grouped into

different families (mono-ester, diester, phosphonates, inorganic mono- and

condensed phosphates…). Curly brackets indicates monoester region which

is highly crowded, which hampers identification of individual P monoesters.

In this spectrum (s. Figure 6), many signals are visible, with varying integrals

and varying positions on the frequency x-axis (NMR frequency expressed as

“chemical shift” with ppm as unit). In principle, each signal can be related to

an individual P-species based on its specific chemical shift values [45]. But

chemical shifts do not only depend on the specific molecule but also on the

soil extract and the conditions present in solution [50, 54, 57]. Extensive

research has resulted in a large body of information on single components

and extracts from model mixtures and natural soils, with the goal to

unambiguously identify P-compounds in NaOH/EDTA extracts [54-57].

Based on these published assignments of P-species, the chemical shift

regions in figure 6 have been defined. Phosphate mono- and diesters are

found in soils all over the world including boreal soils [58]. Furthermore,

the spectrum also reveals less abundant P-species such as phosphonates and

inorganic phosphate compounds. While the grouping displayed in figure 6 is

useful, a mechanistic understanding of P dynamics requires identification of

individual molecular species. One reason for the lacking mechanistic

understanding of P dynamics is the missing identification of individual P-

monoesters due to the severe overlap in this spectral region (s. Figure. 6).

MDPA, internal standard

Monoester region

orthophosphate

Diester region

pyrophosphate and polyphosphate end group

polyphosphate phosphonate

12

With this type of 1D liquid 31P NMR approach on soil extracts, knowledge

have dramatically increased in the last decades about P-speciation, and our

understanding of the biogeochemistry of soil P, and its role in the relevant

ecosystems [47, 48]. This methodology was even applied to other P-

containing matrices ranging from lake sediments to manure, and even to

colloidal particles in aquatic systems [59-61].

Despite its huge success in soil science in the last decades, 1D 31P NMR using

soil extracts has major shortcomings which restrict the exploitation of the

full power of liquid NMR in this field. One main reason is the appearance of

spectral overlap in crowded regions, due to signals with broad line widths

and the small chemical shift dispersion of the 31P nucleus in these

compounds. Because of that analysis of these 1D 31P NMR spectra is often

complicated; a problem which severely hampers e.g. a complete

identification and quantification of all individual components in the

interesting mono- and diester regions. Furthermore, the sample matrix

influences the chemical shifts of P-species to a varying degree; which can

make unambiguous identification difficult. To relieve this problem partially,

spiking experiments have been carried out which enabled at least the

identification of the main components [54, 62-64]. However, this procedure

is not possible for less abundant P-species whose signals would be hidden

under these larger peaks. Furthermore, different species of the same P-

family, such as monoesters can have the same chemical shift, so that the full

spectral integral would be attributed to the P-species used for spiking, while

the other component would be overlooked. Therefore, many NMR studies

report not individual P-species but only the monoester region in total, thus

abandoning all biologically most interesting information on specific,

components of this P- species family.

In general, the occurrence of spectral overlap in these NMR spectra is not

primarily due to the sheer number of individual P-species e.g. in the

monoester region, but due a side effect of the NaOH/EDTA extraction

procedure used routinely for extracting P-species from soil matrices [47, 48].

While this method extracts presumably most of the bioavailable inorganic

and organic P, it also transfers high amounts of paramagnetic metal ions

(e.g. Fe3+, Mn2+) into solution due to the strong complexing properties of

EDTA. Despite being complexed by EDTA, these paramagnetic impurities

still broaden P signals much beyond their intrinsic line width of under 3 Hz,

thus causing overlap and degrading sensitivity, making identification of most

individual P impossible [39].

Finally, the routine extraction protocol itself has biological drawbacks which

have to be carefully considered in any quantitative analysis of organic P by

13

1D liquid 31P NMR. The harsh NaOH/EDTA extraction method with its high

pH and duration causes hydrolysis and degradation of organic P-species

[47]. Depending on the type of P-compound and the time used for

extraction, the degree of degradation varies between P-species [55], making

not only quantitative comparison difficult but also masking the original P-

species present in the soil matrix prior extraction.

Solid state 31P MAS NMR on intact soils

While solution NMR relies on extracts from soil samples, solid state MAS

(magic angle spinning) NMR approaches can probe soil material directly.

This method, still much less used than liquid NMR in soil science, can access

large complexes and molecules adsorbed to immobile matrices, where the

reduced tumbling rates exclude the used of solution NMR. These solid state

NMR methods are able to characterize interactions between molecules and

solid material, and that not only from the point of the adsorbed molecule [8,

65] but (if suitable NMR isotopes and conditions exist) also from the point of

the adsorption matrix [66]. However, due to the missing tumbling and the

presence of heterogeneous material and paramagnetic impurities, the

obtained NMR spectra suffer from poor resolution and often only 2 or 3

major P-species families can be identified (s. Figure 7). Due to the

differential line-broadening effect on various P-groups, only a semi-

quantitative analysis is possible if at all. However, the main advantage of this

method is the probing of the complete P-pool in the soil matrix and not only

the extractable fraction, which might often be less than half of the total P,

with debate ongoing if the extractable fraction can be considered to

represent the bio-available P-pool [37, 48, 49].

Nevertheless, 31P MAS NMR has been employed in studying a wide range of

samples and ecosystems and has generated useful information despite the

limitations of the obtained NMR spectra. Using model minerals the surface

speciation of specific P components could be studied under controlled

conditions, as recently shown with phosphate on Boehmite mineral [8]. This

information could then be transferred to real soil matrices to understand the

dynamics and sorption pattern of individual P-species there; information

essential for understanding the mobility and biological availability of these

P-species to plants [47]. But also dissolved organic matter in aquatic system

has been studied by 31P MAS NMR upon enrichment by ultrafiltration [67].

By investigating dried material from high-molecular weight organic material

originating from various sea depths (0-4000 m) they found a constant ratio

between P esters and phosphonates. They concluded that dissolved organic P

is preferentially remineralized from dissolved organic matter, and its

selective removal form this matter indicates the demand of marine

14

microorganisms for this nutrient. To avoid lyophilization of huge amounts

of water for this type of research, enrichment procedures using small

amounts of water where suspended colloidal particles based on polymeric

ion exchange material adsorb P-species, are investigated after simple

centrifugation as pellets by solid state 31P MAS NMR ([68], ongoing

development in the lab). Nevertheless 31P MAS NMR is not only employed

on natural P-species but is also ideal in tracing the behavior of P-based

xenobiotics (warfare components, pesticides etc) in soil matrices, especially

their mobility there and any transformation processes which can cause

alteration of their toxicity and bioavailability [65].

Figure 7: A comparison of spectral resolution between solid state 31P MAS

NMR (top trace, 4 kHz spinning speed) and liquid (bottom trace) 31P NMR.

In 31P MAS NMR the large peak in the middle consists of several P

components e.g. monoesters, diesters and inorganic P, concealed by the poor

resolution associated with solid state 31P MAS NMR on intact soil samples.

To the left and to the right of this main peak are two emerging peaks that are

the MAS spinning side bands and arise due to the spinning of the sample

during the NMR experiment. The bottom trace reveals several P peaks

although they are not completely resolved. The same chemical shift range is

present in both spectral traces and differences in chemical shift are due to

differences in pH between soil and soil extract.

15

Aim of thesis

While much research has been carried out to elucidate the role of nitrogen as

nutrient in ecosystems, much less knowledge exists about the role of P. The

main reason for this is not only the complexity of soils as the main P source

in terrestrial ecosystems, but also the appearance of P in many different

molecular species. And these P-species can even be adsorbed to soil surfaces

and interact with multivalent metal ions (e.g. Fe3, Al3+, Mn2+ …) present in

these soils. Therefore, advanced experimental molecular techniques are

required for identification of the individual P constituents composing the

organic P-pool in these soils, and for tracking their behavior during soil and

ecosystem development. And one of the most promising method for this

purpose is liquid 31P NMR on soil extracts. This approach provides a wealth

of information on P-speciation and dynamics in soils, but its wider

application in soil sciences is severely hampered by the fact that the presence

of paramagnetic soil impurities causes poor spectral resolution and prevents

application of multi-dimensional NMR methods for identification of

unknown P-components and separation and quantification of all P-species

present in the soil´s organic P-pool.

The general objective of this thesis was therefore to carry out a

multidisciplinary study to develop and implement the necessary 31P NMR

methodology for providing new basic knowledge on a molecular level about

the speciation and dynamics of organic P-species in various ecosystems. The

specific objectives were:

Developing biologically meaningful new methods in organic P specification

in agricultural and forest soils based on solid state/liquid 31P NMR

techniques. In particular, to implement 2D liquid NMR experiments for

unambiguous P-species assignment and quantification; an objective which

requires development of soil extraction methods which remove NMR

interfering paramagnetic impurities.

Analyzing the speciation and dynamics of organic P in soils in different

ecosystems: i) boreal forest productivity gradient, ii) humus soil

chronosequence (7800 years) in Northern Sweden, and iii) Semi-arid

tropical agricultural woodlands with different soil mineralogy.

Understanding the chemical and biological dynamics of organic P-species in

these ecosystems and how these contribute to soil fertility and to plant and

microbial growth.

16

By addressing these objectives we will not only develop new NMR based

molecular techniques for soil science, but we will generate molecular

information important to understand the environmental behavior of organic

P released from the terrestrial environment and its microbial availability; key

issues in future climate related effects on our high-latitude ecosystems and

in combating P-restriction in food/forestry production, especially in tropical

third world countries.

Materials and Methods

NMR spectroscopy

The NMR signal

Most, but not all nuclei have a magnetic spin (I) which allows them to be

susceptible to an external magnetic field. The most commonly used nuclei in

chemistry are 1H, 13C, 15N and 31P which all have I=1/2. Putting the sample in

an NMR spectrometer with a large internal static magnetic field with a

magnetic field strength B0, all magnetically susceptible nuclei align parallel

or antiparallel to this external field much like a compass needle aligning to

the earth’s magnetic field (s. Figure 8). These orientations reflect a lower (α)

and a higher (β) energy state, respectively. The discrete difference between

these two energy states is proportional to the gyromagnetic constant, γ. The

lower energy state is marginally more populated resulting in a bulk

magnetization called Mz or the longitudinal magnetization. However the

energy difference is quite small and this causes NMR to be a rather

insensitive technique that requires a sample with high concentration. In

NMR experiments, radiofrequency (RF) signals induce transitions between

the two states, when the radiofrequency fulfills the resonance condition.

17

Figure 8: In its relaxed state, the macroscopic sample magnetization is

found aligned to B0 parallel to the z- axis in a three dimensional coordinate

system.

When a RF pulse is applied along the x-axis it creates a small magnetic field

perturbing the position of Mz angling it towards the x- y- plane (s. Figure 9).

Here the Mx,y components rotate around the z-axis with the frequency ν,

which is called the Larmor frequency. The rotating magnetization generates

a radiofrequency field which is picked up by a detection coil. The signal

detected is at this point in a time domain and is known as Free Induction

Decay (FID). This can be transformed using a Fourier transformation

converting the time-domain signal into a frequency-domain signal, which is

the NMR spectrum. After the Mz component has been perturbed in an NMR

experiment, is relaxes back to equilibrium with a characteristic time constant

T1, also called the spin-lattice relaxation time, which is experimentally

important to allow quantification of NMR spectra.

Figure 9: Here the magnetization vector, M0, to the left is before the 90˚ RF

pulse and to the right after the RF pulse. From that position it will relax back

to the static magnetic field, B0, at the z axis.

18

The RF pulse is short, generally in the µs time regime and adjusting pulse

length together with pulse power can give maximum signal when Mz is

completely rotated into the x, y plane, by a so-called 90º pulse.

The chemical shift

The chemical shift is the position of one signal in an NMR spectrum, relative

to the position of a specific standard in a ppm scale. The chemical shift is

created by the fact that each nucleus experiences a slightly modified B0

leading to slight variation in , reflecting its chemical surrounding i.e.

electron density and how it is affected by other atoms in the molecule. A

change in the structure of the molecule means a change in the electronic

environment and will move its position along the frequency axis [45].

Because the variation in is small, it is usually given in parts per million

(ppm) relative to a reference compound, according to equation δ = (νmolecule -

νstandard)/ νstandard. This equation can be used to calculate the chemical shift. δ

is the chemical shift, ν is the resonance frequency and ν standard is the

frequency of the reference compound. For 31P NMR, 85 % phosphoric acid is

used as an external standard with chemical shift δ =0.0 ppm.

Experimentally, it is essential that the B0 field is as homogenous across the

sample volume as possible, so that identical molecules in any volume

element will contribute signals with the same frequency, so that (i) the signal

of each P-species has maximal intensity, and (ii) the signals of all P-species

are as sharp as possible for optimal resolution in the spectrum. Homogeneity

of B0 is optimized using a set of coils (“shim coils”) situated around the

sample in the NMR magnet. Currents in these shim coils are manipulated in

such a way that the magnetic field becomes as homogeneous as possible.

Indirect spin-spin couplings

Indirect spin-spin couplings - also known as J-couplings or scalar couplings -

are crucial for structure determination and identification of unknown

compounds. Interaction of one spin with the spin states of other NMR-active

nuclei through up to four connecting bonds causes splitting of the peak of the

observed nucleus. When n is the number of equivalent neighboring nuclei,

this effect causes a splitting of the signal in a pattern of n+1 multiplet lines.

The lines follow a specific pattern intensity ratio that for a spin system of

I=1/2 is given by Pascal´s triangle, [69], which is valuable for structure

determination. Within a group of chemically equivalent nuclei, splitting due

to spin-spin couplings does not occur. The distance between lines within a

multiplet is called the J-coupling constant. This constant is independent of

19

magnetic field strength and it is measured in Hertz (Hz). The size of J

couplings depends on a molecule’s conformation and therefore helps to

identify a molecule and determine its structure. The splitting effect between

different nuclei can be cancelled out using a broadband decoupling sequence

that irradiates the nucleus not being observed continuously. Examples of

such sequences are WALTZ-16 and GARP [70]. Spin-spin couplings are an

important factor in many experiments because they can be used to create

magnetization transfer between nuclei, which is the basis for a large group of

multidimensional correlation experiments.

Line broadening contributing factors

In order to analyze NMR spectra properly all individual resonances have to

be separated, i.e. the spectra have to be fully resolved. Resolution is strongly

affected by the line widths of the signals, which are specified through the

peaks’ width at half height, Δν1/2, measured in Hz. The line width of an NMR

signal is reverse proportional to the spin-spin relaxation time T2, so any

mechanism that accelerates T2 relaxation increases line width. Important

factors affecting relaxation and therefore line width are the size of the

chemical shift anisotropy (CSA), dipolar interactions and paramagnetic

impurities [45]. Dipolar interactions and CSA are molecular properties; their

contribution to relaxation depends on B0, temperature, and the viscosity of

the NMR sample, all parameters that can only be modified in a limited

range. But for small molecules, the contribution of these relaxation

mechanisms to 31P relaxation is small, so that line widths of approximately 1

Hz can be obtained.

Paramagnetic impurities such as iron (Fe3+) and manganese (Mn2+) cause

severe line broadening if present. The unpaired electrons in the outer shell of

these ions have a magnetic momentum that can affect the spin of the

observed nucleus, causing faster relaxation and thus broader lines. This

problem is exacerbated by the high affinity of highly charged metal ions for

phosphate groups.

20

Table 1: Table adapted from Vincent et al. 2013 [58] where the chemical

shifts of both 31P and 1H can be seen. This is a direct result of removing the

paramagnetic ions and thus being able to perform 2D 1H-31P HSQC NMR on

soil extracts. In [39] fine structures are also included for additional

information. Unknowns and internal standards (MPA and MDPA) have been

removed from the table.

Label Compound 31P-shift (ppm)

1H-shift (ppm)

P family

A β glycerophosphate 4.95 4.07 Monoester

B α glycerophosphate 5.27 3.68, 3.75 Monoester

C Myo-Inositol hexakisphosphate 4.65 (C4,6) 4.52 Monoester

Cʹ “ 4.55 (C5) 4.27 “

Cʹʹ “ 5.03 (C1,3) 4.48 “

Cʹʹʹ “ 5.97 (C2) 4.48 “

D Guanosine 2´ monophosphate 4.33 4.89 Monoester

E Guanosine 3´ monophosphate 4.90 4.58 Monoester

F 2-aminoethyl phosphonate 18.98 1.59, 3.28 Phosphonate

G Uridine 2’ monophosphate 4.62 4.54 Monoester

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 5´ monophosphate 4.78 3.84, 3.89 Monoester

M DNA –0.5 to 0.0 3.80–4.20

4.62–4.98

Diester

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

21

Q Adenosine 2´, 5´ diphosphate 4.54 (2´) 4.93 Monoester

R “ 4.81 (5´) 3.88, 3.94 “

T Scyllo-Inositol hexakisphosphate 4.14 4.47 Monoester

A1 Orthophosphate 6.29 Orthophosphate

A4 Polyphosphate (end-chain) –3.70 to–4.27 Polyphosphate

A5 Pyrophosphate -4.14 Pyrophosphate

A6 Polyphosphate (mid-chain) –18.5 to–21.0 Polyphosphate

The 2D 1H-31P HSQC NMR experiment

In 1D 31P NMR, acquisition of the FID starts directly after the RF pulse, and

the FID is Fourier transformed and the spectrum analyzed after suitable

post-processing. For 2D NMR, an evolution time and magnetization mixing

are inserted between the initial excitation pulse and data acquisition. It is of

significant importance that all pulses are accurately calibrated. The HSQC

was developed in 1980 by Bodenhausen and Ruben [71] and has quickly

become the mainstay of analytical tools in protein chemistry. The HSQC also

has potential as a valuable tool in other disciplines such as environmental

research [39]. In a 1H- 31P HSQC experiment, 1H magnetization is excited

and transferred via J couplings to 31P. Then 31P chemical shifts are recorded

in the evolution time, and finally 31P magnetization is transferred back to 1H

for detection. The evolution time, t1, is systematically varied starting with

t1=0 and incrementing for each of the n experiments that create the raw data

of the 2D experiment. Thus, NMR signals are obtained that depend on two

time axes, the “indirect” time domain created by incrementing t1, and the

“real” time domain t2 corresponding to recording of the FIDs. Modulations of

the signals as function of t1 and t2 encode 31P and 1H chemical shifts,

respectively. Two Fourier transformations are performed and the result is a

spectrum with two dimensions [72, 73]. Fourier transformation of t2 creates

the frequency dimension for the more sensitive nucleus, usually 1H, and

transformation along of t1 creates a second frequency dimension of the less

sensitive nucleus e.g. 31P. We now have two chemical shifts, and because the

HSQC transfers magnetization between J-coupled nuclei, the 2D spectrum

correlates the chemical shifts of J-coupled 31P and 1H nuclei. The spectra are

commonly presented as contour plots like topographical maps (s. Figure 10).

22

The combination of two chemical shifts vastly increases resolution compared

to 1D 31P NMR, because P-species with identical 31P chemical shifts can be

resolved based on different 1H chemical shifts. Table 1 lists 1H and 31P

chemical shifts of some typical P compounds found in soils.

Figure 10: A typical 2D 1H-31P HSQC spectrum showing the monoester

region, with a 1D 31P NMR spectrum as overlay. This overlay shows how the

increased resolution of the 2D spectrum allows identification of individual P-

species which are not resolved in the 1D spectrum. The circled peak X is an

unidentified P-species present in both boreal and tropical samples.

Inductively coupled plasma optical emission spectrometry (ICP-OES)

ICP-OES is a well-established and frequently used technique. Today over 70

elements of a total 92 can simultaneously be analyzed in a matter of minutes.

The main feature of this method is the plasma ionization and the optical

emission detector. Samples are introduced generally as a liquid and are

promptly nebulized into an aerosol before entering the plasma torch. The

sample is transported through the center of the plasma torch by argon gas.

This argon gas is also part of the plasma discharge. The argon gas passes a

RF copper coil that applies an oscillating RF current creating an electric and

magnetic field. As argon gas passes through the RF field electrons are

removed from the argon atoms and caused to crash into other argon atoms

23

by the magnetic field creating a continuous ionization process of the argon

gas. This adds energy to the process and is known as inductive coupling. This

converts the otherwise non-reactive gas argon to turn into argon plasma with

temperatures between 6 000 and 10 000 K.

The sample when passing through the center of the plasma torch spends

approximately 2 milliseconds in the center creating optimal conditions for

low matrix interference. As the sample subsequently passes through the

plasma it is first removed of all solvent and then the remaining salts are

vaporized and molecules broken into atoms. These atoms are then excited by

the high consistent temperature in the plasma. Excitation is the process in

which one to several electrons is moved from their ground state into an

excited state. When they go back from the excited state they release energy in

the form of a photon. This energy is specific for each element and each

elements different excited states and this creates a highly accurate method

for analyzing elements. In the optical emission detector the light created by

the released photon is detected and analysis is complete. The detection limit

for ICP-OES is in the parts per billion (ppb) range and this is applicable to

the majority of the more than 70 elements possible to analyze with this

method. ICP-OES, Optima 200 DV (PerkinElmer, Überlingen, Germany);

equipped with Cross-flow nebulizer and double spray chamber was used for

elemental analysis. A portion of 50 mg of lyophilized extract was dissolved in

20 ml of 2% HNO3 (aq.). If needed steps were taken to insure there was no

particulate matter left. Standards with concentrations of 2 ppm and 10 ppm

were made for Al, Fe, Mn and 31-P and used in all ICP measurements

throughout my PhD project.

X-ray fluorescence spectroscopy (XRF)

XRF is a non-invasive analytical technique widely used in archaeological,

industrial, geological, and environmental studies [74]. A problem for XRF is

the matrix effects. That is most commonly caused by the composition and

grain size of the observed sample. By grinding the samples into a fine powder

these effects can be minimized. XRF on powder, pellets, or fused beads

needs quite a large sample. The sample sometimes need to be in the range of

several grams for accurate elemental quantification [75]. The latest of

wavelength-dispersive XRF (WD-XRF) machinery gives the possibility to

minimize the amount of sample by specific calibration procedures on small

sample volumes. The XRF measurements in paper 3 were made on a Bruker

S8 Tiger WWD machine.

24

Other analytical methods used in this thesis

The EA-IRMS elemental analysis for carbon and nitrogen, in paper 3, was

made using an elemental analyzer (Flash 2000 Organic Elemental Analyzer,

Thermo Fisher Scientific Inc., Bremen Germany). Multi variate analysis was

performed on Simca 13.0 32-bits (SIMCA v.13.0 Umetrics, Umeå, Sweden),

with the 1D 31P NMR integration data set combined with elemental analysis

data from XRF and EA-IRMS.

Site descriptions

In this thesis several sites in different ecosystems were investigated. At each

site, there was a hypothesis that P-speciation might change as a function of

variation in site conditions. Therefore the sites allowed us to investigate the

influence of abiotic and biological parameters on P biogeochemistry. The

first site was Betsele, where a study on six boreal humus layer soils was

performed. Betsele is situated in the Umeå river valley northwest of the town

of Betsele in northern Sweden. It is a 90 m productivity gradient with a

groundwater recharge area and a groundwater discharge area. The gradient

area has a slope of 2% and has been thoroughly described in literature [24,

76, 77]. The transition between groundwater recharge and discharge is

associated with a pronounced change in soil properties and in the vegetation.

Therefore the goal of paper IV was to investigate relations between the

parameters changing along the gradient and P biogeochemistry [78].

The second sample area consisted of eight sites on the coast outside Umeå in

northern Sweden. These sites were chosen to perform a chronological

gradient due to the landrising occurring there (9 mm year-1). The coast

outside Umeå is rising as a result of the last ice age. The age of the sites could

therefore be calculated from the elevation above sea level for each site. The

youngest site was approximately 90 years and the oldest 7800 years. Here a

total of eight sites were included and from all sites we collected five boreal

humus layer soil samples. From these chronosequence soil samples were also

used for paper I [39] as well as for paper II [58].

Finally, the last two sites are representative of sub-sahelian tropical soils and

are taken in Burkina Faso, representative for old soils where adsorption of P-

species to surfaces of secondary minerals is thought to strongly limit P-

bioavailability. The two Burkina sites represent one oxisol soil and one

Ferralsol soil. Outside the village of Saponé the soils are sandy and very poor

in P. The other soil comes from Finlande, another village that is situated in

an area where the soils are richer in P and organic material than Saponé. The

samples were taken under tree canopies and outside tree canopies in a

25

previously determined pattern, with the aim to test the hypotheses that soil

type and presence of trees might influence P-speciation. This studies are

described in paper III.

Figure 11: On the left is a typical soil profile from a boreal soil in northern

Sweden. On the right is a soil profile from Saponé in Burkina Faso

containing depleted soil with only small amounts of nutrients. (Pictures

taken by Reiner Giesler, boreal soil; Ulrik Ilstedt Saponé soil).

Sampling and general preparations

At each individual site four to six replicate samples were taken and pooled.

Samples were taken with a soil auger at a depth of 5 to 15 cm except for the

Burkina Faso samples which were taken in 10 cm segments at 0 to 50 cm and

100-120 cm. The Burkina Faso study in paper III focused on the 0 cm to 10

cm layer. The boreal soil samples from Betsele and the land rising coast

chronosequence (LRC) were stored under cold conditions directly after

sampling. These samples were then sieved using a 4 or 5 mm steel mesh and

subsequently dried. After drying, a sufficient portion was ball-milled into a

fine powder ready for extraction or direct analysis. Betsele samples were

milled by hand in a mortar. The Burkina Faso samples were dry already on

sampling and were transported and kept dry prior to analysis. The tropical

samples were sieved and milled in the same fashion as boreal soils.

26

Preparations for solid state NMR

The dried and milled soil sample was carefully packed in a 7.5 mm zirconia

MAS sample rotor without any further treatment. Each rotor was weighted

prior and upon sample loading. Solid state 31P NMR spectra were acquired

on a Chemagnetics 400 MHz spectrometer (Chemagnetics/Varian, Fort

Collins, CO, USA) equipped with a 7.5 mm double resonance probe. A 31P

frequency of 162 MHz together with a proton frequency of 400 MHz was

used for all experiments and a MAS spinning speed of 4 kHz at 293 K. A

single pulse excitation sequence together with proton decoupling was used

[79] All spectral processing was done using the Spinsight software

(Chemagnetics).

Sample preparations for liquid NMR by conventional extraction

For liquid NMR further preparations are necessary. Specifically for liquid 31P

NMR a common extraction scheme using NaOH-EDTA was developed by

Cade-Menun and Preston[50]. The suggested mechanism for solubilizing

and extracting soil organic P is derived from the proposed adsorption of

anions such as organic P on soil mineral surfaces by Russel [80] (s.

Figure.12). The hydroxide ions are drawn into the surface layer by the

metallic cations, rendering the net surface charge more negative with

increasing pH, and thus causing an electrostatic repulsion between the

surface and phosphate groups. At the same time the sodium ions replace the

monovalent cations and also neutralize the net surface charge. In practice, a

portion of 1.5 g of the dried and milled soil is extracted using a 1:20 mass

ratio between soil and extraction solution (250 mM sodium hydroxide and

50 mM ethylenediaminetetraacetic acid disodium salt (EDTA). Soil and

extraction solution were shaken together for either 4 or 16 hours. After

centrifugation, a smaller portion was set aside for other wet chemical

analysis. The majority of the extract was frozen at -80˚C or flash frozen

using liquid nitrogen and then lyophilized. In paper IV the lyophilized

material was directly dissolved in heavy water (D2O) for 31P NMR analysis in

a 10 mm NMR glass tube.

27

Figure 12: Cartoon representing the extraction mechanism of organic P as

proposed in [80]

Sulfide treatment

The conventional NaOH-EDTA extraction co-extracts a significant amount of

paramagnetic metal ion such as Fe and Mn, which are known to cause line

broadening in NMR spectra. Therefore we hypothesized that physical

removal of the paramagnetic ions would strongly improve resolution in NMR

spectra. The method that we developed adds an extra step and by that also

extra time to the NMR sample preparation but is crucial for obtaining

sharper lines in the 31P NMR spectra. The sample preparation towards liquid 31P NMR starts by dissolving the lyophilized material in D2O as stated in the

previous paragraph. In the protocol developed and reported in paper I [39],

we used a total of three D2O solutions for NMR sample preparation: i) The

bulk solvent is pure D2O, ii) an approximated 5 molar equivalents solution of

sodium sulfide (Na2S) in D2O, iii) methyl phosphonic acid (MPA) in D2O

with a concentration giving 50 µg in the NMR sample with a 10 µl addition.

To prepare a sample, 100 mg of the lyophilized sample is weighed into an

Eppendorf cap, and 10 µl MPA solution and 50 µl sulfide solution are added.

The choice of 50 µl is calculated to have sulfide ions in excess, compared to

the expected or measured amount of paramagnetic metals in the sample.

Thirdly, D2O is added to give a total volume of 500 µl. The sample mixture is

homogenized by vortexing the Eppendorf, then sulfide precipitation is

allowed to occur at room temperature overnight, approximately 18 hours.

28

Thereafter the sulfide precipitate is removed by centrifugation of the

Eppendorf cap for 30 minutes at 10 000 rpm in a bench Eppendorf

centrifuge. After centrifugating the supernatant is transferred into a 5 mm

NMR tube and analyzed by NMR.

Figure 13: Cartoon depicting the reaction of the NaOH/EDTA extract with

sulfide. While hydroxide ions liberate P-species from metal association,

sulfide precipitates metal ions, preventing their interaction with phosphate

groups and removing them from solution. EDTA is excluded from the

cartoon, not from the extraction. (Adaption from Figure 12)

NMR experiments

For paper I a Bruker Avance DRX 500 MHz spectrometer operating at

202.456 MHz for 31P and a Bruker Avance DRX 600 MHz spectrometer

(Bruker, Germany) equipped with a 5 mm 1H, 13C, 31P cryo probe, were used.

We used for paper II a Bruker Avance III 400 MHz spectrometer (Bruker,

Germany) operating at 161.76 MHz for 31P for 1D experiments and the 600

MHz spectrometer for 2D experiments.The samples from Burkina, paper III,

was analyzed mainly on the 400 MHz spectrometer. A few samples were

analyzed using the 600 MHz spectrometer. The experiments for paper IV

were made on the 500 MHz spectrometer.

29

SIMCA analysis

A set of difficulties appeared proving to be unreasonably time-consuming to

solve. The inherent factors in the sample affecting pH and thus giving a small

but noticeable change in chemical shift of some P compound peaks in the

NMR spectra. This made alignment between individual sample spectra’s

impossible to achieve within a reasonable timeframe. The lack of alignment

meant that the data from the integration of a full 1D 31P NMR spectra could

not be imported into SIMCA since severe differences in the data appeared

caused by this lack of alignment. We therefore, in paper III, chose to

integrate peaks manually and then export the integration data to SIMCA. A

strategy that worked in the sense that individual peaks assigned to the same

P compound could be matched to each other and as such provide better data

for analysis in SIMCA.

30

Summary of Papers

Paper I; High Resolution Characterization of Organic Phosphorus in

Soil Extracts using 2D 1H-31P NMR Correlation Spectroscopy

Aim of the study

For many years 31P NMR spectra on soil extracts have been suffering from

poor resolution which impedes identification and quantification of soil

organic P-species. In this paper we aimed to resolve and improve the key

factors behind this problem. We intended to: First, to develop a method to

remove the paramagnetic ions that degrade the spectral resolution by

inducing line broadening. Second, to apply 2D 1H-31P correlation

spectroscopy to soil extracts, to improve identification of P-species, in

particular when very similar 31P chemical shifts preclude unambiguous

identification; third, to produce a worldwide accessible table of both 31P

chemical shifts and 1H chemical shifts of common soil P-species.

Methods and Results

Alkaline NaOH/EDTA extraction solution, the most commonly used

extraction for soil organic P, usually extracts between 30 and 85 % of soil P.

However this standard extraction creates several problems for the intended

analysis. Hydrolysis of the phosphate ester bond causes different P-species,

in particular of phosphodiesters, to degrade into smaller components and

other P-species. One example is RNA that degrades into eight

mononucleotides, because isomeric 2’ and 3’ monophosphates are formed

from each nucleotide. This hydrolysis is caused by the high pH in the

extraction solution and has so far not successfully been addressed. The

second big problem is line broadening in the NMR experiments. This is

mainly caused by the co-extracted paramagnetic metal ions. We

hypothesized that the paramagnetic ions had to be removed completely from

solution because they still affect NMR spectra even when complexed by

EDTA. By adding sulfide ions this goal could be achieved. The sulfide first

reduces Fe3+ to Fe2+ which reduces its affinity for forming complexes with

phosphate groups. Secondly, the sulfide forms insoluble metal sulfides

allowing for a convenient removal. The decrease in line width is significant,

from typically above 10 Hz down to 2-3 Hz. The longer T2 relaxation times

associated with sharper line width make it possible for the first time to

obtain good 2D 1H-31P spectra of soil extracts, significantly improving

resolution. The increased resolution is not only from sharper lines but also

from the added 1H dimension. This extra dimension adds an extra set of

31

chemical shifts for identification of each P-species, and also provides fine

structure information extractable from the J-coupling information in the 1H

lines. Now a comprehensive list with 31P and 1H chemical shifts and 1H fine

structures of soil organic P-species is available.

Conclusion

By treating the soil extracts with sulfide and subsequently removing the

paramagnetic metal ions we were able to dramatically reduce the line widths

in 1D liquid 31P NMR spectra. This proves that physically removing the ions

is essential for optimizing NMR resolution. When paramagnetic ions were

removed, the line width for all P-species decreased to 2-3 Hz, which is

comparable to “clean” samples. The decrease in line width also allowed for

the use of two-dimensional 1H-31P NMR. This is a significant breakthrough in

the analysis of the speciation of soil P, as it allows not only for the

unambiguous identification of most P-species but also gives valuable

information on the structure of yet unidentified species. Furthermore,

quantification is more reliable since overlap caused by poor resolution is

avoided.

32

Paper II; Soil organic phosphorus transformations in a boreal forest chronosequence

Aim of the study

It is known that the composition of soil organic P changes in ecosystems, as

they develop over time. We wanted to investigate how that composition

changes over time as a boreal ecosystem develops. We hypothesized that

there would be a decrease in total P and in easily mineralizable P-species

over time. We also hypothesized that P-compounds with a higher sorption

affinity would increase with progressing site age.

Methods and Results

This is the first study of soil organic P where both one-dimensional 31P NMR

and two-dimensional 1H-31P correlation NMR were used in an environmental

study. Humus horizon samples from a chronosequence spanning over 7800

years were analyzed using the commonly used NaOH/EDTA extraction to

prepare samples for 1D 31P NMR, while for the 2D experiments the extracted

material was treated with sulfide to meet the criteria of line width for multi-

dimensional NMR experiments.

Overall no clear trends supporting our hypothesis could be seen. Although

site age is not replicated results are still consistent with other similar studies

in the region. Total P showed no clear trend but was almost constant across

the gradient. Easily mineralizable P increased with the two youngest sites

and after that declined. Although soil Fe and Al content varied with site age,

no significant correlation of P sorption with age was observed.

Conclusion

The key results are that monoesters other than inositol phosphates are

predominantly RNA and phospholipid degradation products, indicating that

approximately 40 % of the extracted soil organic P originates from microbial

biomass. Secondly 2D 1H-31P NMR contains information leading us to the

possible identification of compounds not possible to identify with 1D 31P

NMR alone. These results would not have been obtainable if only 1D 31P

NMR had been used.

33

Paper III; Effect of Trees on Forms of Soil Phosphorus in an

Agroforestry Parkland in Burkina Faso (manuscript)

Aim of the study

We hypothesized that we would see a difference in organic P composition, in

a tropical agroforest parkland in Burkina Faso, between soil samples taken

under the canopies of trees, compared toin the middle between trees, due to

influences of trees on soil properties. In this study we included two different

tree species in two locations that differ in soil iron and clay content. We also

hypothesized that close to trees there would be more P forms of microbial

origin due to changes in the activity of the microbial community as an effect

of increased amounts of organic matter, and that this difference would be

more pronounced in the area with more Al/Fe clays.

Methods and Results

The main analytical techniques in this study were 31P NMR and XRF. The

data from these techniques were analyzed with multivariate analysis.

Between the two locations a clear difference in the amount of P and also in P-

composition was visible. Looking at each location, there is a difference

between under and outside tree canopies which gives good separation in the

multivariate analysis. This separation is driven by different soil parameters

at both locations, but is at both locations linked to an increased fraction of

organic P in the total soil P pool.

Conclusion

The presence of trees gives higher amounts of P and organic matter under,

compared to outside tree canopiesand it also gives a greater diversity of P-

species, probably caused by a larger microbial community. Thus, presence of

trees seems to increase the bioavailability of P.

34

Paper IV; Changes in organic P-composition in boreal forest humus soils: the role of iron and aluminium

Aim of the study

This study aimed to improve the understanding of factors controlling soil

organic P-dynamics and speciation in boreal forest soils. More specific the

objectives were to a) see if compounds with a high adsorption affinity, due to

higher number of phosphate groups creating a larger overall negative charge

on the molecule, dominate in highly sorbing soils in groundwater discharge

areas: amd b) of weakly sorbing compounds, with low number of phosphate

groups and/or sterical hindrance, occur in higher levels in a low adsorption

soils in groundwater recharge areas?

Methods and Results

Liquid- and solid-state 31P NMR methods were used to probe the soil organic

P of the humus layer at six sampling sites covering two groundwater

recharge and groundwater discharge areas in northern Sweden. The dynamic

properties of these areas make them appear as a productivity gradient with

low productivity in the recharge area and higher productivity in the

discharge area. Along this 90-meter gradient a large change in P composition

was observed. Moving from groundwater recharge to groundwater discharge

area the levels of soil organic P compounds with high affinity for adsorption

increased. At the same time the concentration of compounds with low

adsorption affinity declined. The concentrations of myo- and scyllo-, inositol

hexakisphosphates, correlate strongly with soil Al and Fe concentrations.

The correlation between P diesters and Al/Fe concentrations on the other

hand is weak.

Conclusion

The results from this study are consistent with other studies that find

positive correlation between the concentrations of especially myo-inositol

hexakisphosphate and Al/Fe. This furthermore coincides with the fact that

the greater quantity of myo-inositol hexakisphosphate is more strongly

correlated between soil/mineral surface adsorption capacity than with

parameters such as N and organic carbon concentrations and microbial

biomass. In conclusion we can see that soil organic compounds that have a

higher propensity for adsorption are found in higher concentrations in

groundwater discharge areas that have a high adsorption capacity.

Simultaneously we find that in groundwater recharge areas with low

35

adsorption capacity we have a larger relative proportion of compounds with

low propensity for adsorption, such as P diesters. Furthermore,

polyphosphate levels decline along the productivity gradient going from

recharge to discharge which could be connected to the fact that

polyphosphate is found in higher levels in fungi than in bacteria together

with the fact that the microbial community shifts from fungi-oriented

towards bacteria-oriented along the gradient.

36

Conclusions and future work

Although the commonly used extraction with NaOH/EDTA extracts a large

fraction of soil P, it suffers from severe drawbacks. Alkaline conditions with

pH above 13 cause rapid hydrolysis of especially phospholipids and RNA.

The high pH also lyses cells of microorganisms and plants, thus blending the

pools of free soil P and of cellular P. This extraction also co-extracts a

significant amount of paramagnetic metal ions like iron and manganese.

These paramagnetic ions cause a dramatic increase in 31P line width, strongly

reducing spectral resolution. The EDTA that is used to increase

solubilization of soil P also keeps the co-extracted paramagnetic ions in

solution making them constantly close to the extracted P species to be

observed by NMR. The results presented in paper I solve the problem with

paramagnetic ions, so that optimal resolution can be achieved, together with

the possibility to apply 2D 1H-31P NMR for unambiguous assignment or

identification of P-species. The problem of hydrolysis is on the other hand

still very much present and it has been claimed that the extra time added by

the sulfide treatment is of more negative effect since it adds to the hydrolysis

time. However, we consider an increase in the fraction of hydrolysis

irrelevant, because substantial hydrolysis occurs even using the usual

NaOH/EDTA extraction conditions. Given this fact, we consider application

of 2D 1H-31P NMR as a significant advance, because it is possible to

differentiate between naturally occurring metabolites and hydrolysis

products [39]. Still, for some hydrolysis products it is not possible to deduce

the original P species. Optimizing the NaOH/EDTA extraction with the

sulfide treatment, to minimize hydrolysis, may reduce this problem. Future

work should be focused on developing extraction protocols that avoid

hydrolysis, while maintaining the efficiency in extracting soil organic P.

Application of the new methodology produced well-resolved 1D 31P NMR

spectra for all soils that were analyzed. The 2D 1H-31P spectra revealed that

overlap can occur even in seemingly well-resolved 1D 31P spectra, allowed

identification of several new organic P species. Furthermore, 2D spectra gave

hints concerning the structure of some unknown P species, which remain to

be identified to extend the chemical shift collection for soil P species, and to

increase our understanding of soil P speciation. Results on boreal soils

indicated that concentrations of P species can be related to their sorption

properties, and that a large fraction of extractable P originated from living

biomass. Similar conclusions could be inferred from a completely different

system, dry tropical soils from Burkina Faso, where P species originating

from microbes were more abundant under the canopies of scattered trees in

the agroforestry parklands in soils with high sorption properties. Thus, I

finally conclude that despite remaining challenges in the 31P NMR method, it

37

can reveal interesting insight concerning ecosystem properties and

processes.

38

Acknowledgements

Fem år ganska precis har passerat och jag, projektet och mycket annat har

utvecklats och förändrats. Om det är till det bättre låter jag andra avgör. Vi

är ju ändå i en bransch där ”granskning av ens jämlikar” utgör en av

hörnstenarna. Under denna tid har jag träffat på många människor, sett nya

platser av vår planet och även fått delta i olika aktiviteter som äggskjutning,

hundspann och konferenser.

Inkörsporten till allt det här kommer från en av mina studiekompisar som

också sökte sig till det akademiska livet som doktorand. Shahin

Noorbakhsh, som han heter, hade i maj 2009 blivit kallad till intervju hos

professor Gerhard Gröbner men hade samtidigt också blivit erbjuden en

doktorandtjänst på SLU i Uppsala. Han visste att jag också letade tjänst och

ringde därför mig och frågade om han skulle lämna mitt namn istället. Jag

svarade naturligtvis ja. Efter ett tag fick jag ett samtal eller ett mail från

Gerhard som undrade om jag kunde komma förbi universitetet på en

intervju med honom och Ulrik Ilstedt, en av mina assisterande handledare.

På den vägen är det. I början på juni erbjöds jag tjänsten, tackade ja och

kunde sedan börja planera för ett nytt kapitel i mitt liv. Tillsammans med

tjänsten var det viktigaste för mig just då att jag hann berätta nyheten för

min pappa som då var svårt sjuk och senare dog i mitten på juli. Jag är

mycket tacksam mot Shahin för att han tänkte på mig och lämnade mitt

namn till Gerhard. I samband med det vill jag också uttrycka min

tacksamhet för Centrum för Miljöforskning som slängt upp en ansenlig

summa pengar för att betala för åtminstone de första fyra åren av min tid

som doktorand. Ett tack också till andra donatorer.

Gerhard, du tog in mig i ett projekt som utvecklades på vägen till att handla

om nästan allt annat än ditt eget huvudområde, fast fas NMR. Trots det har

det alltid funnits en generös, snäll och rolig handledare nära till hands med

”immer” något att säga om vad som än avhandlades. Jag fick en chans till

något spännande av dig och jag hoppas att du inte blivit besviken utan att jag

på något sätt bidragit till dig och ditt arbete på samma sätt som du har gett

mig.

Jag har över åren kommit att jobba mycket med Jürgen Schleucher då

flera av delprojekten inneburit att han är inblandad från start. Han har i

perioder varit huvudhandledare tillsammans med Gerhard kan man säga och

för det är jag tacksam.

39

Redan från början var det bestämt att ett av mina delprojekt skulle innefatta

prover från Burkina Faso och det var Ulrik Ilstedts bord. Tyvärr har det

projektet hamnat lite i bakvattnet av de andra projekten och har aldrig riktigt

fått fart. Nu är det med ändå med ett manuskript i avhandlingen och

kommer snart vara klart. Ulrik tog med mig till Burkina en vecka och det var

mycket intressant och något jag kommer att minnas med glädje.

När jag kom till universitetet i oktober 2009 fick jag börja jobba direkt

tillsammans med en post doc från Costa Rica. Ett intensivt energiknippe vid

namn Andrea Vincent. I followed her around from place to place, from

building to building in our daily work sampling, preparing, extracting,

cleaning and drying and all the different things needed to be done in our

project. I learned a lot from you and for that I am grateful.

Solomon Tesfalidet, vill jag tacka för hjälpen med ICP delarna. En

värdefull hjälp som mottagits med tacksamhet.

I mitt kontor satt det en kille vid namn Marcus Wallgren som jämt

jobbade och som i framtiden definitivt kommer bli professor. Han är en

riktigt bra kille som har hjälpt mig med mycket under min tid här och vi har

tagit en eller två öl tillsammans vid olika tillfällen. Trots att du själv

doktorerat, Marcus och flyttat till annan avdelning finner du ändå tid att

hjälpa mig och andra med allt möjligt, t.ex. korrekturläsning av min

avhandling. Jag har också haft stort utbyte av alla diskussioner då vi pratat

om allt mellan himmel och jord. Jag hoppas att det också har varit av nytta

för dig. Det har varit ett lufthål för mig.

Med tiden flyttade Martin Lidman in i kontoret, in i Gerhards grupp och

han sitter här än. Väldigt kunnig med många tankar i huvet. Roligt att du gav

dig på att brygga öl också. Det ger oss ju möjligheten till en ”collaboration

brew” i framtiden. Tack Martin och lycka till.

Till Gerhards grupp har det hört till flera andra personer med tiden. Artur

Dingeldein som är nyaste doktoranden. En glad och trevlig kille med huvet

på skaft. Lycka till i fortsättningen, Artur.

When I started in Gerhards group, Tofeeq Ur-Rehman was with us. A

modest, pleasant and bright man from Pakistan whom with his family spent

a number of years in Umeå, Sweden for his PhD. We had some interesting

discussions him and me. I wish him all the best.

Recently came Ilona Dudka to our group. Yes, she is another person not

working with soil and stuff like me, but a nice easy going person. She also

40

jumps up fast and eager when we come in the corridor when it is fika time. A

good swedish quality. Good luck, Ilona.

Ximena Aguilar en härlig tjej som efter ett tag försvann upp till Med chem

vilket var en förlust för oss men en vinst för Med chem. Vi peppade varandra

när det gick tungt och en klapp på axeln när det gick bättre. Jag hade ändå

turen att få ta del av Ximenas kunskaper från hennes expertisområde i

pikobryggeriet Umeå Brygghus där hon gjorde ett positivt avtryck, tack

Ximena.

Alexander Christiansen som tillbringade ett par trevliga nyårsaftnar hos

oss och Moritz Niemiec hans kompanjon som fortfarande jobbar på. Tack

för den här tiden.

Lina Nilsson som kom, såg och for vidare. En till som helt plötsligt

hamnade hos Med chem. Glad och trevlig och saknad hos oss. Lycka till,

Lina.

Jörgen Åden och jag har spenderat mången lunch och fikastund

snackandes om allt från vapen till takplåt. Hjälp med både det ena och det

andra har han också kunnat erbjuda. Tack för den här tiden Jörgen och lycka

till.

Therese Mikaelsson och jag hamnade till slut i samma kontor och glad för

det är jag. Vi har också spenderat många luncher vid samma bord och det

har blivit mycket jaktprat med tiden. Lycka till, Therese och jag hoppas att

du hittar något bra jobb i Vindeln eller närområdet.

Jag har också fått spela lite innebandy under åren. Med betoning på lite. Det

var Tobias Sparrman, NMR-oumbärlig och fika-kungen som tyckte att jag

skulle vara med och spela och javisst tänkte jag. Sen bröt jag knät och sen var

det slut med den aktiviteten. Jag är skyldig Tobias ett stort tack för all hjälp

jag fått av honom under åren med oscilloskop, prober, och allt annat NMR

relaterat du hjälpt mig med. I owe you, som man säger i vissa delar av

världen. Tack för allt, Tobias.

Thank you, Ina Ehlers for the conversations here, in Germany and in

Austria. We have both been under the supervision of Jürgen and we started

almost the same time. I hope your work moves along and that your

dissertation is just around the corner.

Sen är det en hel del andra personer som passerat under åren. Xiang, Linh,

Dat, Konrad, Agnieszka, Lenka, Janek, Robert, Matteus, Ulrika, Christoph,

41

osv osv. Kan vara någon som jag just nu inte kommer ihåg som jag efter

tryck kommer att tänka på och då känns det jobbigt men vet detta;

Någonstans är det någon som kommer ihåg dig.

När jag började var det mycket folk i korridoren. Vi var ett helt gäng

forskargrupper som fikade, samtalade, hade kul och arbetade tillsammans.

Trots att det inte var någon som jobbade med jord lärde jag känna många av

er och kommer att komma ihåg många av er med värme.

Till sist vill jag också tacka min familj, min fru Karin mina söner David,

Hannes och Adam för att ni inte gjort det hela för svårt för mig utan

funnits där i vått och i torrt.

”Chansen kom och jag tog den……..”

42

References

1. Carpenter, S.R., et al., Nonpoint Pollution Of Surface Waters With Phosphorus And Nitrogen. Ecological Applications, 1998. 8(3): p. 559-568.

2. Ryther, J.H. and W.M. Dunstan, Nitrogen,Phosphorus, And

Eutrophication In Coastal Marine Environment. Science, 1971. 171(3975): p. 1008.

3. Elser, J.J., et al., Global Analysis of Nitrogen and Phosphorus Limitation of Primary Producers in Freshwater, Marine and Terrestrial Ecosystems. Ecology Letters, 2007. 10(12): p. 1135-1142.

4. Hinsinger, P., Bioavailability Of Soil Inorganic P In The Rhizosphere As Affected By Root-Induced Chemical Changes: A Review. Plant and Soil, 2001. 237(2): p. 173-195.

5. Brown, G.E., et al., Metal Oxide Surfaces And Their Interactions

With Aqueous Solutions And Microbial Organisms. Chemical Reviews, 1999. 99(1): p. 77-174.

6. Goldberg, S. and G. Sposito, On The Mechanism Of Specific Phosphate - Adsorption By Hydroxylated Mineral Surfaces - A Review. Communications in Soil Science and Plant Analysis, 1985. 16(8): p. 801-821.

7. Frossard, E., et al., Processes Governing Phosphorus Availability In Temperate Soils. Journal of Environmental Quality, 2000. 29(1): p. 15-23.

8. Li, W., et al., Surface Speciation of Phosphate on Boehmite

(gamma-AlOOH) Determined from NMR Spectroscopy. Langmuir, 2010. 26(7): p. 4753-4761.

9. Wyant, K.A., J.R. Corman, and J.J. Elser, eds. Phosphorus, Food, And Our Future. 2013, Oxford University Press: New York.

10. Ashley, K., D. Cordell, and D. Mavinic, A Brief History Of Phosphorus: From The Philosopher's Stone To Nutrient Recovery And Reuse. Chemosphere, 2011. 84(6): p. 737-746.

11. Tilman, D., et al., Forecasting Agriculturally Driven Global

Environmental Change. Science, 2001. 292(5515): p. 281-284.

43

12. Magid, J., H. Thiessen, and L.M. Condron, Dynamics Of Organic Phosphorus In Soils Under Natural And Agricultural Ecosystems., in Humic Substances In Terrestrial Ecosystems, A. Piccolo, Editor. 1996, Elsevier: Amsterdam. p. 429-466.

13. Cordell, D., J.O. Drangert, and S. White, The Story Of Phosphorus:

Global Food Security And Food For Thought. Global Environmental Change-Human and Policy Dimensions, 2009. 19(2): p. 292-305.

14. Gilbert, N., The Disappearing Nutrient. Nature, 2009. 461(7267): p. 1041-1041.

15. Wardle, D.A., L.R. Walker, and R.D. Bardgett, Ecosystem Properties And Forest Decline In Contrasting Long-Term Chronosequences. Science, 2004. 305(5683): p. 509-513.

16. Walker, T.W. and J.K. Syers, Fate Of Phosphorus During

Pedogenesis. Geoderma, 1976. 15(1): p. 1-19.

17. Chadwick, O.A., et al., Changing Sources Of Nutrients During Four Million Years Of Ecosystem Development. Nature, 1999. 397(6719): p. 491-497.

18. Sanchez, P.A., Ecology - Soil Fertility And Hunger In Africa. Science, 2002. 295(5562): p. 2019-2020.

19. Giesler, R., et al., Phosphate Sorption In Aluminum- And Iron-Rich

Humus Soils. Soil Science Society of America Journal, 2005. 69(1): p. 77-86.

20. Giesler, R., T. Petersson, and P. Hogberg, Phosphorus Limitation In Boreal Forests: Effects Of Aluminum And Iron Accumulation In The Humus Layer. Ecosystems, 2002. 5(3): p. 300-314.

21. Giesler, R., et al., Microbially Available Phosphorus In Boreal Forests: Effects Of Aluminum And Iron Accumulation In The Humus Layer. Ecosystems, 2004. 7(2): p. 208-217.

22. FAO, Production Yearbook 1999, in Statistical Series. 1999, Food

And Agricultural Organisation Of The United Nations: Rome.

23. Pettersson, E., Predictive Functions For Impact Of Nitrogen Fertilization On Growth Over Five Years. 1994, Forest Research Institute of Sweden. p. 1-56.

44

24. Giesler, R., M. Hogberg, and P. Hogberg, Soil Chemistry And Plants In Fennoscandian Boreal Forest As Exemplified By A Local Gradient. Ecology, 1998. 79(1): p. 119-137.

25. Liu, Y.-T. and D. Hesterberg, Phosphate Bonding on Noncrystalline

Al/Fe-Hydroxide Coprecipitates. Environmental Science & Technology, 2011. 45(15): p. 6283-6289.

26. Lindegren, M. and P. Persson, Competitive Adsorption Between Phosphate And Carboxylic Acids: Quantitative Effects And Molecular Mechanisms. European Journal of Soil Science, 2009. 60(6): p. 982-993.

27. Turner, B.L., et al., Soil Organic Phosphorus Transformations During Pedogenesis. Ecosystems, 2007. 10(7): p. 1166-1181.

28. Turner, B.L., et al., Inositol Phosphates In The Environment.

Philosophical Transactions of the Royal Society of London Series B-Biological Sciences, 2002. 357(1420): p. 449-469.

29. Jansson, M., et al., Terrestrial Carbon And Intraspecific Size-Variation Shape Lake Ecosystems. Trends in Ecology & Evolution, 2007. 22(6): p. 316-322.

30. Cross, A.F. and W.H. Schlesinger, A Literature-Review And Evaluation Of The Hedley Fractionation - Applications To The Biogeochemical Cycle Of Soil-Phosphorus In Natural Ecosystems. Geoderma, 1995. 64(3-4): p. 197-214.

31. Turner, B.L., Soil Organic Phosphorus In Tropical Forests: An

Assessment Of The NaOH-EDTA Extraction Procedure For Quantitative Analysis By Solution P-31 NMR Spectroscopy. European Journal of Soil Science, 2008. 59(3): p. 453-466.

32. Celi, L. and E. Berberis, Abiotic Stabilization Of Organic Phosphorus In The Environment, in Organic Phosphorus In The Environment, B.L. Turner, E. Frossard, and D.S. Baldwin, Editors. 2005, CAB International: Wallingford. p. 113-132.

33. Lagerstrom, A., et al., Soil Phosphorus And Microbial Response To A Long-Term Wildfire Chronosequence In Northern Sweden. Biogeochemistry, 2009. 95(2-3): p. 199-213.

34. Sugden, A., R. Stone, and C. Ash, Ecology In The Underworld -

Introduction. Science, 2004. 304(5677): p. 1613-1613.

45

35. Martin, C.J., Reaction Of The Coordinate-Complexes Of Inositol Hexaphosphate With First Row Transition Series Cations And Cd(Ii) With Calf Intestinal Alkaline-Phosphatase. Journal of Inorganic Biochemistry, 1995. 58(2): p. 89-107.

36. Martin, C.J. and W.J. Evans, Phytic Acid - Divalent-Cation

Interactions .3. A Calorimetric And Titrimetric Study Of The Ph-Dependence Of Copper(Ii) Binding. Journal of Inorganic Biochemistry, 1986. 28(1): p. 39-55.

37. Condron, L.M., B.L. Turner, and B.J. Cade-Menun, Chemistry And Dynamics Of Soil Organic Phosphorus, in Phosphorus: Agriculture And The Environment, J.T. Sims and A.N. Sharpley, Editors. 2005, American Society of Agronomy: Madison, WI. p. 87-121

38. Simpson, A.J., D.J. McNally, and M.J. Simpson, NMR Spectroscopy In Environmental Research: From Molecular Interactions To Global Processes. Progress in Nuclear Magnetic Resonance Spectroscopy, 2011. 58(3-4): p. 97-175.

39. Vestergren, J., et al., High-Resolution Characterization Of Organic

Phosphorus In Soil Extracts Using 2D 1H-31P NMR Correlation Spectroscopy. Environ Sci Technol, 2012. 46(7): p. 3950-3956.

40. Van Emmerik, T.J., et al., P-31 Solid-State Nuclear Magnetic Resonance Study Of The Sorption Of Phosphate Onto Gibbsite And Kaolinite. Langmuir, 2007. 23(6): p. 3205-3213.

41. Lindstrom, F., P.T.F. Williamson, and G. Grobner, Molecular Insight Into The Electrostatic Membrane Surface Potential By N-14/P-31 MAS NMR Spectroscopy: Nociceptin-Lipid Association. Journal of the American Chemical Society, 2005. 127(18): p. 6610-6616.

42. Nordgren, A., A Method For Determining Microbially Available-N

And Available-P In An Organic Soil. Biology and Fertility of Soils, 1992. 13(4): p. 195-199.

43. Ilstedt, U. and S. Singh, Nitrogen And Phosphorus Limitations Of Microbial Respiration In A Tropical Phosphorus-Fixing Acrisol (Ultisol) Compared With Organic Compost. Soil Biology & Biochemistry, 2005. 37(7): p. 1407-1410.

44. Bonev, B., et al., Electrostatic Peptide-Lipid Interactions Of Amyloid-Beta Peptide And Pentalysine With Membrane Surfaces

46

Monitored By P-31 MAS NMR. Physical Chemistry Chemical Physics, 2001. 3(14): p. 2904-2910.

45. Derome, A.E., Modern Nmr Techniques For Chemistry Research.

Tetrahedron Organic Chemistry Series. Vol. 6. 1991: Pergamon Press.

46. Newman, R.H. and K.R. Tate, Soil-Phosphorus Characterization By P-31 Nuclear Magnetic-Resonance. Communications in Soil Science and Plant Analysis, 1980. 11(9): p. 835-842.

47. Cade-Menun, B. and C.W. Liu, Solution Phosphorus-31 Nuclear Magnetic Resonance Spectroscopy of Soils from 2005 to 2013: A Review of Sample Preparation and Experimental Parameters. Soil Science Society of America Journal, 2014. 78(1): p. 19-37.

48. Turner, B.L., et al., Extraction Of Soil Organic Phosphorus. Talanta,

2005. 66(2): p. 294-306.

49. Hedley, M.J., J.W.B. Stewart, and B.S. Chauhan, Changes In Inorganic And Organic Soil-Phosphorus Fractions Induced By Cultivation Practices And By Laboratory Incubations. Soil Science Society of America Journal, 1982. 46(5): p. 970-976.

50. Cade-Menun, B.J. and C.M. Preston, A Comparison Of Soil Extraction Procedures For P-31 NMR Spectroscopy. Soil Science, 1996. 161(11): p. 770-785.

51. Cade-Menun, B.J., et al., Soil And Litter Phosphorus-31 Nuclear

Magnetic Resonance Spectroscopy: Extractants, Metals, And Phosphorus Relaxation Times. Journal Of Environmental Quality, 2002. 31(2): p. 457-465.

52. Cade-Menun, B.J., et al., Refining P-31 Nuclear Magnetic Resonance Spectroscopy For Marine Particulate Samples: Storage Conditions And Extraction Recovery. Marine Chemistry, 2005. 97(3-4): p. 293-306.

53. Dong, L., et al., Investigation Into Organic Phosphorus Species in Sediments of Baiyangdian Lake in China Measured by Fractionation and P-31 NMR. Environmental Monitoring and Assessment, 2012. 184(9): p. 5829-5839.

54. Doolette, A.L., R.J. Smernik, and W.J. Dougherty, Spiking Improved

Solution Phosphorus-31 Nuclear Magnetic Resonance Identification

47

of Soil Phosphorus Compounds. Soil Science Society of America Journal, 2009. 73(3): p. 919-927.

55. Turner, B.L., N. Mahieu, and L.M. Condron, Phosphorus-31 Nuclear

Magnetic Resonance Spectral Assignments Of Phosphorus Compounds In Soil NaOH-EDTA Extracts. Soil Science Society of America Journal, 2003. 67(2): p. 497-510.

56. Makarov, M.I., L. Haumaier, and W. Zech, Nature Of Soil Organic Phosphorus: An Assessment Of Peak Assignments In The Diester Region Of P-31 NMR Spectra. Soil Biology & Biochemistry, 2002. 34(10): p. 1467-1477.

57. McDowell, R.W. and I. Stewart, Peak Assignments For Phosphorus-31 Nuclear Magnetic Resonance Spectroscopy In Ph Range 5-13 And Their Application In Environmental Samples. Chemistry and Ecology, 2005. 21(4): p. 211-226.

58. Vincent, A.G., et al., Soil Organic Phosphorus Transformations In A

Boreal Forest Chronosequence. Plant and Soil, 2013. 367(1-2): p. 149-162.

59. Hupfer, M., B. Rube, and P. Schmieder, Origin And Diagenesis Of Polyphosphate In Lake Sediments: A P-31-NMR Study. Limnology and Oceanography, 2004. 49(1): p. 1-10.

60. Paytan, A., et al., Selective Phosphorus Regeneration Of Sinking Marine Particles: Evidence From P-31-NMR. Marine Chemistry, 2003. 82(1-2): p. 55-70.

61. He, Z.Q., et al., Phosphorus Forms In Conventional And Organic

Dairy Manure Identified By Solution And Solid State P-31 NMR Spectroscopy. Journal of Environmental Quality, 2009. 38(5): p. 1909-1918.

62. Cade-Menun, B.J., J.A. Navaratnam, and M.R. Walbridge, Characterizing Dissolved And Particulate Phosphorus In Water With P-31 Nuclear Magnetic Resonance Spectroscopy. Environmental Science & Technology, 2006. 40(24): p. 7874-7880.

63. Doolette, A.L., R.J. Smernik, and W.J. Dougherty, Overestimation Of The Importance Of Phytate In NaOH-EDTA Soil Extracts As Assessed By (31)P NMR Analyses. Organic Geochemistry, 2011. 42(8): p. 955-964.

48

64. Smernik, R.J. and W.J. Dougherty, Identification Of Phytate In Phosphorus-31 Nuclear Magnetic Resonance Spectra: The Need For Spiking. Soil Science Society of America Journal, 2007. 71(3): p. 1045-1050.

65. Chamignon, C., et al., Mobility Of Organic Pollutants In Soil

Components. What Role Can Magic Angle Spinning NMR Play? European Journal of Soil Science, 2008. 59(3): p. 572-583.

66. Pascaud, P., et al., Interaction between a Bisphosphonate, Tiludronate, and Biomimetic Nanocrystalline Apatites. Langmuir, 2013. 29(7): p. 2224-2232.

67. Clark, L.L., E.D. Ingall, and R. Benner, Marine Phosphorus Is Selectively Remineralized. Nature, 1998. 393(6684): p. 426-426.

68. Vestergren, J., et al., Adsorption Dynamics Of Phosphate Species On

Ion Exchange Resin – A 31p Nmr Study. in preparation.

69. Lash, T.D. and S.S. Lash, The Use Of Pascal-Like Triangles In Describing 1st-Order NMR Coupling Patterns. Journal of Chemical Education, 1987. 64(4): p. 315-315.

70. Smith, S.A. and N. Murali, Relaxation Effects In A System Of A Spin-1/2 Nucleus Coupled To A Quadrupolar Spin Subjected To RF Irradiation: Evaluation Of Broadband Decoupling Schemes. Journal of Magnetic Resonance, 1999. 136(1): p. 27-36.

71. Bodenhausen, G. and D.J. Ruben, Natural Abundance N-15 NMR by

Enhanced Heteronuclear Spectroscopy. Chemical Physics Letters, 1980. 69(1): p. 185-189.

72. Aue, W.P., E. Bartholdi, and R.R. Ernst, 2-Dimensional Spectroscopy - Application To Nuclear Magnetic-Resonance. Journal of Chemical Physics, 1976. 64(5): p. 2229-2246.

73. Braunschweiler, L. and R.R. Ernst, Coherence Transfer By Isotropic Mixing - Application To Proton Correlation Spectroscopy. Journal of Magnetic Resonance, 1983. 53(3): p. 521-528.

74. West, M., et al., Atomic Spectrometry Update-X-Ray Fluorescence

Spectrometry. Journal of Analytical Atomic Spectrometry, 2010. 25(10): p. 1503-1545.

49

75. Cultrone, G., et al., Iberian Ceramic Production From Basti (Baza, Spain): First Geochemical, Mineralogical And Textural Characterization. Archaeometry, 2011. 53: p. 340-363.

76. Nordin, A., P. Hogberg, and T. Nasholm, Soil Nitrogen Form And

Plant Nitrogen Uptake Along A Boreal Forest Productivity Gradient. Oecologia, 2001. 129(1): p. 125-132.

77. Högberg, P., Interactions Between Hillslope Hydrochemistry, Nitrogen Dynamics And Plants In Fennoscandian Boreal Forest, in Global Biogeochemical Cycles In The Climate System, E.D. Schulze, M. Heimann, and S. Harrison, Editors. 2001, Academic Press: San Diego, CA. p. 237-256.

78. Vincent, A.G., et al., Changes In Organic Phosphorus Composition In Boreal Forest Humus Soils: The Role Of Iron And Aluminium. Biogeochemistry, 2012. 108(1-3): p. 485-499.

79. Shand, C.A., et al., Solid-Phase P-31 NMR Spectra Of Peat And

Mineral Soils, Humic Acids And Soil Solution Components: Influence Of Iron And Manganese. Plant and Soil, 1999. 214(1-2): p. 153-163.

80. Russel, E.W., Soil Conditions and Plant Growth. 11th Ed. ed. 1988, Harlow, UK: Longman Scientific & Technical.