Introduction
On the global scale, readily available sources of phosphorus (P), a crucial
macronutrient element for agricultural production, are being faced with
scarcity and overpricing (Scholz et al., 2013; Van Vuuren et al., 2010).
Environmental pollution frequently arises from their impurities (Cd, U; Hartley et al., 2013; Kratz et al., 2016) and from over-fertilization
(Rubaek et al., 2013). Further problems are the quick formation of stable
and inaccessible compounds that limit plant P uptake (Shen et al., 2011) and
the low agronomic efficiency of no more than 15 % of fertilizer P in the
first year of application (Schnug et al., 2003). Many recent studies have
targeted sustainable agriculture through improving P availability from
applied fertilizers (Delgado et al., 2002; Schröder et al., 2011),
increasing P-uptake efficiency from organic and inorganic P pools in the
soil (Kaur and Reddy, 2014) and developing new technologies for P recycling
from human and animal waste (Siebers and Leinweber, 2013; Herzel et al.,
2016). Particular attention has been paid to the oxidation process, e.g., by
thiobacilli of elemental sulfur to sulfuric acid, in order to enhance the
solubility of non-water-soluble P from rock phosphates (Powers, 1923; Lee et
al., 1987; Fan et al., 2002) or meat and bone ashes (Schnug et al., 2003).
As an economically and environmentally attractive example, pyrolyzed animal
bone chips branded as “bone char” (BC), a slow-release apatite-based
P fertilizer, have been surface modified by sulfur (S) compounds to enhance
its solubility in neutral to alkaline soils. Incubation-leaching and pot
experiments confirmed that surface-modification was an effective approach in
P-release promotion from BC fertilizer (Morshedizad et al., 2016; Zimmer, D.
and Panten, K., personal communication). Such an in situ digestion of an
apatitic phosphate with elemental S was first described by Fan et al. (2002, 2012).
Despite these attempts to raise the dissolution and use efficiency of BC in
supplying P for crop requirements, a considerable fraction of applied BC–P
to the soil remains insoluble in the short term and is not taken up by
plants over the entire cropping period. A detailed P speciation can clarify
the fate of insoluble P from BC, which has not been done before.
Chemical speciation is described as the analytical identification of chemical
species of defined elements and measuring their quantities in the system
(Templeton et al., 2000). The precise characterization of various P species
in the soil as a dynamic response to nonequilibrium conditions imposed by
human activities, such as fertilization, can support a better understanding of
reactivity, stability and particularly the plant accessibility of different
P forms and provide a basis for best management practices. Several
techniques, such as sequential fractionation (Dieter et al., 2010; Condron
and Newman, 2011), nuclear magnetic resonance (NMR) spectroscopy (Liu et
al., 2009; Vestergren et al., 2012; Ahlgren et al., 2013), Raman
spectroscopy (Lanfranco, 2003; Vogel et al., 2013) and chromatography
coupled to mass spectroscopy (De Brabandere et al., 2008; Paraskova et al.,
2015), have been developed for P-speciation analysis in soil and sediments.
Each one of these techniques can offer specific advantages and disadvantages
depending on the phase and complexity of sample matrixes (Kruse et al., 2015).
Complementarily, X-ray absorption near-edge structure (XANES) spectroscopy
is well suited for the identification of various P species through
the fingerprinting of molecular structures in solid and heterogeneous mediums
based on fine features and the position of absorbing edges (Kelly et al., 2008;
Kizewski et al., 2011). The advantages of XANES spectroscopy for soil
samples make it a promising technique for direct and in situ P speciation with no
pretreatment and minimal sample manipulation (Toor et al., 2006; Kelly et
al., 2008).
According to the best of our knowledge, no studies have characterized
P-speciation changes in BC particles over nonequilibrium conditions in the
soil system, and only few investigations have been reported on the P release
from BC and alteration in P species of the soil. Siebers et al. (2013)
investigated K-edge XANES spectroscopy on BC-incubated soil samples and
provided evidence that the increase in extractable Ca and Mg phosphate
fractions were related to the contribution of hydroxyapatite (HAP) increase
after BC application. Accordingly, the objective of this study was to
provide practical information on the fate and alteration of P species in BC
and novel surface-modified BC (BCplus) particles and their treated
soils under incubation-leaching and ryegrass cultivation practices using
sequential P fractionation and P XANES spectroscopy.
Materials and methods
Incubation-leaching experiment
Two particle size fractions (1–2 and 2–4 mm) of bone chars were incubated with a silt loam soil
(BC produced by the pyrolysis of degreased animal bone chips at 800 ∘C and
BCplus as a surface-modified BC obtained by blending with reduced
S-containing compounds composed of 60 % elemental S, 30 % calcium
sulfate dehydrate and 10 % methanesulfonate (Zimmer et al., unpublished
results of S X-ray absorption near-edge fine structure spectroscopy) in a
commercial biogas desulfurization process; patent application DE 212012000046U1;
http:www.google.com/patents/DE212012000046U1?cl=en&hl=de).
The soil was classified as Dystric Cambisol (FAO) with a
pH of 4.7 (measured in 0.01 molL-1 CaCl2 solution) and total
(digestion with HNO3 and analyzed using ICP-OES; USEPA, 1997) and
available (extracted by 1 molL-1 NH4NO3 and analyzed using
ICP-OES; He and Singh, 1993) P contents of 1.6 gPkg-1 and
14 mgPkg-1, respectively. The BC and BCplus contained a total P of 149 and
123 gkg-1, total calcium (Ca) of 185 and 265 gkg-1 and total S of 6 and
199 gkg-1 with average pHCaCl2 values of 7.8 and 4.9,
respectively.
The BCs were added to 30 g of air dry soil (< 2 mm) at levels of
0 mgPkg-1 soil (control) and 500 mgPkg-1 in five
replicates. The soil and BC–BCplus mixture was homogenized and packed into glass
columns with a 10 cm length and an inner diameter of 2 cm. A P-free filter (MN 616 G; Macherey-Nagel GmbH & Co., KG Düren, Germany) was placed at
the bottom of each column to avoid any particle losses. The amended soils
were incubated for 70 days at 20 ∘C in the dark and
constant soil moisture between 60 and 70 % of soil-water-holding capacity.
During the incubation period, the soil columns were leached with three pore
volumes of deionized water added by a droplet irrigation simulator system.
The leaching process was repeated in five steps, each one after 1, 5, 13, 34
and 70 days. The P concentrations in collected leachates were measured using
inductively coupled plasma–optical emission spectrometry (ICP-OES). The outcomes
of the leaching experiment were described in Morshedizad and Leinweber (2017). After the incubation-leaching experiment, the treated soil samples
were carefully removed from the glass columns and air-dried, and the BC–BCplus particles
were manually separated from the soils very gently. The BC–BCplus particles were
delicately washed with deionized water to remove adhered soil particles,
allowed to dry completely at ambient conditions and finely ground for
further analyses.
Pot experiment with annual ryegrass
The same BC and BCplus as described for the incubation-leaching
experiment were used in original sizes (mostly between 1 and 5 mm) for the P
fertilization of annual ryegrass in a pot experiment. The experiment was
set up using an acidic sandy silt soil with an available P content of
24.2 mgPkg-1 and a pH of 5.2. The pot experiment was set up by
adding BC and BCplus at the levels of 0 mgPkg-1
(control) and 280 mgPkg-1 into the 6 kg of the soil dry
matter in each pot and in four replicates arranged in a completely randomized
block. After 4 weeks of incubation at field capacity water content and
ambient temperature conditions, 30 seeds of annual ryegrass per pot were
sown on 13 May 2016. The experiment was conducted in a glasshouse
under ambient air and temperature conditions and the soil moisture was
maintained at field capacity during the whole experiment. All other essential
nutrients were sequentially added at sufficient levels before seeding and
after six cuts of ryegrass between 23 June and 3 November 2016. Finally, after the last harvest (the seventh), plant parts
(shoots and roots) were dried at 60 ∘C and BC particles were
manually separated from the soils (as they could be detected visually by their
size and dark color) very gently using tweezers. Then these particles were
washed delicately with deionized water to remove attached soil particles,
allowed to dry completely at ambient conditions and finely ground to fine
powders for further analyses.
Sequential phosphorus fractionation
Soil samples were sequentially extracted based on chemical solubility in
order according to a modified Hedley et al. (1982) procedure. After BC–BCplus
particle detachment, duplicate 0.5 g fine-ground and air-dried soil samples
were weighed into 50 mL centrifuge tubes. In summary, chemical P
fractionation included the following steps.
The mobile and readily available P fraction was extracted with resin
strips (saturated in 0.5 M NaHCO3) after 18 h of end-over-end shaking
in 30 mL of deionized water. The resin strips were separated from
solids and the solution and washed using 50 mL of 1 M HCl to remove absorbed P. The
soil suspension was centrifuged at 2500 × g for 20 min and the
supernatant was decanted.
Next, the labile inorganic and organic fractions weakly absorbed to
mineral surfaces and some microbial P were extracted by 30 mL of
0.5 M NaHCO3, 18 h of end-over-end shaking and centrifugation at
2500 × g for 20 min. The supernatant was filtered (Whatman no. 42
filter) and collected for measurements.
The inorganic P adsorbed and bound to Al- and Fe-oxide minerals and
organic P from humic substances were extracted using 30 mL of 0.1 M NaOH
solution and repeating the second step as described above.
The relatively insoluble fraction of P bound to Ca and Mg minerals and
apatite was extracted by 30 mL of 1 M HCl in the same way as in the
previous steps.
Total P concentrations (Pt) and inorganic P (Pi) in all extracts
were measured by ICP-OES and colorimetrically (molybdenum blue method;
Murphy and Riley, 1962), respectively. The organic P (Po)
concentrations were calculated by using Pt-Pi.
Phosphorus K-edge XANES analysis
The XANES data collection for characterizing P species in all soil samples
and BC–BCplus particles was acquired at the Synchrotron Light Research Institute
(SLRI) in Nakhon Ratchasima, Thailand on the beamline 8 (BL8) of the
electron storage ring with a covering photon energy from 1.25 to 10 KeV,
electron energy operated at 1.2 GeV and a beam current of 80–150 mA (Klysubun
et al., 2012). The P K-edge XANES spectra were collected from dried and very
finely ground treated soils and particulate BC–BCplus samples that had been
diluted to P concentrations < 10 mgPkg-1 with SiO2
powder (to eliminate self-absorption effects; Prietzel et al., 2013), again
ground in an agate stone mini-mortar and spread uniformly as a thin layer on
P-free kapton tape (Lanmar Inc., Northbrook, IL, USA). Data collection was
operated in standard conditions with energy calibration by
standard pure elemental P and allocating the reference energy (E0) at
2145.5 eV using the maximum peak of the first-derivative spectrum. All spectra were recorded at
photon energies between 2045.5 and 2495.5 eV in step sizes of 5 eV (2045.5
to 2105.5 eV and 2245.5 to 2495.5 eV), 1 eV (2105.5 to 2135.5 eV and 2195.5
to 2245.5 eV) and 0.25 eV (2135.5 to 2195.5 eV) with a 13-channel germanium
detector in fluorescence mode. At least three scans were collected and
averaged for each sample.
Distribution of inorganic P (Pi), organic P (Po) and
total P (Pt) concentrations (mgPkg-1 soil) of sequentially
extracted P fractions in the soils as affected by different treatments
(treated with two particle size fractions (1–2 and 2–4 mm) and original
sizes of BC and BCplus or unfertilized soils (control) after
incubation-leaching and ryegrass cultivation experiments).
Treatment
Resin P
NaHCO3–P
NaOH–P
HCl–P
Pi
Po
Pt
Pi
Po
Pt
Pi
Po
Pt
Pi
Po
Pt
Incubation leaching
Control
47
5
52
160
99
259
565
294
859
113
10
123
BC1-2mm
56NS
7NS
63NS
163NS
108NS
271NS
578NS
303NS
881NS
140NS
56NS
196NS
BC2-4mm
50NS
7NS
57NS
161NS
105NS
266NS
574NS
301NS
875NS
121NS
40NS
161NS
BC1-2mmplus
61∗
7NS
68NS
172NS
111NS
283NS
593∗
313NS
906∗
131NS
37NS
170NS
BC2-4mmplus
50NS
7NS
57NS
160NS
104NS
264NS
574NS
298NS
872NS
115NS
21NS
135NS
Ryegrass cropping experiment
Control
4
4
8
25
27
52
75
121
196
28
5
33
BC
2NS
5NS
7NS
24NS
32NS
56NS
79NS
125NS
204NS
30NS
6NS
36NS
BCplus
6∗
5NS
11∗
35NS
27NS
62NS
85∗∗
128NS
213NS
34NS
7NS
41NS
∗ Significant at P<0.05; ∗∗ Significant at
P<0.01; NS Nonsignificant difference (treatment vs. control;
Tukey's test)
The P XANES spectra were normalized, and after merging replicates a linear
combination fitting (LCF) was performed using the ATHENA software package
(Ravel and Newville, 2005). All XANES spectral data were baseline corrected
in the pre-edge region between 2115 and 2145 eV and normalized in the post-edge
region of 2190–2215 eV. The same ranges were used for the reference P
K-edge XANES spectra to achieve consistency in the following fitting analysis
(Prietzel et al., 2016). To achieve the best compatible set of references
with each specified sample spectrum, LCF analysis was performed in the
energy range between -20 eV and +30 eV relative to the E0 using the
combinatorics function of ATHENA software to attain all possible binary,
ternary and at most quaternary combinations between all 19 P reference
spectra. The following set of reference P K-edge XANES spectra, all recorded
in SLRI under the same adjustments by Werner and Prietzel (2015) and
Prietzel et al. (2016), were used for fitting and calculations; Ca, Al and
Fe phytate, noncrystalline and crystalline AlPO4, noncrystalline and
crystalline FePO4⋅2H2O, Ca hydroxyapatite
(Ca5(OH)(PO4)3), inositol hexakisphosphate (IHP),
ferrihydrite–IHP, montmorillonite–Al–IHP, soil organic matter Al–IHP (SOM–Al–IHP),
ferrihydrite–orthophosphate, montmorillonite–Al–orthophosphate,
SOM–Al–orthophosphate, IHP, orthophosphate, CaHPO4,
Ca(H2PO4)2 and MgHPO4. To select the best possible
combination fit between the sample spectrum and the P reference spectra, the
lowest reduced chi value (χ2) and R factor were chosen.
Results
Effect of BCs on sequentially extracted P after
incubation leaching
After 70 days of incubation leaching, the sequential P fractionation of
amended soils showed variations in the amount and distribution of various
P fractions between different treatments (Table 1). For all treatments, NaOH
extracted the majority of fractionated P (62.4 to 66.5 % of total
fractionated P), followed by the labile P fraction (NaHCO3, 19.2 to
20.0 %), HCl–P (9.5 to 13.9 %) and the readily available P (resin
strips, 4.1 to 4.8 %). The BC–BCplus addition increased the total soil P pools
although the difference was significant only for the
BC1-2mmplus and BC1-2mm
treatments. The largest increase in total fractionated Pt
(resin Pt + NaHCO3–Pt + NaOH–Pt + HCl–Pt)
occurred in BC1-2mmplus
(133.8 mgPkg-1 soil), followed by BC1-2mm
(118.6 mgPkg-1 soil), BC2-4mm
(67.1 mgPkg-1 soil) and
BC2-4mmplus (35.7 mgPkg-1 soil)
compared to the control soil.
The proportion of P enrichment in each fraction varied between different
treatments in the order
NaOH–P > HCl–P > NaHCO3–P > resin P for
BC1-2mmplus and
BC2-4mmplus. For the BC1-2mm and
BC2-4mm treatment the order was
HCl–P > NaOH–P > NaHCO3–P > resin P. In all treatments,
the Pi proportions in each of the P fractions were greater than
the Po proportions. Compared to the control soil, the most
Pi increase was observed in NaOH–Pi and
resin Pi in response to BC1-2mmplus
application (Table 1). Moreover, after 70 days of incubation leaching, soil
pH increased in BC treatments whereas BCplus amendments had an
acidifying effect. Soil pH levels of BC1-2mm and BC2-4mm
increased by 0.07 and 0.05 units and decreased for
BC1-2mmplus and BC2-4mmplus
treatments by 0.21 and 0.15 units compared to unamended control soil
(pH = 5.06).
Effect of BCs on sequentially extracted P after ryegrass
cropping
Sequentially extracted P fractions in soil varied between different
treatments after 230 days of ryegrass cropping (Table 1). In all treatments
(control, BC and BCplus), NaOH–P was the largest P pool mainly
associated with Al- and Fe-oxide minerals and humic substances (65.0 to
67.5 % of total fractionated P), followed by the NaHCO3–P (18.2 to
19.0 %), HCl–P (11.5 to 12.6 %) and resin P (2.2 to 3.4 %)
fractions. Enrichments of P fractions in BCplus treatments were
more pronounced than in treated soils with BC particles. In this treatment
the concentrations of readily available and labile inorganic P fractions were
insignificantly smaller than in the control. Additionally, a significant
increase in P concentration was obtained only in resin Pi and
NaOH–Pi fractions of the BCplus-treated soil
(Table 1). The maximum increase in total fractionated P was obtained in
the BCplus treatment (37.6 mgPkg-1 soil). In
comparison to incubation-leaching results, a similar sequence was observed
for the order of increasing magnitude of P fractions in response to BC and
BCplus amendments
(NaOH–P > NaHCO3–P > HCl–P > resin P). However, for BC
treatment, the total P extracted by resin strips was lowered in comparison
with the control. In the control and BC treatments, Po was the
predominant form in NaOH–P and NaHCO3–P fractions, while for
BCplus it was only in the NaOH–P fraction. Each P fraction was
highest under BCplus application, except for
NaHCO3–Po in the BC treatment.
Separately, the effect of BC and BCplus application on ryegrass
yield parameters was examined in the 230-day pot experiment. The results
indicated that the P uptake, ryegrass yield and apparent nutrient recovery
efficiency (ANR) of BCplus treatments exceeded those of the BC and
control treatments and increased to values comparable with triple superphosphate (TSP) fertilizer (Zimmer, D. and Panten, K., personal
communication). The addition of BC and BCplus did not significantly
change the bulk soil pH, although local acidification around
BCplus particles (pH 4.9; Morshedizad and Leinweber, 2017)
can probably lower soil pH in small-scale areas compared to BC treatments (pH
about 8).
Normalized P K-edge XANES spectra of different BC and BCplus
particle sizes (1–2 and 2–4 mm) before (control) and after a 70-day
incubation-leaching experiment compared to the reference compounds selected
using the LCF method.
P K-edge XANES spectra of BC and BCplus particles before
(control) and after 230 days of ryegrass cultivation compared to the
reference compounds selected by the LCF method.
XANES analysis of BC–BCplus particles
All spectra from BCs were characterized by an intense white-line peak,
a post-edge position and no distinct pre-edge, which corresponded to
calcium phosphate compounds including Ca hydroxyapatite, dicalcium phosphate
(CaHPO4) and Ca phytate (Fig. 1). The P K-edge XANES results
indicated no obvious alterations in the spectral features of BC–BCplus particles after
the incubation-leaching experiment. After 70 days of incubation leaching, the
BC spectra were shifted towards Ca hydroxyapatite, and this was more
pronounced for the 2–4 mm than for the 1–2 mm BC particles. The opposite
trend was the case for BCplus particles; the white-line
signal intensity decreased after the incubation-leaching period and the post-edge
of the spectra tended more to dicalcium phosphate. This effect was stronger for
BCplus particle size reduction from 2–4 mm to 1–2 mm.
To quantify the P speciation of BC and BCplus particles, LCF
analyses using all possible combinations were performed on all
P K-edge XANES spectra (Table 2). The fitting results indicated that
untreated BC and BCplus particles before the experiment
contained on average 61 and 60 % Ca hydroxyapatite, 22 and 30 %
CaHPO4 and 18 and 10 % Ca phytate. After 70 days of
incubation leaching, the proportion of Ca hydroxyapatite increased to the
average of 80 % in BC, while it remained unchanged in
BCplusparticles. The CaHPO4 proportion increased in
BCplus particles to the average of 34 %, whereas the lower
content was assigned in the spectra of BC particles accounting for 10 %
of total P species. Moreover, the Ca phytate proportion decreased slightly in
BC and BCplus particles from about 18 and 10 % to averages
of 11 and 7 %, respectively.
P K-edge XANES spectra of unfertilized (control) and fertilized
soils with BC and BCplus particles under a 70-day incubation-leaching
experiment compared to the reference compounds selected by the LCF method.
Results of linear combination fitting (LCF) conducted on P K-edge
XANES spectra of bone char (BC) and surface-modified bone char (BCplus)
particles before and after a 70-day incubation-leaching period. These best
fits were achieved using all possible combinations with 19 spectra of
P reference compounds.
Reference compound
Before experiment
After 70 days of incubation leaching
BC
BCplus
BC
BCplus
1–2 mm
2–4 mm
1–2 mm
2–4 mm
1–2 mm
2–4 mm
1–2 mm
2–4 mm
Ca hydroxyapatite (%)
58 ± 6
64 ± 5
62 ± 5
58 ± 5
75 ± 4
85 ± 3
59 ± 5
60 ± 6
CaHPO4 (%)
24 ± 5
19 ± 4
28 ± 4
32 ± 6
14 ± 3
5 ± 2
33 ± 4
35 ± 5
Ca phytate (%)
18 ± 4
17 ± 4
10 ± 3
10 ± 3
11 ± 3
10 ± 2
8 ± 4
5 ± 4
R factor
0.012
0.008
0.007
0.009
0.005
0.002
0.009
0.010
Results of linear combination fitting (LCF) conducted on P K-edge
XANES spectra of bone char (BC) and surface-modified bone char (BCplus)
particles before and after 230 days of ryegrass cultivation in a pot
experiment. These best fits were achieved using all possible combinations
with 19 spectra of P reference compounds.
Reference compound
After 230 days
Before experiment
ryegrass cultivation
BC
BCplus
BC
BCplus
Ca hydroxyapatite (%)
63 ± 6
70 ± 4
75 ± 4
49 ± 8
CaHPO4 (%)
29 ± 5
29 ± 3
17 ± 4
43 ± 6
Ca phytate (%)
8 ± 4
1 ± 3
8 ± 3
8 ± 5
R factor
0.012
0.005
0.006
0.018
Spectra of BC and BCplus particles before and after 230 days
of ryegrass cultivation were characterized by a sharp white line, followed by
a shoulder and then a post-edge feature between 2160 and 2175 eV, which was
divided into two peaks (Fig. 2). These features were most similar to the
P K-edge XANES spectra of Ca hydroxyapatite, CaHPO4 and Ca phytate
standard compounds. Treated BC particles had a white line with higher
intensity that appeared more similar to the Ca hydroxyapatite spectrum. In
contrast, BCplus particles under ryegrass cultivation showed a
weaker white line exhibiting the shoulder and post-edge feature more
comparable to the CaHPO4 spectrum.
Some differences in the proportions of P species observed between BC–BCplus particles
before and after the cropping period in the ryegrass pot experiment are
presented in Table 3. The LCF results revealed overall contributions of 63
and 70 % Ca hydroxyapatite, 29 and 29 % CaHPO4 and 8 and 1 %
Ca phytate in the original BC and BCplus, respectively. After the
cropping period, the percentage of Ca hydroxyapatite was increased in BC
particles. In the BCplus treatment, the percentage of CaHPO4
increased from 29 to 43, while the percentage of Ca hydroxyapatite was
reduced from 70 to 49 %. The Ca phytate proportion remained unchanged in
BC particles, while that of BCplus increased from 1 to 8 % after the
ryegrass cultivation period.
XANES analysis of soil samples
The P K-edge XANES spectra of soil samples from the incubation-leaching
experiment showed two dominant features: (1) a strong white line
lacking a pre-edge and shoulder and (2) a tailed post-edge feature (Fig. 3).
The most similarity to these features was seen in XANES spectra of amorphous
AlPO4, FePO4 and SOM–Al–IHP compounds. Distinct differences
appeared between the control and treated soil with BCplus, not
with BC treatments. This was reflected by slightly lower intensities of both
white-line and post-edge features.
The P species of treated soils in the incubation-leaching experiment were
determined by LCF analysis to select at most four reference compounds in
the combinatorics of all possible fitting combinations (Table 4). The fitting
results indicated that P in the control soil and BC treatments occurred
dominantly as amorphous AlPO4 (≈ 40 %), FePO4
(≈ 30 %) and SOM–Al–IHP (≈ 20 %) compounds. In
BCplus-treated soils, the average proportion of amorphous
AlPO4 decreased to 26 %, and instead Ca(H2PO4)2 was
identified with an average of 25 %, which did not appear in the control
and BC treatments. The LCF results showed that the soil treated with
BCplus had no detectable Ca hydroxyapatite, which was found in
the control and BC treatments.
Results of linear combination fitting (LCF) conducted on P K-edge
XANES spectra of unfertilized (control) and fertilized soils with bone char
(BC) and surface-modified bone char (BCplus) particles in the 70-day
incubation-leaching experiment. These best fits were achieved using all
possible combinations with 19 spectra of P reference compounds.
Reference compound
Control
BC treatment
BCplus treatment
1–2 mm
2–4 mm
1–2 mm
2–4 mm
Ca hydroxyapatite (%)
8 ± 1
4 ± 1
8 ± 1
0
0
AlPO4 amorphous (%)
42 ± 1
42 ± 2
40 ± 1
27 ± 1
24 ± 1
FePO4 (%)
29 ± 1
31 ± 2
31 ± 1
27 ± 1
26 ± 1
SOM–Al–IHP (%)
21 ± 2
23 ± 4
21 ± 3
26 ± 2
21 ± 1
Ca(H2PO4)2 (%)
0
0
0
20 ± 1
29 ± 1
R factor
0.0003
0.0007
0.0003
0.0005
0.0004
P K-edge XANES spectra of unfertilized (control) and fertilized
soils with BC and BCplus particles under 230 days of ryegrass
cultivation compared to the reference compounds selected by the LCF method.
The XANES spectra recorded from treated soil samples in the ryegrass pot
experiment showed the presence of an intense white line in the energy range
of 2152 to 2158 eV and a stretched post-edge feature approximately from 2165
to 2178 eV (Fig. 4). Decreases in white-line and post-edge intensities of
the soil samples appeared as an effect of BCplus application.
Visual inspection of P K-edge spectra revealed no indication of specific
alteration in spectral features in response to the BC treatment.
Amorphous AlPO4 was identified by LCF analysis as the dominant component
(≈ 35 %) in all treated soil samples from the ryegrass pot
experiment (Table 5). The second major P form in the control soil was IHP
(29 %), followed by Ca phytate (27 %), with the latter also as
pronounced as that observed for BC and BCplus treatments. All
treated soils varied in proportions of free or bound IHP forms. The
Mont–Al–PO4 and Ca(H2PO4)2 compounds were only assigned in the
control and BCplus treatments, respectively.
Results of linear combination fitting (LCF) conducted on P K-edge
XANES spectra of unfertilized (control) and fertilized soils with bone char
(BC) and surface-modified bone char (BCplus) particles under 230 days
of ryegrass cultivation in a pot experiment. These best fits were achieved
using all possible combinations with 19 spectra of P reference
compounds.
Reference compound
Control
BC treatment
BCplus treatment
AlPO4 amorphous (%)
35 ± 3
35 ± 3
34 ± 1
Ca phytate (%)
27 ± 3
28 ± 3
27 ± 1
IHP (%)
29 ± 5
21 ± 7
0
Mont–Al–IHP (%)
0
16 ± 1
0
SOM–Al–IHP (%)
0
0
25 ± 2
Mont–Al–PO4 (%)
9 ± 1
0
0
Ca(H2PO4)2 (%)
0
0
14 ± 2
R factor
0.0006
0.0008
0.0006
Discussion
P availability as revealed by sequential fractionation
The sequence of P distribution between sequentially extracted P fractions was
in accordance with findings by many studies (Cross and Schlesinger, 1995;
McDowell and Stewart, 2006; Hashimoto and Watanabe, 2014), reflecting the
general status of different P pools in acidic soils. The results indicated
that the largest P proportion was found in the NaOH fraction, reflecting P
fixed to Fe and Al oxides, followed by the NaHCO3–P fraction
assigned to P weakly absorbed on crystalline Fe and Al oxides or surface of
minerals. Guo et al. (2000) reported that the NaOH–P fraction may support the
labile NaHCO3–P fraction as a buffering P pool in highly weathered
and acidic soils. According to soil pH values (4.7 and 5.2), the larger
proportions of NaHCO3–P even than HCl–P can be explained by the
abundance and surface loadings of Fe and Al oxides that support the
electrostatic binding of phosphate ions and a scarcity of Ca and Mg minerals
or soluble ions. As expected, the lowest P proportions were found in the
mobile and readily available P fraction extracted by resin strips in
agreement with many comparable studies (Cross and Schlesinger, 1995; Bauchemin
et al., 2003; Sharpley et al., 2004; Siebers et al., 2013). Among the two
soils that were used in the two different experiments, the largest
proportions of inorganic P were achieved in the soil after the
incubation-leaching experiment, while the organic P forms were considerably
more abundant in the soil samples after ryegrass cultivation (Table 1). These
differences may be due to the microbial activities in the rhizosphere of
grasses and the transformation of Pi to more stable
Po fractions during the longer plant cultivation period (230 days)
than in the non-cropped incubation-leaching experiment (70 days).
In general, all P-fraction concentrations were elevated by adding BC and
BCplus particles, which appeared to follow the same pattern in
both soils under two different experimental conditions. However, significant
differences were found only between the control and BCplus-treated soils (1–2 mm in the incubation-leaching experiment) for the
resin-P and NaOH–P fractions. Since the BC–BCplus particles were separated from the
soils before chemical analysis, it was expected that partly dissolved BCs
would have a limited impact on different P fractions rather than totally
ground and mixed BCs. This is consistent with the study of Siebers et
al. (2013) according to which the BC application (< 90 µm BC
thoroughly mixed in soil) significantly increased the insoluble P proportion
(H2SO4–P). Additionally, our study confirmed previous findings
concerning the effect of particle sizes on the P release from BCs
(Morshedizad and Leinweber, 2017) and consequently the P status of treated
soils (Ma and Matsunaka, 2013). Sequentially extracted P contents increased
with the decreasing size of BC particles whereby BCplus treatments
appeared more dependent on particle size than BC treatments. The results of
the sequential P fractionation of BCplus treatments in the
incubation-leaching experiment indicated that the P increase was more
pronounced for P fixed to Al and Fe oxides (NaOH–P) than other fractions,
whereas for BC treatments the largest increase occurred in P bound to Ca and
Mg minerals (HCl–P). It seems that local pH changes in soil associated with
BC and BCplus amendments could eventually lead to a different
distribution of released P into different soluble or insoluble P pools,
which are generally controlled by pH (Arai and Sparks, 2007). However, due to
a lower fertilization level and a longer period of experiment in ryegrass
cultivation compared to incubation leaching, it appears that the chemical
equilibrium has been established in the soil (no significant change in bulk
soil pH), and accordingly the soil P fractions were altered minimally.
P speciation of BC–BCplus particles by XANES
The predominance of Ca hydroxyapatite in BCs as evidenced by P K-edge XANES
analysis is consistent with findings reported by previous studies (Warren et
al., 2009; Siebers et al., 2013). The mineral phase of bone consists mainly
of hydroxyapatite, and its contribution to bone and bone char compositions
depends on species, the age of animals (Wu et al., 2003), carbonization
temperature and residence time (Novotny et al., 2012). Bone crystallinity
might be improved through structural modifications on poorly crystalline
fresh bone samples (such as mineral maturity over periods of time or
intensive carbonization), which can also result in increased proportions of
hydroxyapatite and accordingly a decrease in P solubility (Novotny et al.,
2012). Based on LC fittings, the second major component of BC–BCplus particles was
CaHPO4, in good agreement with the results of Rajendran et al. (2013)
who indicated that heated bones at 400 ∘C contained some more soluble
phosphates, such as CaHPO4 and CaH2PO4, in addition to the
hydroxyapatite fraction. The authors reported that spectra of calcined bone
samples at 700 ∘C had a white line at 2154 eV and two post-edge peaks
at 2162 eV and 2169 eV, with no pre-edge peaks, and appeared similar to
CaHPO4 and CaH2PO4 spectra. Our LCF also assigned Ca phytate
in BC–BCplus samples, which seems to be controversial as a component of animal bone
materials. The P K-edge spectrum of Ca phytate is very similar to other
Ca-bound P compounds with a distinct white line and lack of a pre-edge
feature, although it is likely distinguishable due to the specific shape of
white-line tailing and the absence of a post-edge signal at 2164 eV (Prietzel et
al., 2016). Moreover, some inaccuracies in LCF estimations have to be
considered because of (1) uncertainty in the speciation of organic P forms by
K-edge XANES, (2) lack of reference compounds representing all P forms in
BCs and (3) smaller Ca phytate proportions than the proposed 10 to 15 % of
Pt as a detection limit for reliable XANES fittings (Beauchemin
et al., 2003). Therefore, the P proportions assigned to Ca phytate could also
originate from a range of other Ca–P compounds.
In both experiments, incubation leaching and ryegrass cropping, changes in
the proportions of Ca hydroxyapatite and CaHPO4 in BC particles followed an
opposite trend than in BCplus particles. After the placement of BC
particles in the soil, Ca phosphate seemed to be released gradually over
time, which provides a locally lime-saturated condition. Due to elevated pH
surrounding the BC particles, dissolved P can be resorbed to maintain
solubility and the Ca–P equilibrium constant, which likely resulted in a
decreased proportion of soluble CaHPO4 and possibly transformation
into the relatively insoluble Ca hydroxyapatite fraction. In contrast, if
BCplus particles were applied to soils, larger proportions of
CaHPO4 at the expense of Ca hydroxyapatite could be explained by soil
acidification through the microbial oxidation of released S (Lee, et al.
1987; Fan et al., 2002). This effect was more pronounced over the longer
time period in the ryegrass cropping pot experiment, favoring a greater
CaHPO4 than Ca hydroxyapatite fraction. This implies that BCplus
can actively supply P with a predominance of soluble over insoluble P forms in
the long term and thus meet crop requirements.
P speciation of treated soils by XANES
Differences between the characteristics of the two soils, dissimilar mechanisms of
incubation leaching and plant uptake, and different experiment time
durations complicate the joint interpretation of the P XANES data. In
the unfertilized soil of the incubation-leaching experiment, the proportions of P
species followed the order AlPO4 > FePO4 > SOM–Al–IHP > Ca hydroxyapatite, which did not vary despite partial changes in some
proportions after the application of both size fractions of BC particles. In
general, these results concur with the findings by Siebers et al. (2013) that
Ca hydroxyapatite proportion was slightly increased by BC application. This
could be attributed to irreversibly mixing finely ground BC in the soil
samples, whereas in the present experiments the BC particles were separated
from the soils before P speciation. Furthermore, these XANES data (Table 4
and Table 5) are in agreement with sequential P-fractionation results
(Table 1), which indicated the dominance of inorganic over organic P forms and
showed the P fractions almost unchanged after BC application. Implications of
the low solubility of BC particles observed in this work are consistent with
previous studies showing a slow release of P from BCs (Warren et al., 2009;
Siebers et al., 2013; Morshedizad et al., 2016). Besides reducing the
AlPO4 and Ca hydroxyapatite proportions, BCplus
particles introduced highly soluble Ca(H2PO4)2 to soils in the
incubation-leaching experiment. These results imply that considerable changes
in P speciation were more attributed to pH reductions, and accordingly
leaching losses of solubilized P forms compared with P enrichment by
BCplus dissolution. This is supported by results from a
previous publication in which two particle sizes of BCplus gave
a significant rise in the leached P concentration after 1, 5, 13, 34 and
70 days of incubation along with reductions in soil pH (Morshedizad and
Leinweber, 2017). This is in line with Sato et al. (2005) who found that
increasing soil pH in a naturally acidic soil (pH = 4.32) was an
effective approach to minimize P leaching, while pH decrease resulted in
the transformation of stable to soluble and more leachable P species. Regarding
the XANES results of the ryegrass cultivation experiment (Table 5), the
effect of BCplus treatment can be better explained. In the
control soil, the presence of AlPO4 and the increasing abundance of
organic P forms (Ca phytate and IHP compounds; Table 5) were consistent with
the appearance of NaOH–P and HCl–P fractions by sequential extraction
(Table 1). In the BC treatment the proportions of AlPO4 and
Ca phytate did not change compared to the control, but the contribution of
organic P increased by Mont–Al–IHP formation. The stability of different P
fractions can be favored by the pH effect (Gustafsson et al., 2012) and likewise
the dependence of BC particle solubility on the soil pH (Siebers et al.,
2013). In agreement with the incubation-leaching results (Table 4),
Ca(H2PO4)2 was detected as a result of BCplus
amendment even though similar proportions of AlPO4 and Ca phytate
were observed between the control and the BCplus treatment.
However, the data in Table 5 on the presence or absence of Ca(H2PO4)2
in soils of ryegrass experiment may have been influenced by small proportions
(< 10–15 %; reliable detection limit by XANES; Beauchemin et al.,
2003) of other simple calcium phosphates that have a spectrum similar to the
one of Ca(H2PO4)2 in the LCF analysis. The results of sequential P
fractionation and XANES analyses on treatments in the two different
experiments presented here demonstrated that the surface modification of BC
particles effectively improved soluble P fractions in BCplus
particles and consequently in amended soils.