Introduction
Phosphorus (P) is an essential nutrient for plant growth and limits
terrestrial ecosystem productivity in many arable and grassland soils (Vance
et al., 2003). The availability and transport of P depend on the speciation
and concentration of P in the soil solution, which contains both
“dissolved” and colloidal P forms (Shand et al., 2000; Hens and Merckx,
2002; Toor and Sims, 2015). Dissolved orthophosphate is generally the main P
species in solution and can be directly taken up by plant roots (Condron et
al., 2005; Pierzynski et al., 2005). However, colloidal P in the size range
of 1–1000 nm (Sinaj et al., 1998) may also contribute significantly to
total P content in the soil solution (Haygarth et al., 1997; Shand et al.,
2000; Hens and Merckx, 2001). Recent studies found that fine colloids
(< 450 nm fraction) in soil water extracts consisted of nano-sized
(< 20 nm) and small-sized (20 < d < 450 nm) particles with
different organic matter and elemental composition (Henderson et al., 2012;
Jiang et al., 2015a). Very fine nano-sized P colloids, around 5 nm are even
prone to plant uptake (Carpita et al., 1979). In addition, the presence of
fine colloids alters the free ionic P content in the soil solution through
sorption processes (Montalvo et al., 2015). After diffusion-limited uptake
depletes the free ionic P in the soil solution, these fine colloids disperse
in the diffusion layer and therewith re-supply free ionic P species for roots
(Montalvo et al., 2015). Because water-dispersible colloids (WDCs) can be
easily released from soil in contact with water (Jiang et al., 2012; Rieckh
et al., 2015), they have also been suggested as model compounds for mobile
soil colloids (de Jonge et al., 2004; Séquaris et al., 2013). However, little
is known about the chemical composition of P species in different-sized WDCs.
Recent studies have started to characterize natural fine colloidal P in
freshwater samples and soil water extracts using asymmetric flow field-flow
fractionation (AF4) coupled to various detectors (e.g., ultraviolet, UV, and
inductively coupled plasma mass spectrometer, ICP-MS) for improved size
fractionation of colloids and online analysis of their elemental composition
(Henderson et al., 2012; Regelink et al., 2013; Gottselig et al., 2014; Jiang
et al., 2015a). These analyses are increasingly combined with solution
31P-nuclear magnetic resonance (NMR) spectroscopy, which offers low
detection limits and can quantify different inorganic and organic P compound
groups (Cade-Menun, 2005; Cade-Menun and Liu, 2014) in isolated colloidal
materials (e.g., Liu et al., 2014; Jiang et al., 2015a, b; Missong et al.,
2016). However, we are not aware of studies that have applied these methods
systematically to WDCs obtained from different major reference soils. Here,
we focus on the comparison of Cambisols and Stagnosols. In contrast to
Cambisols, Stagnosols are soils with perched water forming redoximorphic
features. Due to temporary water saturation and resulting oxygen limitation,
the reduction of iron (FeIII) is accompanied by the dissolution of its
oxides and hydroxides (Rennert et al., 2014), and the P associated with these
Fe minerals should correspondingly be redistributed in the soil solution.
The objective of this study was to elucidate how stagnant water conditions
alter the potential release of different P compounds in colloidal and
“dissolved” fractions of the soil solution. For this purpose, water-extractable
P was obtained from a transect of Cambisols to Stagnosols in a German
temperate grassland, and characterized using both solution 31P-NMR and
AF4 coupled online with UV and organic carbon detector (OCD) or ICP-MS
analyses.
General soil characteristics and concentrations (g kg-1 soil)
of total organic carbon (TOC), total Fe, Al, P, and Si in bulk S1 (Cambisol),
S2 (Stagnic Cambisol), and S3 (Stagnosol). The lowercase letters indicate
significant differences among soil sites (significant difference of soil site
1 and 3 was tested by t test; p < 0.05).
Soil
pHd
Water
Elevation
TOC
Fee
Al
P
Si
content (%)
(m a.s.l.)
(g kg-1)
(g kg-1)
(g kg-1)
(g kg-1)
(g kg-1)
S1a
4.90 ± 0.12a
46.5 ± 2.9
512.9
35.6 ± 2.3a*
23.0 ± 1.1a*
52.6 ± 2.9a
1.2 ± 0.1a
320 ± 7.6
S2b
4.90
45.3
507.5
35.8
24.0 ± 0.4
54.0 ± 2.0
1.3 ± 0.1
320 ± 7.0
S3c
5.36 ± 0.20b
59.0 ± 7.6
505.1
71.1 ± 15.1b*
12.8 ± 0.4b*
38.7 ± 1.1b
1.8 ± 0.4b
312 ± 12.1
a The mean of samples S1-1, S1-2, and S1-3 ± standard deviation.
b The mean of three replicates of sample S2 ± standard deviation.
c The mean of samples S3-1, S3-2, and S3-3 ± standard deviation.
d The mass ratio of soil: water = 1 : 2.5.
e Data were log transformed before t test analyses because of unequal
variances.
Materials and methods
Site description
The grassland test site in Rollesbroich is located in the northern part of
the Eifel in North Rhine-Westphalia, Germany (50∘62′ N,
06∘30′ E). The grassland vegetation is dominated by perennial
ryegrass (Lolium perenne L.) and smooth meadow grass (Poa pratensis L.). According to the soil map of the geological service of North
Rhine-Westphalia (Fig. 1), the dominant soil types on the test site are
Cambisols (extensive meadow with three to four cuts per year, no cattle
grazing), Stagnic Cambisols (cattle pasture but with less frequent grazing
than the Stagnosols), and Stagnosols (intensively used as pasture with
frequent cattle grazing followed by harrowing with a tire-drag harrow and
application of organic manure (cattle slurry): classification according to
IUSS Working Group WRB (2015). The elevation along the transect generally
decreases from south to north, with the highest elevation of 512.9 m a.s.l.
at plot 1 and the lowest point of 505.1 m a.s.l. at plot 3 (Fig. 1,
Table 1). The catchment mean annual precipitation was 103.3 cm for the
period from 1981 to 2001, and the highest runoff occurred during winter due
to high precipitation and low evapotranspiration rates, as well as overland
flow due to saturation excess (Gebler et al., 2015). The topsoil samples
(2–15 cm) of plot 1 (S1-1, S1-2, and S1-3, Cambisol), 2 (S2, Stagnic
Cambisol), and 3 (S3-1, S3-2, and S3-3, Stagnosol) were taken as a
representative transect across the site in early March 2015 (Fig. 1). It is
worth noting that Stagnic water conditions do not mean that the soils are
under reduced conditions for the whole year – only for some significant time
of the year. We sampled a Stagnosol, but only the topsoil (2–15 cm), which
was not under perching water, i.e., it was aerobic at time of sampling. However, at other times of the year, these soils experienced periods of reducing conditions that did not occur in the
other samples along the transect. Surface turf (0–2 cm) was removed as it
contained predominantly grass roots and little mineral soil. Removal of this
very surface turf may also help minimizing effects from recent manure input
on soil properties. Stones and large pieces of plant material were removed by
hand. All samples were sieved immediately to < 5 mm and stored at
5 ∘C.
Excerpt from the soil map of the test site at Rollesbroich (modified from Geologischer Dienst Nordrhein-Westfalen, 2008).
Numbered red dots indicate location of plots.
Water-dispersible fine colloids (WDFCs) separations and
AF4–UV–ICP-MS/ AF4–UV–OCD analyses
The WDCs of Rollesbroich grassland soil samples with three field replicates
in S1 and S3 were fractionated using the soil particle-size fractionation
method of Séquaris and Lewandowski (2003), but with moist soils. In
brief, moist soil samples (100 g of dry soil basis) were suspended in
ultrapure water (Mill-Q; pH: 5.5) in a soil: solution mass ratio of 1 : 2,
and shaken for 6 h. Thereafter, 600 mL of ultrapure water were added and
mixed. The WDC suspensions were collected using a pipette after a 12 h
sedimentation period. These WDC suspensions were subsequently centrifuged
for 15 min at 10 000 × g and filtered through 0.45 µm
membranes (cellulose mixing ester) to produce the suspension containing WDFCs
sized below 0.45 µm. It is worth noting that Mill-Q water was used
here to extract soil colloids instead of rain water or pore water, since
total amounts of WDFCs will likely be larger when using Mill-Q water, i.e.,
we consider these WDFCs as potentially water-dispersible colloids. In
addition, the use of Mill-Q water facilitates subsequent sample processing
with AF4 and NMR. It is inevitable that Mill-Q water would result in the
release of P due to desorption and dissolution of poorly crystalline
authigenic mineral phases. Additionally, living cells within the soil would
also certainly undergo significant osmotic stress, likely resulting in
osmotic rupture and releasing organic and inorganic P found in intracellular
components. It is also worth noting that the experimental procedure with
Mill-Q water under oxic conditions may have an impact on oxidation of aqueous
iron (Fe2+) and colloidal ferrous particles. However, at time of
sampling, the very surface soils were not fully water saturated to what is
allowed,
even for Stagnosols at this time of the year. Thus, the analyzed species and
size fractions are representative of differences in response to the
extraction procedure based on different soil redox conditions that reflect a
kind of legacy of former redox cycles, but at time of sampling and analyses
the soils were aerobic.
Asymmetric flow field-flow fractionation (AF4) fractograms of water-dispersible fine colloids (WDFCs) of S1, S2, and S3. The fractograms show
the organic carbon (OC) and ultraviolet (UV) signal intensities (a, b, c)
and the Fe, Al, P, and Si mass flow (d, e, f) monitored by
inductively coupled plasma mass spectrometer (ICP-MS) of S1 (Cambisol), S2
(Stagnic Cambisol), and S3 (Stagnosol). The sizes of peaks were according to
the AF4 result of sulfate latex standard particles and dynamic light
scattering results. The OC and UV peaks occurred with elements (ICP-MS)
peaks at the same time and the slight delay among these peaks is due to the
different length of tubes to different detectors, which cause slightly
different internal volume and retention time.
An AF4 system (Postnova, Landsberg, Germany) with a 1 kDa polyethersulfone
(PES) membrane and 500 µm spacer was used for size fractionation of
the soil sample WDFCs. It is a separation technique that provides a
continuous separation of colloids. The retention time of the colloids can be
converted to hydrodynamic diameters of the colloids using AF4 theory or
calibration with suitable standards (Dubascoux et al., 2010). The AF4 was
coupled online to an ICP-MS system (Agilent 7500, Agilent Technologies,
Japan) for monitoring of the Fe, aluminum (Al), silicon (Si), and P contents
of the size-separated particles (Nischwitz and Goenaga-Infante, 2012) and to
OCD and UV detectors for measuring organic carbon (OC). These elements were
analyzed as part of the main soil minerals (e.g., clay minerals and Fe oxides)
that can be associated with P (Jiang et al., 2015a). The OCD is a promising
technique for monitoring OC concentrations for liquid-flow-based separation
systems with the advantages of high selectivity and low detection limits
(Nischwitz et al., 2016). Briefly, the operation principle is that the
acidification of the sample flow removes inorganic C and subsequently the OC
is oxidized in a thin film reactor to carbon dioxide, which can be quantified
by infrared detection (Nischwitz et al., 2016). A 25 µM NaCl
solution at pH 5.5, which provided good separation conditions for the WDFCs,
served as the carrier. The injected sample volume was 0.5 mL and the
focusing time was 15 min with 2.5 mL min-1 cross-flow for the
AF4–UV–OCD system while 2 mL injected volume and 25 min focusing time were
used for the AF4–ICP-MS system. Thereafter, the cross-flow was maintained at
2.5 mL min-1 for the first 8 min of elution time, then set to
decrease linearly to 0.1 mL min-1 within 30 min, and maintained for
60 min. It then declined within 2 min to 0 mL min-1, and remained at
this rate for 20 min to elute the residual particles. The detection limit of
the ICP-MS system was 0.1–1 µg L-1 for the elements analyzed
in this study. The AF4 characteristics of WDFCs did not change significantly
in the 6-month period of the investigation.
Particle separations of WDFCs and solution 31P-NMR
spectroscopy
The soil samples were treated as described in Sect. 2.2 to obtain the
suspension containing WDFCs < 450 nm. We pooled the WDFC suspensions of
the field replicates in order to receive sufficient samples for solution
31P-NMR. The first peak fraction after AF4 separation has a particle
size smaller than ∼ 20 nm (approximately 300 kDa; Jiang et al.,
2015a; Fig. 2). Therefore, the suspension containing WDFCs < 450 nm of
these three samples were separated into three size fractions:
300 kDa–450 nm, 3–300 kDa, and < 3 kDa (nominally 1 nm; Erickson,
2009). The 300 kDa–450 nm particle fractions were separated by passing
∼ 600 mL of the WDFC suspension through a 300 kDa filter (Sartorius,
Germany) by centrifugation. The 3–300 kDa particle fractions were
subsequently isolated by passing the < 300 kDa supernatant through a
3 kDa filter (Millipore Amicon Ultra) by centrifugation. Finally, the final
supernatant containing the < 3 kDa particles as well as the electrolyte
phase was frozen and subsequently lyophilized.
The bulk soil samples (1 g) and the three fractions of soil water extracts
were respectively mixed with 10 mL of a solution containing 0.25 M NaOH and
0.05 M Na2EDTA (ethylenediaminetetraacetate) for 4 h, as a variation
of the method developed to extract P for 31P-NMR (Cade-Menun and
Preston, 1996; Cade-Menun and Liu, 2014; Liu et al., 2014). Extracts were
centrifuged at 10 000 × g for 30 min and the supernatant was
frozen and lyophilized. Each NaOH–Na2EDTA-treated lyophilized extract,
and the < 3 kDa fraction without NaOH–Na2EDTA treatment, were
dissolved in 0.05 mL of deuterium oxide (D2O) and 0.45 mL of a
solution containing 1.0 M NaOH and 0.1 M Na2EDTA (Turner et al.,
2007). A 10 µL aliquot of NaOD was added to the < 3 kDa
fraction without NaOH–Na2EDTA treatment to adjust the pH. The prepared
samples were centrifuged at 13 200 × g for 20 min (Centrifuge
5415R, Eppendorf).
Solution 31P-NMR spectra were obtained using a Bruker Avance 600 MHz
spectrometer equipped with a prodigy probe (a broadband CryoProbe, which uses
nitrogen (N)-cooled RF coils and preamplifiers to deliver a sensitivity
enhancement over room temperature probes of a factor of 2 to 3 for X-nuclei
from 15N to 31P), operating at 242.95 MHz for 31P. Extracts
were measured with a D2O field lock at room temperature. Chemical shifts
were referenced to 85 % orthophosphoric acid (0 ppm). The NMR parameters
generally used were 32 K data points, 3.6 s repetition delay, 0.7 s
acquisition time, 30∘ pulse width and 10 000 scans. Compounds were
identified by their chemical shifts after the orthophosphate peak in each
spectrum was standardized to 6.0 ppm during processing (Cade-Menun et al.,
2010; Young et al., 2013). Peak areas were calculated by integration on
spectra processed with 7 and 2 Hz line broadening, using NUTS software (2000
edition; Acorn NMR, Livermore, CA) and manual calculation. Peaks were
identified as reported earlier (Cade-Menun, 2015), and by spiking a select
sample with myo-inositol hexakisphosphate (myo-IHP; McDowell et al., 2007).
Statistical analyses
Elemental concentrations in bulk soils, soil water extracts, and AF4
fractograms of soil colloidal particles were tested for significant
differences (set to P < 0.05) using Sigmaplot version 12.5. A t test
was conducted to determine the significance of differences among soil sites,
whereas one-way Repeated Measures (RM) ANOVA followed by post hoc separation of means using the Fisher LSC procedure to test for significant differences
among soil fractions and AF4 fractograms for Cambisol and Stagnosol. Data
were assessed with Shapiro–Wilks and Brown–Forsythe tests to meet the
criteria of normal distribution and homogeneity of variances respectively;
those which had unequal variance data were log10 transformed before
statistical analyses.
Results and discussion
Colloid and colloidal P distribution in different size fractions
based on AF4-fractograms
The AF4–UV–OCD and AF4–ICP-MS results of the WDFCs showed different OC, Si,
P, Fe, and Al concentrations in different-sized colloid fractions as a
function of elution time (Fig. 2). Before the first peak, an initial small
void peak occurred at 1 min (Fig. 2d, e, f). Thereafter, three different
colloid-sized fractions occurred individually as three peaks in the WDFCs of
all samples (Fig. 2). The first peak of the fractograms corresponded to a
particle size below 20 nm according to the calibration result using latex
standards (Jiang et al., 2015a). The third peak, which was eluted without
cross-flow, contained only small amounts of residual particles or particles
possibly previously attached on the membrane during focus time; it had
similar OC and element distributions as the second peak in all samples
(Fig. 2). Therefore, we considered these two fractions together as a whole, and the size ranges from 20 to 450 nm from here onward are described as
the “second size fraction”.
For the first fraction representing nano-sized colloids of the three field
sites, the OCD and UV signals indicated increasing OC concentration in the
order of S1 (Cambisol; Fig. 2a), S2 (Stagnic Cambisol; Fig. 2b), and S3
(Stagnosol; Fig. 2c). Distinct peaks of Fe, Al, and P in the first size
fraction (< 20 nm) were only present in the Stagnosol (S3; Fig. 2f),
suggesting that under stagnant water conditions, Fe/Al may more readily be
involved in nano-sized soil particles than under other soil conditions. In
contrast, negligible amount of P, Al, and Fe were detected in the first
fraction of S1 and S2 (Fig. 2d and e, Table S1). While it is sometimes
difficult to determine whether this peak is real or just the tailing of the
void signal (Fig. 2d and e), solution 31P-NMR results confirmed the
presence of P in this size fraction (see next section). The nano-sized
colloids from the Cambisol contained OC and negligible P, Fe, and Al; those
from the Stagnosol contained significantly higher concentrations of OC, P,
Fe, and Al (Table S1). We therefore assumed that the nano-sized colloidal P
forms in the Stagnosol mainly consisted of OC–Fe(Al)–P associations.
Nanoparticulate humic (organic matter)–Fe (Al) (ions/(hydr)oxide)–phosphate
associations have recently been identified both in water and soil samples
(Gerke, 2010; Regelink et al., 2013; Jiang et al., 2015a). Our results
suggest that the formation of these nano-sized specific P associations is
favored by the stagnant water conditions with high OC and water contents in
Stagnosol but not in the other soil types along the grassland transect.
Concentrations (mg kg-1 soil) of P, Al, Fe, and Si in soil water
extracts < 450 nm, < 300 kDa, and < 3 kDa, respectively. Different
lowercase and uppercase letters indicate significant differences among soil sites and
soil fractions, respectively (significant difference of soil sites 1 and 3
was tested by t test; one-way RM ANOVA for soil fractions with Fisher LSD
post hoc test; P < 0.05).
Soil
DOC (g kg-1)
P (mg kg-1)
Al (mg kg-1)
Fe (mg kg-1)
Si (mg kg-1)
< 450 nm
< 450 nm
< 300 kDa
< 3 kDa
< 450 nm
< 300 kDa
< 3 kDa
< 450 nm
< 300 kDa
< 3 kDa
< 450 nm
< 300 kDa
< 3 kDa
S1a
0.18
0.3 ± 0.1ae
0.2 ± 0.2ae
0.1 ± 0.1
2.0 ± 0.4Af
0.6 ± 0.0daBf
0.6 ± 0.0daBf
2.1 ± 0.5A
0.2 ± 0.0daB
0.2 ± 0.0daeB
8.1 ± 0.6aA
6.8 ± 0.3aB
6.6 ± 0.4aB
S2b
0.17
1.3 ± 0.9
0.5 ± 0.6
0.4 ± 0.3
7.3 ± 0.3
1.1 ± 0.2
1.1 ± 0.2
9.2 ± 0.5
0.4 ± 0.1
0.4 ± 0.1
14.1 ± 0.5
7.3 ± 0.0d
7.8 ± 0.8
S3c
0.23
4.4 ± 2.0be
3.3 ± 2.7be
4.1 ± 2.6
4.1 ± 3.1
0.7 ± 0.1b
0.7 ± 0.0b
4.6 ± 3.3
0.4 ± 0.1b
0.5 ± 0.1be
14.6 ± 1.3b
10.6 ± 2.1b
11.4 ± 2.5b
a The mean of samples S1-1, S1-2, and S1-3 (Cambisol) ± standard
deviation. b The mean of three replicate extracts of sample S2 (Stagnic Cambisol) ± standard deviation.
c The mean of samples S3-1, S3-2, and S3-3 (Stagnosol) ± standard deviation.
d Standard deviation of 0.0 means value < 0.05.
e Data were log transformed before t test analyses because of unequal
variances.
f Data were log transformed before one-way RM ANOVA analyses
because of unequal variances.
The second size fraction (Fig. 2a, b, c, i.e., the small-sized colloids)
contained significantly more OC than the smaller nano-sized colloids for all
studied soils (Table S1). Notably, the OC contents of the second fraction
increased in the order Cambisol < Stagnic Cambisol < Stagnosol; the
UV signal therein supporting the results obtained with the OC detector. The
larger-sized colloids were significantly richer in Al, Fe, Si, and P than the
smaller-sized ones (Table S1), though again with differences among subsites;
the Stagnic Cambisol showed the largest Fe, Al, and Si contents in the second
fraction, as if there were a gradual change from low WDFC release in the
Cambisol to the formation of larger WDFC in the Stagnic Cambisol and finally
to the formation of smaller WDFC in the Stagnosol. Though this trend warrants
verification by more sites, it appeared at least as if the increasing oxygen
limitation from Cambisols via Stagnic Cambisols to Stagnosols promoted an
increasing formation of small C-rich P-containing nanoparticles with
additional contributions from Fe- and Al-containing mineral phases.
Stagnosols like S3 are characterized by a dynamic reduction regime with
dissolution of reactive Fe oxides (Rennert et al., 2014), which led to a
decrease in the content of Fe oxides in the second colloidal fraction
(Table S1 in the Supplement). Correspondingly, the dissolution of Fe oxides
in the second fraction under stagnant water may also liberate OC from the
organo-Fe mineral associations, thus releasing some OC to the nano-sized
first fraction (Jiang et al., 2015a). This could be an additional reason for
the higher concentration of OC in the first peak of S3 (Table S1), apart from
a generally slower degradation of organic matter under limited oxygen supply
(Rennert et al., 2014). Hence, the AF4 results indicated that the composition
and distribution of particulate P varied among the different-sized colloidal
particles, and that its properties were impacted by the soil type and related
properties. However, AF4–ICP-MS results do not provide information about the
elemental concentrations of the “dissolved” P fraction of these grassland
soils. We cannot rule out any effects from sample storage or from the use of
Mill-Q water, as discussed in the Methods section, However, although all
samples were treated the same way, differences among the samples were
consistent with soil characteristics at each site. This suggests that the
influences of treatment and storage were minimal, but further investigation
is warranted in future studies.
The proportion (%) of phosphorus speciesa determined by
solution 31P-NMR for the different soil fractions of S1 (Cambisol), S2
(Stagnic Cambisol), and S3 (Stagnosol).
Soil fractions
Pi
Po
Ortho-P
Pyro-P
Poly
P-mono
P-monob
P-diest
P-diestb
Phon-P
%
S1 bulk
43.4
56.6
41.2
1.5
0.7
52.9
44.5
2.2
10.6
1.5
S2 bulk
47.8
52.2
46.4
0.9
0.5
48.6
43.7
1.4
6.3
2.2
S3 bulk
63.7
36.3
63.0
0.2
0.5
31.2
27.0
1.5
5.7
3.6
S1 300 kDa–450 nm
22.8
77.2
22.8
–c
–
56.7
49.5
11.1
18.3
9.4
S2 300 kDa–450 nm
56.8
43.2
53.1
1.0
2.7
29.9
26.9
5.2
8.2
8.1
S3 300 kDa–450 nm
70.2
29.8
59.7
9.2
1.3
24.2
19.9
2.8
7.1
2.8
S1 3–300 kDa
100
–
100
–
–
–
–
–
–
–
S2 3–300 kDa
100
–
100
–
–
–
–
–
–
–
S3 3–300 kDa
100
–
61.5
38.5
–
–
–
–
–
–
S1 < 3 kDa
13.5
86.5
–
–
13.5
26.9
26.9
1.9
1.9
57.7
S2 < 3 kDa
21.3
78.7
9.5
5.1
6.7
29.3
13.8
24.2
34.6
25.2
S3 < 3 kDa
22.2
77.8
8.8
6.0
7.4
29.4
27.4
8.2
10.2
40.2
a Inorganic P (Pi), organic P (Po),
orthophosphate (Ortho-P), pyrophosphate (Pyro-P), polyphosphate (Poly),
orthophosphate monoesters (P-mono), orthophosphate diesters (P-diest),
phosphonates (Phon-P). b Recalculation by including diester
degradation products (α glycerophosphate, β glycerophosphate,
and mononucleotides) with P-diest rather than P-mono (Liu et al., 2014; Young
et al., 2013). c Below detection limit, i.e., < 0.05 %.
Soil total, colloidal, and dissolved P contents based on
fractionation by filtration
Soil water extracts < 450 nm, < 300 kDa, and < 3 kDa were
obtained by filtration to determine total elemental contents by ICP-MS
analysis. Data did not have to be pooled for these analyses; therefore, we
could test statistical differences. We considered the soil water extract
< 3 kDa in this paper to be the “dissolved” fraction. Significant
differences (P < 0.05) were ascertained for elevated concentrations of
total organic carbon (TOC), total P, as well for lower concentrations of total Al and Fe in the
Stagnosol relative to the Cambisol (Table 1). Furthermore, the Stagnosol had
significantly higher concentrations of Si and P in the individual size
fractions of soil water extracts (except marginally significantly higher P in
< 3 kDa, p=0.06), as well as higher Fe and Al concentrations in
< 300 kDa and < 3 fractions than the corresponding fractions of the
Cambisol (Table 2). The Stagnic Cambisol generally resembled the Cambisol
rather than the Stagnosol in bulk soil analysis, but this was not the case
for the soil water extracts. This implied that the stagnic properties have a
greater impact on the colloidal particles and “dissolved” fraction compared
to bulk soil.
The oxygen limitation and reduction regime of the Stagnosol probably also
favored the accumulation of OC and dissolution of Fe oxides both in bulk soil
and colloids (Rennert et al., 2014). Dissolution of Fe oxides in turn results
in a disaggregation of colloidal particles (Jiang et al., 2015a). As the
released oxides are main carriers for P, these processes may explain why the
distribution of colloidal and dissolved P also changed across the different
grassland soils. As Table 2 shows, large proportions of P in the < 450 nm
fraction of the Stagnosol were dissolved P (i.e., recovered here in the
< 3 kDa fraction), whereas colloidal P dominated in the Cambisol and
Stagnic Cambisol.
Inorganic and organic P species in the different-sized soil
colloidal and the “dissolved” fractions
Solution 31P-NMR was used to elucidate the speciation of P in bulk soil
and soil water extracts separated by ultrafiltration into the size fractions
300 kDa–450 nm, 3–300 kDa, and < 3 kDa for each of the three soils
(Figs. 3 and S1 in the Supplement, Table 3). The identified P included
inorganic P forms (orthophosphate, pyrophosphate, and polyphosphate), and
organic P in phosphonate, orthophosphate monoester, and diester compound
classes. Phosphonates included 2-aminoethyl phosphonic acid (AEP) and several
unidentified peaks (Table S3). Orthophosphate monoesters included four
stereoisomers of inositol hexakisphosphate (myo-, scyllo-,
neo-, and D-chiro-IHP), diester degradation products
(α glycerophosphate, β glycerophosphate and mononucleotides),
choline phosphate, and unidentified peaks at 3.4, 4.2, 4.7, 5.0, 5.3, and 5.9 ppm.
Orthophosphate diesters were divided into deoxyribonucleic acid (DNA)
and two categories of unknown diesters (OthDi1 and OthDi2, respectively).
Orthophosphate, pyrophosphate, orthophosphate monoesters, and diesters have
also been detected in other studies of grassland, arable, and forest
Cambisols and Stagnosols (e.g., Murphy et al., 2009; Turrion et al., 2010;
Jarosch et al., 2015).
Solution phosphorus-31 nuclear magnetic resonance spectra of
NaOH–Na2EDTA extracts of bulk soil, 300 kDa–450 nm, 3–300 kDa, and
< 3 kDa fractions in soil water extracts < 450 nm of S3 (Stagnosol).
For the bulk soil samples and colloidal fractions of 300 kDa–450 nm of our
soil samples, orthophosphate and orthophosphate monoesters (mainly
myo-IHP) were the main P compounds in all samples (Figs. 3 and S1,
Tables 3 and S2). These main P compounds in these two soil fractions showed
similar trends among the soil samples: the proportions of organic P (e.g.,
orthophosphate monoesters and diesters) decreased in the order of
Cambisol > Stagnic Cambisol > Stagnosol (Table 3). The similarity in
this trend for the different organic P forms can likely be attributed to
similarities in the mineral components of bulk soil and colloidal fractions:
i.e., similar element concentrations and thus likely also similar clay
mineralogy, Fe oxide signature and OC content of bulk soil, and respective
colloid fraction according to the AF4–OCD and AF4–ICP-MS results (Fig. 2 and
Table S1). Orthophosphate, orthophosphate monoesters, and diesters are
predominantly stabilized by association with these mineral components
(Solomon and Lehmann, 2000; Turner et al., 2005; Jiang et al., 2015a). We
assume that most of the relatively higher proportion of orthophosphate and
lower percentage of organic P in the Stagnosol may be attributed to the
dissolution of Fe oxides, which likely released organic P. Additionally, the
higher concentrations of OC in both bulk soil (Table 1) and large colloids of
the Stagnosol probably favored the formation of OC–Fe/Al–PO43-
complexes (see above). However, we cannot rule out the effects of differences
in grazing and manure application on the P forms in these soils. Cattle
grazing and the application of cattle slurry would be expected to add P that
is predominantly orthophosphate, with lower concentrations of organic P forms
including myo-IHP (Cade-Menun, 2011, and references therein). Thus, this
may have contributed to the increased orthophosphate and decreased
organic P we observed on these sites.
Our study is the first to distinguish the chemical P composition in colloidal
fractions of 3–300 kDa and 300 kDa–450 nm. We found different P
speciation and distribution between these two fractions. This is probably
related to differences in their element composition, which are dominated by
OC–P/OC–Fe(Al)–P associations in the 3–300 kDa soil fraction and by clay–Fe
oxides–OC–P associations in the 300 kDa–450 nm size fraction (Fig. 2).
Intriguingly, we did not find any organic P but only inorganic P in the
3–300 kDa of all three soils (orthophosphate in Cambisol and Stagnic
Cambisol, orthophosphate and pyrophosphate in the Stagnosol; Table 3).
Furthermore, the Stagnosol nanoparticle fraction 3–300 kDa had a higher
proportion of pyrophosphate than the 300 kDa–450 nm size fraction.
When comparing the solution 31P-NMR results of the < 3 kDa soil
fractions with and without NaOH–Na2EDTA treatments (Figs. 3 and S1), we
observed that most of the phosphonates, orthophosphate monoesters, and
diesters were lost after NaOH–Na2EDTA treatment (Figs. 3 and S1). There
were two possible explanations: (1) “dissolved” organic P in the
NaOH–Na2EDTA solution is sensitive and easily hydrolyzed to
orthophosphate (Cade-Menun and Liu, 2014); or (2) in absence of
NaOH–Na2EDTA, most orthophosphate was removed by adsorption on
sedimentary material in the re-dissolved solution after centrifugation when
preparing the samples for NMR analysis (Cade-Menun and Liu, 2014), resulting
in elevated portions of organic P in the NMR sample. The second possibility
may also explain the observation that there was no orthophosphate in the
“dissolved” fraction of the Cambisol without NaOH–Na2EDTA treatment
(Fig. S1). Almost all the orthophosphate may have been removed with the
sedimentary phase due to the extremely low concentration of dissolved P in
this soil. Therefore, we will focus on the discussion of results obtained
from the < 3 kDa soil fractions without NaOH–Na2EDTA treatment, as
they provide better information on the origin of Po species than the other
samples that received this treatment.
The composition of P species in the < 3 kDa soil fractions (i.e.,
“truly” dissolved P) differed among the three soils (Table 3). The majority
of observed P in the < 3 kDa soil fraction of the Cambisol was organic P,
comprised mainly of phosphonates and orthophosphate monoesters. The
< 3 kDa soil fraction of the Stagnic Cambisol contained various P species
from all compound classes, including orthophosphate, orthophosphate
monoesters, orthophosphate diesters, pyrophosphate, polyphosphates, and
phosphonates. The < 3 kDa soil fraction of the Stagnosol contained
similar P species as the Stagnic Cambisol, with relatively higher proportions
of orthophosphate monoesters and phosphonates, but a lower proportion of
orthophosphate diesters (Table 3). It is worth noting that there were more
species of phosphonates in the < 3 kDa fraction than other fractions of
each soil (Figs. 3 and S1). The larger signal at ∼ 21–23.5 ppm was
assigned to AEP (Doolette et al., 2009; Cade-Menun, 2015), which occurred in
both the soil particles and the < 3 kDa fraction. However, the small
signals at ∼ 36–39 and 45–46 ppm existed only in the < 3 kDa
fraction of soil samples (Figs. 3 and S1). The resonance at 36–39 ppm might
be assigned to dimethyl methyl phosphonic acid, based on Cade-Menun (2015).
However, spiking experiments were not conducted to identify peaks in this
region, so their specific identity and origins remain unknown.
The solution 31P-NMR results showed that P species composition in the
two colloidal fractions and the electrolyte phase differed among all three
soil samples, with more phosphonates potentially existing in the electrolyte
phase. However, in the study of Missong et al. (2016), more phosphonates and
orthophosphate diesters were found in colloidal fractions rather than the
electrolyte phase of two forest Cambisols. Missong et al. (2016) used
centrifugation while we used filtration to separate these particle sizes and
phases. Additionally, Missong et al. (2016) worked with forest soils while we
worked with grassland soils. McLaren et al. (2015) recently confirmed that
the speciation of organic P is markedly different between high (> 10 kDa)
and low (< 10 kDa) molecular weight fractions of soil extracts. In any
case, both colloidal aggregation and changes in soil order paralleled P
forms. However, other soil properties (like pH) and former redox states,
as well as variations in anthropogenic, site-adapted management may be
additional covariates affecting P colloids and composition.
Distribution of orthophosphate monoesters and pyrophosphate
With variations in overall P species composition, the proportions of certain
species of orthophosphate monoesters were also distributed differently among
the investigated fractions of the three soils. For example, the proportion of
various IHP stereoisomers (i.e., myo-, scyllo-,
D-chiro-IHP) decreased with decreasing colloid size (Table S2). This
suggests that the majority of IHP was associated with soil mineral particles
but did not exist in the dissolved form in our soil samples. The
myo-IHP stereoisomer is the principal input of inositol phosphate to
soil in the form of plant material (Turner et al., 2002) and the other
stereoisomers may come from plants or may be synthesized by soil organisms
(Caldwell and Black, 1958; Giles et al., 2015). Inositol phosphate is
stabilized mainly through strong adsorption on the surface of amorphous metal
oxides and clay minerals (Celi and Barberis, 2007). Shang et al. (1992) found
myo-IHP sorbed onto Al and Fe oxides to a greater extent than
glucose 6-phosphate. Several orthophosphate monoesters such as unknown peaks
at 3.4, 4.7, and 5.9 ppm were only detected in the electrolyte phase of soil
samples (Table S2). The differences in orthophosphate monoester species
distribution between soil particles and the electrolyte phase show that soil
minerals such as clay minerals and Fe(Al) oxides are only associated with
certain species of orthophosphate monoesters such as IHP, while other species
of orthophosphate monoesters exist only in the electrolyte phase. Further
research is warranted to fully understand the factors controlling Po in these
different size fractions.
It is worth noting that although the proportion of pyrophosphate in bulk soil
was very low, there was more pyrophosphate in the colloidal and electrolyte
phases of the Stagnic Cambisol and the Stagnosol than in the Cambisol, and
mostly in the electrolyte and nano-sized colloidal fraction (Table 3). Our
former study (Jiang et al., 2015b) indicated that Fe/Al oxides were not the
main bonding site for pyrophosphate adsorption in different-sized fractions
of an arable soil. Considering that a high proportion of pyrophosphate
(38.5 %) existed in the 3–300 kDa fraction of the Stagnosol, which
contained P mainly in OC–Fe(Al)2/3+–P associations (see above), it seems
reasonable to assume that pyrophosphate existed as a colloidal
OC–Fe(Al)2/3+–pyrophosphate complex. In this regard, the accumulation of
pyrophosphate may have been favored by the larger OC contents in this soil
(Fig. 2c).
This study shows for the first time that P species composition varies among
the electrolyte phase and colloids of different size, with the specific
distribution being related to the stagnic water regime of the soil. It could
potentially promote P availability by a mechanism that results in a loss of
colloids, thus providing less surface area for the immediate bonding of
inorganic P to minerals, while at the same time potentially releasing
organic P from mineral bonding so that it is more prone to decomposition.
Relating the static differences in P species composition among the different
soils and fractions to true dynamics of P transformations, e.g., by
performing controlled mesocosm experiments, now warrants further attention.