Chemical Geology 397 (2015) 51–60
Contents lists available at ScienceDirect
Chemical Geology
journal homepage: www.elsevier.com/locate/chemgeo
Influence of natural organic matter on the bioavailability and
preservation of organic phosphorus in lake sediments
Yuanrong Zhu a,b, Fengchang Wu a,⁎, Zhongqi He c, John P. Giesy d,e,f, Weiying Feng a,b, Yunsong Mu a,
Chenglian Feng a, Xiaoli Zhao a, Haiqing Liao a, Zhi Tang a
a
State Key Laboratory of Environment Criteria and Risk Assessment, Chinese Research Academy of Environmental Sciences, Beijing 100012, China
College of Water Sciences, Beijing Normal University, Beijing 100875, China
USDA-ARS Southern Regional Research Center, 1100 Robert E Lee Blvd, New Orleans, LA 70124, USA
d
Department of Biomedical Veterinary Biosciences and Toxicology Centre, University of Saskatchewan, Saskatoon, Saskatchewan, Canada
e
Department of Biology and Chemistry, City University of Hong Kong, Kowloon, Hong Kong, China
f
State Key Laboratory for Marine Pollution, City University of Hong Kong, Kowloon, Hong Kong, China
b
c
a r t i c l e
i n f o
Article history:
Received 25 August 2014
Received in revised form 12 January 2015
Accepted 13 January 2015
Available online 21 January 2015
Editor: Carla M Koretsky
Keywords:
Enzymatic hydrolysis
31
P NMR
Organic phosphorus
Bioavailability
Preservation
Natural organic matter
a b s t r a c t
Information about the bioavailability and sequestration of organic phosphorus (Po) in sediments is fundamental
to understanding biogeochemical cycling of phosphorus (P) in eutrophic lakes. However, the processes
governing preservation of Po in sediments are still poorly understood. Sequential extraction of Po by H2O
(H2O-Po) and NaOH–EDTA (NaOH–EDTA Po), in combination with enzymatic hydrolysis/31P NMR, was applied
to estimate the bioavailability of Po in sediments of Lake Tai (Ch: Taihu), China. Of H2O-Po and NaOH–EDTA Po,
45.5–89.4% and 30.4–71.3% respectively were hydrolyzed by phosphatase, and therefore considered to be biologically available. Of NaOH–EDTA Po, 28.7–69.6% could not be hydrolyzed by phosphatase; this portion was
characterized by 31P NMR as monoester P and/or diester P. Simulation experiments of hydrolysis of model Po
compounds in the presence of humic acids (HA), which were used as a model for natural organic matter
(NOM), and metals, including Al, Ca, and Fe, have demonstrated that enzymatic hydrolysis of labile monoester
P was weakly reduced by HA or metal ions. Condensed phosphate (e.g., pyrophosphate) and phytate-like P
(e.g., inositol phosphates) were resistant to enzymatic hydrolysis in the presence of HA and/or metal ions,
which indicated that they may be possibly preserved in sediments. These observations suggest that NOM in
sediments can be a significant factor determining the bioavailability and preservation of Po in sediments.
The presence of metals would enhance the effect of NOM on preservation of Po in sediments. Formation
of Po–metal–HA or Po–metal complexes might be mechanisms responsible for these processes.
© 2015 Elsevier B.V. All rights reserved.
1. Introduction
Eutrophication, a process where water bodies receive excess nutrients due to activities of humans, which then stimulates excessive
growth of plants, has become a worldwide concern and its causes
and strategies to control it are areas of active research (Smith and
Schindler, 2009; McMahon and Read, 2013). Continuous allochthonous
inputs and internal recycling of nutrients are both significant factors
contributing to eutrophication of lakes. Phosphorus (P) is the primary
controllable, limiting nutrient, especially with respect to long-term control of blooms of nuisance algae in most lakes (McMahon and Read,
2013). As external inputs of P have gradually been reduced over the
last few decades, biogeochemical cycling of internal P that has accumulated in sediments has become the primary factor maintaining the
⁎ Corresponding author. Tel.: +86 10 84915312; fax: +86 10 84931804.
E-mail address: wufengchang@vip.skleg.cn (F. Wu).
http://dx.doi.org/10.1016/j.chemgeo.2015.01.006
0009-2541/© 2015 Elsevier B.V. All rights reserved.
trophic status of lakes (Søndergaard et al., 2003; Zhu et al., 2013a,b).
Organic P (Po), which can constitute a substantial pool of internal P
from sediments, has received much less attention than inorganic P (Pi)
in the past decades (Zhang et al., 2008; Baldwin, 2013; Zhu et al.,
2013a). In fact, the biogeochemical cycle of Po might play an important
role in maintaining eutrophic status for lakes, especially after external
sources of P have been controlled (Zhang et al., 2008; Zhu et al.,
2013a). Thus, knowledge of the composition, bioavailability, and preservation of Po in sediments is necessary to understand P dynamics in
eutrophic lakes.
Phosphorus nuclear magnetic resonance (31P NMR) spectroscopy
has become a preferred technique that is widely used to characterize
forms of P present in sediments that has significantly advanced knowledge of Po in sediment from lakes (Baldwin, 2013). This technique
distinguishes P compounds, including orthophosphate, pyrophosphates, polyphosphate, phosphate monoester, diester phosphate, and
phosphonates (Hupfer et al., 2004; Cade-Menun, 2005; Zhang et al.,
2009). However, techniques for processing of samples, including
52
Y. Zhu et al. / Chemical Geology 397 (2015) 51–60
pretreatment of samples with chemicals, such as HCl, extraction with a
basic solution of NaOH–EDTA, and preconcentration methods such as
lyophilization, are needed for solution 31P NMR spectroscopy analysis,
and these result in some degradation of labile Po compounds (Turner
et al., 2003; Cade-Menun et al., 2006). Enzymatic hydrolysis, a biochemical analysis procedure, provides a relatively mild approach for characterizing the lability of Po in environmental samples (He et al., 2006c,
2008; Monbet et al., 2007; Zhu et al., 2013a). Organic P has been characterized previously by a combination of enzymatic hydrolysis and 31P
NMR (He et al., 2007, 2008). Both methods have been found to be suitable for characterization of Po compounds from environmental samples.
With solution 31P NMR, most P compounds that have different functional groups can be identified and quantified (Turner et al., 2003;
Cade-Menun, 2005). Though a well designed procedure for enzymatic
hydrolysis cannot identify all Po contained in samples, this technique
can provide an estimate of hydrolyzable, and thus bioavailable Po
such as labile monoester P, diester P, and phytate-like P in sediments
(Bünemann, 2008; Zhu et al., 2003a). Since both enzymatic hydrolysis
and 31P NMR have advantages and disadvantages, characterization of
Po using both methods concurrently allows for a more comprehensive
characterization of P in sediments.
Humic acids (HA) have been used as representatives of natural
organic matter (NOM) in studies of interactions of Po with NOM
and metals (He et al., 2006a, 2009a). Most naturally occurring Po
compounds are present in either the mono- or diester form, which
leaves one or two non-ester hydroxyl groups to bind with NOM or
metal bridging to form complexes (Laarkamp, 2000; He et al., 2006b;
Zhu et al., 2013a), HA as a model for NOM, such as Po combined
with HA (Po–HA) (Brannon and Sommers, 1985a) and Po combined
with HA with metal bridging (Po–metal–HA) (Laarkamp, 2000;
Benitez-Nelson et al., 2004; Monbet et al., 2007) have been widely
discussed and investigated. However, solution 31P NMR spectroscopy
cannot distinguish whether monoester P or diester P is linked to a carbon chain like HA or metal ions in complex compounds. For example,
the complexes of HA–DNA would be detected as DNA only in solution
31
P NMR spectroscopy. Compared with 31P NMR, enzymatic hydrolysis
is a potential tool to assess the effect of NOM or metals on the reactivity
of Po, because results of previous studies have shown that hydrolysis of
Po by enzymes is likely to be influenced by NOM or metal ions present in
lake sediments (De Groot and Golterman, 1993; Zhu et al., 2013a). Results of investigations of the stability of phosphate esters, including
phosphoserine and phosphoethanolamine, incorporated into model
humic polymers, indicate that Po–humic materials are resistant to
both chemical and enzymatic hydrolysis (Brannon and Sommers,
1985b). For diester P, DNA-P bound to HA was protected more strongly
against degradation by DNase than free DNA (Crecchio and Stotzky,
1998). Therefore, enzymatic hydrolysis alone (or combined with 31P
NMR) is a valuable (or comprehensive) tool to investigate the interactions of Po with NOM or metals. Incorporation of Po into NOM and the
association of Po with metal ions might both be mechanisms by which
Po is protected from enzymatic hydrolysis in sediments (Laarkamp,
2000; Benitez-Nelson et al., 2004; Bai et al., 2009; Zhu et al., 2013a).
Therefore, preservation of some Po in sediments could be enhanced by
these interactions, which influences the biogeochemical cycle of P in
lakes. Few studies have investigated mechanisms for preservation of
Po or quantified them in lake sediments (Carman et al., 2000; Reitzel
et al., 2007, 2012; Ding et al., 2013), hence further and more detailed
clarification on these processes was warranted.
In this study, enzymatic hydrolysis and 31P NMR were used to
characterize Po in sediments from algae-dominated and macrophytedominated regions of Lake Tai. The influence of NOM on enzymatic
hydrolysis was also investigated. Finally, enzymatic hydrolysis of
model Po–HA, Po–metal, and Po–metal–HA complexes were analyzed
to confirm observations about the influence of NOM on Po hydrolysis,
which has implications for bioavailability and preservation of Po in
sediments.
2. Materials and methods
2.1. Study site and sampling
Lake Tai (Ch: Taihu), located in the Yangtze River delta, Jiangsu
Province (Fig. 1), is the third largest freshwater lake in China. It's a typical shallow lake with a surface area of 2338 km2, and an average depth
of 1.9 m (Qin et al., 2007). With economic development and changes in
land use of this area, large loads of pollutants and nutrients have been
input to the lake. Currently, eutrophication is one of the main problems
in Lake Tai (Zhao et al., 2013). Algal blooms have occurred with increasing frequency and intensity in the western and northern parts of the
lake, especially in algal-dominated regions including Zhushan Bay and
Meiliang Bay since the 1980s, and in recent years, blooms of phytoplankton, including those of cyanobacteria have expanded to Gonghu
Bay (Qin et al., 2007; Bai et al., 2009; Duan et al., 2009). In contrast,
large amounts of vegetation, including submerged vegetation,
floating-leaf vegetation and emergent vegetation dominate the east
and southeast regions of Lake Tai, including south of Gonghu and,
Xukou Bays and the southeastern part of Lake Tai (called “East Lake
Tai”) (Zhao et al., 2013). These regions have better water quality.
However, eutrophication has been accelerated by increasing nutrient
concentrations in the water and sediments of East Lake Tai (Qin et al.,
2007). Especially, there were large amounts of organic matter accumulated in sediments from the macrophyte-dominated East Lake Tai.
In May 2009, surface sediments (top 3 cm) were collected by use of
a gravity core sampler from four regions of Lake Tai (Fig. 1). Of these
sites (identified as T1–T5), T1 (31°28′0.95″N, 120°10′37.86″E) and T2
(31°24′32.17″N, 120°8′41.59″E) are located in Meiliang Bay, T4
(31°26′34.80″N, 120°2′39.03″E) is located in Zhushan Bay, which is a region dominated by algae. T3 (31°24′50.64″N, 120°21′5.86″E) is located
in Gonghu Bay, which is a transitional region between macrophytes
and algae. T5 (31°5′51.65″N, 120°32′54.97″E) is located in the East
Lake Tai, which was in a macrophyte-dominated region. Sediments
were transported to the laboratory in air-tight plastic bags and placed
in cold storage on dry ice. Sediments were lyophilized and ground to
powder and stored at −20 °C until analysis.
2.2. Analysis of sediment properties
Total concentrations of Al, Ca, Fe and Mn were measured by the use
of inductively coupled plasma optical-emission spectrometry (ICP-OES)
after micro-acid (HNO3–HCl–HF) wet digestion of sediments. Chinese
standard reference samples of sediment (GSD-12) were analyzed
simultaneously in order to check the accuracy of results. General characteristics of forms of P were determined by methods that had been
harmonized and validated by use of the Standards, Measurements and
Testing (SMT) program of the European Commission (Ruban et al.,
1999, 2001). The operationally defined scheme was composed of five
steps to sequentially extract the different forms of phosphorus (P):
total P (TP), inorganic P (Pi), Po, inorganic P soluble in NaOH (Fe/Al-P,
P bound to Al, Fe and Mn oxides and hydroxides), and inorganic P
extractable by HCl (Ca-P, P associated with Ca). For each fraction of P,
concentrations were analyzed by the use of the molybdenum blue
method (Murphy and Riley, 1962). Sediments were pretreated by an
excess of 1 mol/L HCl to remove carbonates, then analyzed for total
organic carbon (TOC) and total nitrogen (TN) using an elemental
analyzer (Vario EL Ш, Elementar, Germany). Properties of sediments
from different regions of Lake Tai are shown (Table 1).
2.3. Extraction of organic P
Sediments were extracted by a modified NaOH–EDTA procedure.
Briefly, for each sample of sediment sample, 3 g was extracted via shaking with 60 mL of deionized water (2 h) in the first step to characterize
water-soluble Po (H2O-Po). The residue was then pretreated with 0.1 M
Y. Zhu et al. / Chemical Geology 397 (2015) 51–60
53
Fig. 1. Map of sampling sites in Lake Tai (Ch: Taihu).
HCl by shaking for 1 h at room temperature to remove cations and thus
reduce interferences in NMR spectroscopy of Fe and Mn (Turner et al.,
2005). Before enzymatic hydrolysis, it was important to obtain a concentrated fraction of Po by removing as much Pi as possible. The residue
was then extracted with a solution of 0.25 M NaOH–25 mM EDTA (16 h)
to obtain NaOH–EDTA-Po for enzymatic hydrolysis and 31P NMR analysis. Though 0.25 M NaOH–50 mM EDTA has been widely used for
extraction of Po from sediments and soils (Cade-Menun, 2005; Turner
et al., 2005), this relatively large concentration of EDTA could inhibit enzyme activity, such as alkaline phosphatase (Chen et al., 1996) and
phosphodiesterase (Wang et al., 2001), during enzymatic hydrolysis.
Therefore, 0.25 M NaOH–25 mM EDTA was used. This solution exhibited
efficiencies of extraction of Po that were consistent with those obtained
with 0.25 M NaOH–50 mM EDTA (Xu et al., 2012). H2O-Po was loosely
adsorbed to sediment particles or in the interstitial water of sediments,
which was transferred easily across the water–sediment interface
(Zhu et al., 2013a). Compared with NaOH–EDTA extractable P o ,
H2 O-Po , a generally small but mobile Po fraction in sediments, was
also analyzed. Therefore, H2O-Po and NaOH–EDTA Po were obtained
through this procedure. Dissolved reactive phosphorus (DRP) in extracts was analyzed by the molybdenum blue method (Murphy and
Riley, 1962). TP in extracts was determined after digestion with potassium persulfate (K2S2O8) in an autoclave at 121 °C for 30 min. Organic
P in extracts was then determined by calculation of the difference
between TP and DRP. An aliquot was taken for the measurement of Al,
Ca, Fe and Mn by ICP-OES. The remaining NaOH–EDTA extracts were
used for subsequent experiments.
2.4. Enzymatic hydrolysis and 31P NMR spectroscopy
Alkaline phosphatase (APase, P7640), phosphodiesterase (PDEase,
P4506), and crude phytase from wheat (P1259) were purchased from
Sigma. H2O-Po and NaOH–EDTA Po were analyzed by enzymatic hydrolysis, details of which have been described previously (Zhu et al.,
2013a). APase and PDEase were prepared in a Tris–HCl buffer (0.1 M,
pH 9.0) at concentrations of 2 and 0.02 unit/mL. Crude phytase was
purified to remove DRP. Aliquots of 200 mg of enzyme were dissolved
in 30 mL of an 80% saturated (NH4)2SO4 solution, then purified by
precipitation at 4 °C overnight. The precipitate was collected by centrifugation (10,000 g) for 20 min at 4 °C. The precipitate was dissolved
in 10 mL of 10 mM NaAc–HAc buffer (pH 5.15) and dialyzed six
times using a Spectra/Por Float-Lyzer (MWCO: 3500–5000, Spectrum
Laboratories, Inc.) for 16 h with 2 L buffer. The dialyzed enzyme solution
was then centrifuged (10,000 g, 4 °C) for 20 min. The purified phytase
was prepared in a NaAc–HAc buffer (0.1 M, pH 5.15) or a Tris–HCl buffer
(0.1 M, pH 7.0) to obtain a concentration of approximately 0.1 unit/mL.
Table 1
Physical and chemical properties of sediments.
Location
Al
Ca
Fe
Mn
g kg−1 d.w.
T1
T2
T3
T4
T5
50.5
50.5
23.3
50.3
38.8
TP
Pi
Po
Fe/Al-P
Ca-P
mg kg−1 d.w.
7.2
14.0
3.7
11.2
5.4
28.7
29.7
22.5
28.0
18.3
1.0
0.7
0.4
1.2
0.5
595.4
390.2
437.0
1168.5
618.3
TOC
TN
% d.w.
411.8
188.4
266.6
929.0
366.1
183.5
201.8
170.4
239.6
252.2
170.1
126.2
85.9
703.5
161.2
257.9
104.2
209.3
278.9
263.9
1.10
0.36
1.00
1.36
3.04
TOC/TN
Molar ratio
0.21
0.10
0.16
0.21
0.43
6.1
4.2
7.1
7.6
8.2
54
Y. Zhu et al. / Chemical Geology 397 (2015) 51–60
A 0.3 mL aliquot of NaOH–EDTA extracts was neutralized by 0.1 M HCl,
diluted, added to 2 mL of buffer solution and 0.44 mL enzyme solution
(pH 9.0 for APase, and PDEase combined with APase; and pH 7.0 for
the APase, PDEase and phytase mixtures) to a final volume of 8 mL in
a 10-mL tube, then incubated at 37 °C for 16 h. Samples containing an
enzyme-free buffer were simultaneously incubated to monitor and
correct for any non-enzymatic hydrolysis and effects of the matrix
through the use of a blank. Each extract was analyzed in triplicate and
means were reported. DRP concentrations were quantified by the molybdenum blue/ascorbic acid method. Standards for DRP quantification
were analyzed simultaneously with each procedure to correct for interferences induced by enzymes and matrices (Bünemann, 2008). Through
this procedure (Zhu et al., 2013a), Po was classified into four species:
(1) labile monoester P (hydrolyzed by APase), (2) diester P (hydrolyzed
by APase + PDEase minus labile monoester P), (3) phytate-like P
(hydrolyzed by APase + PDEase + phytase minus labile monoester
and diester P), (4) unidentified Po (the portion of Po that was not hydrolyzed by APase, PDEase and phytase).
Freeze-dried untreated NaOH–EDTA extracts were redissolved in
2 mL of deionized H2O, then shaken by hand and ultrasonic vibration.
Aliquots of 0.5 mL were transferred to NMR tubes, and then 50 μL of
D2O was added for use as a signal lock. 31P NMR spectra were measured
at 161.98 MHz by a Bruker AV 400 MHz spectrometer equipped with a
5 mm broadband observe probe. The conditions used to collect spectra
included a 12 μs pulse (90°), 2-s pulse delay, and acquisition time of
0.2 s, with approximately 24,000 transients. Temperature was regulated
at 20 °C. Based on the ratio of P to Fe and Mn concentrations (w/v) in the
final NMR sample, the total delay time used was adequate to obtain
quantitative spectra of the extracts (McDowell et al., 2006). A 5 s
pulse delay, and an acquisition time of 0.5 s were tested again to analyze
T5 sample to check this. Chemical shifts were referenced to 85% H3PO4
via the signal lock. Peaks were assigned based on literature values
(Turner et al., 2003; Cade-Menun, 2005), integrated to obtain peak
areas, and converted to concentrations of P relative to concentrations
of TP in extracts. Spectral processing was done using MestReNova
(MNova) software version 9.0.1 (Mestrelab Research SL).
2.5. Enzymatic hydrolysis of model P compounds in the presence and
absence of humic acids and metals
Four model P compounds representing a variety of molecular sizes
and functional types were selected and purchased from Sigma-Aldrich
Chemicals (Shanghai, China). They consisted of labile monoester
phosphate (glucose-6-phosphate, Glu6P), condensed-P compounds
(tetra-sodium pyrophosphate, PP), diester phosphate (DNA), and
phytate (inositol hexakisphosphate, IHP6). Solutions of model P compounds containing approximately 2 mM (P) were prepared and accurate concentrations were determined after digestion by H2SO4/K2S2O8.
Commercially available HA (No. 53680) was also purchased from
Sigma-Aldrich, then purified according to a modified IHSS purification
procedure as described elsewhere (Hong and Elimelech, 1997). The
characteristics of HA here could be found in Fetsch and Havel (1998)
and Rigol et al. (1998). The composition of the purified solid HA was
51.3% C, 1.2% N, 3.3% H, 36.6% O, 1.7% S, and 6.0% Ash. The HA solution
(200 mg C/L) was prepared. There were trace concentrations of Al, Ca,
Fe, Mn and P in the prepared HA solutions, which were quantified
(Table S1). Four cationic metal ion (Mn+) solutions, containing AlCl3
(Al3+: 10 mM), CaCl2 (Ca2+: 40 mM), FeCl3 (Fe3+: 2 mM), and FeCl2
(Fe2+: 2 mM) respectively, were also prepared.
The scheme used to study the interactions of model P compounds
with HA, Mn+ and combinations of HA and Mn+ is described below. A
volume of 5 mL of individual model P compounds was added, then
5 mL of a solution containing HA and/or Mn+ were added. The mixture
was diluted to 50 mL, and the final concentrations of metal ions were
matched to those measured in the NaOH–EDTA extracts of sediments
(Table S2). The pH values of mixtures were maintained at 7.0 ± 0.1.
After 1, 24, 48, and 96 h, 1 mL of the reaction solution was removed
and analyzed by enzymatic hydrolysis. Solutions containing Glu6P and
PP respectively were hydrolyzed by APase at pH 9.0, 37 °C; solutions containing DNA were hydrolyzed by PDEase and APase at pH 9.0, 37 °C; and
solutions containing IHP6 were hydrolyzed by phytase at pH 5.15, 37 °C.
To determine the effects of HA, Al3+, Ca2+, Fe3+ and Fe2+ on quantification of DRP, DRP (prepared by KH2PO4) was quantified as well as model P
compounds according to this scheme; DRP was then quantified by the
molybdenum blue/ascorbic acid method. In this study, some Fe2+
could be oxidized to Fe3+ by O2. There was no detectable DRP released
from purified HA by enzymatic hydrolysis.
3. Results and discussion
3.1. Properties of sediments from various regions in Lake Tai
Distributions of P, TOC, TN and TOC/TN were significantly different
between regions of Lake Tai (Table 1). The greatest concentration of TP
was observed in sediments from Zhushan Bay (T4) (1168.5 mg kg−1),
while the concentration was low in the sediments from outer Meiliang
Bay (T2). Inorganic P, including Fe/Al-P and Ca-P was the primary
form of TP in sediments from Lake Tai. Compared with TP, Fe/Al-P is a
good indicator of sediment polluted by external P inputs (Zhu et al.,
2013b). In sediments from Zhushan Bay, the concentration of Fe/Al-P
was 703.5 mg kg−1, which accounted for 75.7% of Pi. This result indicates
a relatively large external input of P to this region. Fe/Al-P is a bioavailable form of P in sediments, which could be an important source of P for
assimilation by algae/bacteria during blooms in Meiliang Bay. At other
locations, Ca-P, which is a relatively stable fraction of sedimentary P
and contributes to the permanent sequestration of P in sediments
(Gonsiorczyk et al., 1998; Zhu et al., 2013c), was the primary form of
Pi. Compared with bioavailable Fe/Al-P, internal cycling of Po is more
likely an important source of internal P in sediments, especially in
Meiliang Bay, Gonghu Bay, and East Lake Tai (Table 1).
TOC content in the sediments ranged from 0.36% to 3.04%. Content of
TOC was greatest in East Lake Tai, which is dominated by emergent
macrophytes, which is characterized by high amounts of organic matter
accumulation in sediments. The molar ratio of TOC/TN ranged from 4.2
to 8.2, which is in the range of typical autochthonous sources, rather
than allochthonous sources (Meyers and Ishiwatari, 1993). These findings indicate that organic matter in sediments of Lake Tai is derived
mainly from decomposition of aquatic macrophytes, algae, and bacteria.
The molar ratio of TOC/TN was greatest in sediment at T5 in East Lake
Tai. This is possibly because of the predominance of aquatic macrophytes in East Lake Tai.
3.2. Enzymatic hydrolysis and bioavailability of water-soluble Po
Concentrations of water-soluble TP (H2O-TP) ranged from 2.0 to
2.8 mg kg− 1, 40.7% to 72.8% of which were H2O-Po (Table S3). In
sediments, concentrations of H2O-Po increased as the TOC content
increased, which is possibly due to greater decomposition of NOM as
a result of greater microbial activity in spring and summer when temperatures are higher (De Vicente et al., 2003). Based on enzymatic hydrolysis of H2O-Po (Fig. 2a), concentrations of individual forms of Po
identified in the H2O-Po were: labile monoester P, 0.2–0.6 mg kg−1; diester P, 0–0.4 mg kg−1; phytate-like P, 0–1.4 mg kg−1 and unidentified
Po, 0.1–1.0 mg kg−1 (Fig. 2b).
Labile monoester P was an important constituent of H2O-Po, and
accounted for 8.2% to 44.0% (average, 28.5%) of H2O-Po (Fig. 2b
and Table S3). For Lake Dianchi, a highly eutrophic lake, the mean concentration and proportion were 1.0 mg kg− 1 and 36.7% respectively
(Zhu et al., 2013a). Concentrations and proportions of labile monoester
P were lesser in H2O-Po from Lake Tai. This indicates that eutrophication
has enhanced the accumulation of labile monoester P of H2O-Po in sediments. Labile monoester P in H2O-Po was an important autochthonous
Y. Zhu et al. / Chemical Geology 397 (2015) 51–60
Fig. 2. Total and enzymatically released Po (a) and derived Po species (b) in water extracts of
sediments from Lake Tai. Data are presented as the average value with standard deviation
(n = 3).
source of P within Lake Tai because of its lability and bioavailability. Labile monoester P would be released and hydrolyzed by enzymes during
periods when minimal DRP is available (Zhou et al., 2002; Zhu et al.,
2013a). Diester P is derived mainly from algae, bacteria, and aquatic
macrophytes, which decompose faster than monoester P in Lake Tai
(Ding et al., 2013). Concentrations and proportions (average, 12.8%) of
bioavailable diester P in H2O-Po were also lesser than those in H2O-Po
from Lake Dianchi (Zhu et al., 2013a). Phytate-like P, which accounted
for 0 to 45.3% (average, 28.7%) (Table S3), was also an important constituent of H2O-Po in sediments from Lake Tai. Compared with algaedominated regions (T1, T2, and T4), concentrations and proportions of
phytate-like P in the H2O-Po were greater from the macrophyte-algae
transitional regions (T3) and macrophyte-dominated regions (T5).
Inositol phosphates and the P bonding in NOM that is similar to the
chemical structure of inositol phosphates were likely the primary
constituents of phytate-like P (He et al., 2011; Zhu et al., 2013a).
Macrophytes in lakes and terrestrial inputs are potential sources of
inositol phosphates in sediments of lakes (Turner et al., 2002). Organic
matter accumulated in sediments from Lake Tai was derived mainly
from aquatic macrophytes, algae and bacteria. Therefore, aquatic macrophytes (Suzumura and Kamatani, 1995; Turner et al., 2002) are possibly a significant source of inositol phosphates in sediments from
macrophyte-algae transitional regions and macrophyte-dominated
regions. Phytate-like P, which would be hydrolyzed by phytase in aquatic environments, was found to be an important form of bioavailable Po
to the overlying water in lakes such as Lake Dianchi, a eutrophic
lake in China (Zhu et al., 2013a). Certain forms of phytate-like P, such
as inositol phosphates, may also be assimilated by algae directly
(Whitton et al., 1991). The portion of unidentified Po that was potentially unavailable to biota accounted for 10.6% to 54.5% (average, 31.0%) of
H2O-Po, while the enzymatically hydrolyzable, thus bioavailable Po of
H2O-Po, would be a readily available source of P for internal cycling
from sediments.
55
was removed by pretreatment with HCl (Table S4). Therefore, recoveries of P extracted by this procedure were between 66.1% and 84.8%.
Furthermore, little Po was removed by pretreatment of HCl (Table S4).
Concentrations of NaOH–EDTA Po ranged from 11.6 to 141.1 mg kg−1.
Concentrations of extracted Po were significantly and positively
correlated with TOC (%) in sediments from Lake Tai (R2 = 0.962,
P b 0.01, n = 5). Additionally, unextractable P could be inert Pi or refractory Po, which might not be bioavailable (Shinohara et al., 2012).
Based on NaOH–EDTA Po hydrolyzed by phosphatase (Fig. 3a),
bioavailable and unidentified Po forms were characterized (Fig. 3b).
Concentrations of labile monoester P ranged from 2.2 to 49.4 mg kg−1,
and accounted for 13.2% to 35.0% of NaOH–EDTA Po (Fig. 3b and
Table S5). Concentrations of diester P ranged from 0 to 9.7 mg kg−1,
and accounted for 0 to 7.6% of NaOH–EDTA Po. Concentrations of
phytate-like P ranged from 1.3 to 33.3 mg kg−1, and accounted
for 11.6% to 51.5% of NaOH–EDTA Po. Concentrations of labile monoester
P of NaOH–EDTA Po were directly proportional to TOC contents of sediments from Lake Tai (Fig. S1a), which indicates that NOM accumulation
could increase the accumulation of labile monoester P. In general,
greater phosphatase activity (e.g., alkaline phosphatase) corresponded
to greater amounts of NOM and Po in the sediments (Zhou et al., 2002,
2008). Therefore, the readily available, labile monoester P in the sediments from regions with greater amounts of NOM would maintain
and accelerate the internal cycling of P (Zhu et al., 2013a). Although
concentrations of total hydrolysable Po were positively correlated with
TOC content (Fig. S1b), there were no relationships between concentrations of diester P, or phytate-like P, and TOC contents in sediments from
different types of lakes (Fig. S1c). Also, phytate-like P was not correlated
with TOC in NaOH extracts of sediments from Lake Dianchi (Zhu et al.,
2013a). Additionally, a large portion of NaOH–EDTA Po (unidentified
Po) could not be hydrolyzed by APase, PDEase, or phytase. Concentrations of unidentified Po ranged from 8.1 to 62.9 mg kg−1, and accounted
for 28.7% to 69.6% (average, 42.3%) of NaOH–EDTA Po. The proportion of
unidentified Po was similar to that of P associated with humic materials
in soils (33–73%) (He et al., 2009b, 2011), but the proportion was greater
than that of nonhydrolyzable P in NaOH–EDTA extracts of animal
manure (9–26%) (He et al., 2007). It was postulated that the unidentified
Po was present in more complex Po forms, such as a phytate-like P associated with HA or metals (Celi et al., 1999; Dao, 2003; He et al., 2004;
3.3. Enzymatic hydrolysis and bioavailability of NaOH–EDTA extractable Po
Concentrations of TP in NaOH–EDTA extracts (NaOH–EDTA TP)
ranged from 213.3 to 749.7 mg kg− 1, and accounted for 54.7% to
67.1% of TP in sediments. In addition, 11.5% to 21.3% of TP, mainly Pi,
Fig. 3. Total and enzymatically released Po (a) and derived Po species (b) in NaOH–EDTA
extracts of sediments from Lake Tai. Data is presented as the average value with standard
deviation (n = 3).
56
Y. Zhu et al. / Chemical Geology 397 (2015) 51–60
Monbet et al., 2007). Both absolute concentrations and relative proportions of phytate-like P were lesser in the typical macrophyte-dominated
East Lake Tai (Fig. 3 and Table S5). However, the concentration of
unidentified Po was 62.9 mg kg− 1, which accounted for 44.1% of
NaOH–EDTA Po in sediments from East Lake Tai (T5). The sum of the
concentrations of phytate-like P and unidentified Po was positively
correlated with TOC content in sediments (Fig. S1d). Therefore, we
further speculate that some phytate-like P would be a constituent of
unidentified Po in sediments.
3.4. Comparison of Po forms and bioavailability by 31P NMR and enzymatic
hydrolysis
Results of 31P NMR spectroscopy of NaOH–EDTA extracts from sediments of Lake Tai are shown in Table 2 and Fig. S2. All extracts contained
Ortho-P, monoester-P, lipid-P, DNA-P and pyro-P. Trace amounts of
phosphonates were found in extracts of sediments from Meiliang Bay
(T1) and Zhushan Bay (T4). Ortho-P, which mainly exists in sediments
in phosphate form (e.g., Fe- and Ca-bound Pi), ranged in concentration
from 202.8 to 682.5 mg kg− 1. Ortho-P was the main constituent of
NaOH–EDTA TP, which accounted for 74.6% to 95.1%. Concentrations
of monoester-P ranged from 7.9 to 78.3 mg kg−1, accounting for 3.7%
to 18.9% of NaOH–EDTA TP extracted from sediments. Monoester-P
includes a range of Po compounds, such as mononucleotides, sugar
phosphates, by-products of decomposition of phospholipids, and inositol phosphates (Cade-Menun, 2005; Jørgensen et al., 2011). In this
study, it was not possible to identify specific Po compounds in the
monoester P region by 1D 31P NMR spectroscopy, as shown in Fig. S2;
these could possibly be distinguished and identified by the use of 2D
1
H–31P NMR spectroscopy (Vestergren et al., 2012). Lipid-P, which
would be expected to quickly degrade to α-glycerophosphate and
β-glycerophosphate (Shinohara et al., 2012), and then be hydrolyzed
by APase, occurred in trace amounts in sediments. Concentrations of
lipid-P ranged from 0.3 to 4.7 mg kg−1, which accounted for only 0.1%
to 0.6% of NaOH–EDTA TP in sediments. Concentrations of DNA-P
ranged from 1.8 to 22.9 mg kg−1, which accounted for 0.9% to 5.5% of
NaOH–EDTA TP extracted from sediments (Table 2). DNA-P, which originated primarily from bacterial DNA, decomposing phytoplankton and
macrophytes, could be an indicator of bacterial abundance in sediments
(Zhang et al., 2009; Baldwin, 2013). Concentrations of DNA-P were positively correlated with TOC in sediments from Lake Tai (R2 = 0.987,
P b 0.01, n = 5, Fig. S3b). These results suggest that the origin of DNA
or microbial cells was closely related with NOM in sediments from
Lake Tai. Concentrations of pyro-P ranged from 0.8 to 4.0 mg kg− 1,
and accounted for only 0.3% to 1.0% of NaOH–EDTA TP extracted from
sediments. Pyro-P, which can be synthesized by algae, bacteria, and
fungi as a response to oxic conditions, is considered the most labile of
the P compound groups, and is responsible for a significant portion of P
recycling in sediments (Hupfer et al., 2004; Ahlgren et al., 2006). Pyro-P
can be quickly hydrolyzed by APase (Zhu et al., 2013a). Poly-P was not
found in sediments from Lake Tai. Additionally, phosphonates are a
group of Po compounds containing a direct C\P bond that is resistant
to chemical, thermal, and photolytic degradation (Benitez-Nelson et al.,
2004). Phosphonates also could not be hydrolyzed by phosphatase,
such as APase, PDEase and phytase (Monbet et al., 2007; Bünemann,
2008; Zhu et al., 2013a). It has also suggested that phosphonates would
be an unrecognized source of P for certain microorganisms in the aquatic
environment (Benitez-Nelson et al., 2004; Monbet et al., 2007; Baldwin,
2013). However, phosphonates were seldom detected in the NaOH–
EDTA extracts of sediments of Lake Tai (Table 2), which is consistent
with results of studies of other lakes in China (Zhang et al., 2009; Ding
et al., 2010). Therefore, further work is needed to elucidate the bioavailability and preservation of phosphonates in the sediments from lakes.
A comparison of the various forms of NaOH–EDTA extractable P,
characterized by enzymatic hydrolysis and 31P NMR is shown in Fig. 4.
For enzymatic hydrolysis, Ortho-P and total Po were directly determined
by the molybdenum blue method, or in combination with digestion by
K2S2O8. Concentrations of Ortho-P determined by 31P NMR were greater
than those determined by enzymatic hydrolysis (Fig. 4a), while the opposite trend was observed for the concentrations of total Po (Fig. 4b).
The differences are the result of experimental artifacts of both 31P
NMR and enzymatic hydrolysis. First, Po was possibly hydrolyzed
through lyophilization of NaOH–EDTA extracts prior to 31P NMR
analysis (Cade-Menun et al., 2006), and analyzed under highly alkaline
conditions (pH N 13) over a relatively long duration (approx. 15 h):
this is widely accepted as an unavoidable limitation to characterization
of P by solution 31P NMR spectroscopy (Turner et al., 2003; He et al.,
2008). Second, concentrations of NaOH–EDTA Po would possibly be
overestimated by the use of molybdate colorimetry, because a portion
of Ortho-P associated with NOM would be determined as Po in the
extracts (Turner et al., 2006).
The sum of the concentrations of monoester P, pyro-P, and poly-P
determined by 31P NMR, which Po compounds, including pyro-P and
poly-P here, are likely to be hydrolyzed by APase and phytase (Zhu
et al., 2013a), was compared with the sum of concentrations of labile
monoester P and phytate-like P characterized by enzymatic hydrolysis.
It should be noted that some proportion of labile monoester P characterized by enzymatic hydrolysis, such as glucose phosphates, would be
degraded into Ortho-P when analyzed by solution 31P NMR with
NaOH–EDTA pretreatment (Cade-Menun et al., 2006). Some diester P,
such as RNA, would also be rapidly degraded to labile monoester P
when characterized by solution 31P NMR with NaOH–EDTA pretreatment (Turner et al., 2003). Though it would be imprecise to compare
concentrations of monoester/pyro/poly-P between these two methods,
it was clear that large portions of monoester/pyro/poly-P characterized
by 31P NMR could not be hydrolyzed by enzymes in the sediments from
the macrophyte-dominated East Lake Tai (Fig. 4c). In a previous study of
Po in environmental samples characterized by solution 31P NMR and
enzymatic hydrolysis (He et al., 2007), it was found that concentrations
of phytate-like P and labile monoester P released by phosphatase were
lower than the concentrations of inositol phosphates and other monoester P quantified by solution 31P NMR spectroscopy. These observations suggest that the monoester P cannot be hydrolyzed totally by
phosphatase, and therefore may not be totally bioavailable for microorganisms. The results of a comparison of DNA-P quantified by enzymatic
hydrolysis and solution 31P NMR have also shown that some DNA in
extractants could not be totally hydrolyzed by phosphatase, and thus
is possibly non-bioavailable (Fig. 4d). In general, Po determined in the
Table 2
Concentrations (mg kg−1) and percentage (%) of P characterized in the NAOH–EDTA extracts from sediments by solution 31P NMR spectroscopy.
Location
Phosphonates
Ortho-Pa
Monoester-P
Lipid-P
DNA-P
Pyro-P
Poly-P
T1
T2
T3
T4
T5
0.7 (0.2)
ND
ND
6.8 (0.9)
ND
347.8 (89.8)b
202.8 (95.1)
227.8 (84.3)
682.5 (91.0)
309.4 (74.6)
31.6 (8.2)
7.9 (3.7)
36.2 (13.4)
46.4 (6.2)
78.3 (18.9)
0.3 (0.1)
0.4 (0.2)
0.7 (0.3)
4.7 (0.6)
0.6 (0.1)
7.0 (1.8)
1.8 (0.9)
5.2 (1.9)
10.6 (1.4)
22.9 (5.5)
1.0 (0.3)
0.8 (0.4)
0.9 (0.3)
3.4 (0.5)
4.0 (1.0)
NDc
ND
ND
ND
ND
a
b
c
Ortho-P, orthophosphate; monoester-P, orthophosphate monoesters; lipid-P, phospholipids; pyro-P, pyrophosphate; poly-P, polyphosphate.
Values in parentheses are percentages of total P in the extracts.
ND, not detected.
Y. Zhu et al. / Chemical Geology 397 (2015) 51–60
57
Fig. 4. Comparison of P species in NaOH–EDTA extracts identified by 31P NMR analysis and enzymatic hydrolysis. The sum of labile monoester P and phytate-like P characterized by
enzymatic hydrolysis was compared with monoester P, pyro-P, and poly-P determined by 31P NMR (c).
extracts could not be totally hydrolyzed by phosphatase; this was
especially true for samples from parts of the lake dominated by primary
productivity of macrophytes (Fig. 5). Some Po may be contained in more
complex forms, such as in association with humic material or metals
(Crecchio and Stotzky, 1998; He et al., 2004; Monbet et al., 2007),
which would likely be resistant to biotic or abiotic hydrolysis
(Brannon and Sommers, 1985b; He et al., 2009b, 2011), and possibly
resist degradation in sediments (Reitzel et al., 2007).
3.5. Influence of humic acids and metals on enzymatic hydrolysis of model P
compounds
There were no effects of HA or metals on the quantification of
phosphate in this study (Table S6), which indicates that the quantification of phosphate that was released from Po by enzymes in the presence
Fig. 5. Organic P (including condensed P) determined by K2S2O8 digestion/molybdate
colorimetry (a), 31P NMR (b), and enzymatic hydrolysis (c).
of HA and metals was accurate. Results of enzymatic hydrolysis of
model Po compounds in the presence of HA and/or metal ions show
that some Po could not be hydrolyzed by enzymes, though free Po compounds could be hydrolyzed totally by enzymes (Fig. 6). These results
suggest that Po associated with HA and metals (Al3 +, Ca2 +, Fe3 +,
Fe2+) protect some Po, especially condensed P (e.g., PP) and phytatelike P (e.g., IHP6), from hydrolysis by phosphatase, thus leading to
lower availability than free Po compounds. Enzymatic hydrolysis of PP
was more affected by HA, Al3+, Fe3+, and Fe2+, while Glu6P was less influenced by HA and metals. There were still trace amounts of metals,
such as Al and Fe, in purified HA solutions (Table S1). Therefore, some
PP could possibly be combined with HA in the presence of trace metals,
and thus be rendered resistant to hydrolysis by APase. Pyrophosphates
were good complexing agents for metal ions, which were combined
strongly with Al3 + and became recalcitrant to hydrolysis by APase.
Though pyrophosphates were also good complexing agents for Ca2+,
Fe3+, and Fe2+, a larger proportion of PP was hydrolyzed by APase, especially for Ca2+. For metal ions including Ca2+, Fe3+, and Fe2+ alone,
the HA–Mn+–PP complex was more resistant to enzymatic hydrolysis
than the Mn+–PP complex. These results indicate that the association
with NOM and metals is a likely mechanism for preservation of some
PP in surface sediments. Concentrations of PP were positively correlated
with concentrations of TOC in sediments (Fig. S3c). In a previous study,
PP was found to be significantly correlated with content of the loss on
ignition (LOI) in surface sediments of Lake Tai (R2 = 0.301, P b 0.05,
n = 18) (Bai et al., 2009). PP was not a constituent of NOM in the sediments of lakes. Therefore, the significantly positive correlation between
PP and NOM in the sediments would further indicate that NOM is responsible for the preservation of some PP in the sediments. However,
PP was rapidly degraded by chemical hydrolysis after 24 h in solution
(Fig. S4a, b), which means that some PP was likely quickly degraded
in sediments of lakes (Ahlgren et al., 2005). For Glu6P, a representative labile monoester P, the HA–Mn+–Glu6P model was hydrolyzed to a lesser
extent by APase (Fig. 6b), which could protect a small proportion of labile
monoester P from enzymatic hydrolysis. Additionally, labile Glu6P was
chemically hydrolyzed in the absence of enzymes (Fig. S5a, b).
Compared with free DNA, only a small portion of DNA could resist
enzymatic hydrolysis in the presence of HA and/or metal ions
(Fig. 6c). Even if the reaction times of HA, metal ions, and DNA were
58
Y. Zhu et al. / Chemical Geology 397 (2015) 51–60
Fig. 6. Effect of HA and metal ions (Mn+) on enzymatic hydrolysis of Po compounds: (a), condensed P, PP; (b), labile monoester P, Glu6P; (c), diester P, DNA; and (d), phytate-like P, IHP6.
Solution prepared including Po, mixture of Po and HA (Po + HA), and mixtures of Po, HA and Mn+ (Po + HA + Mn+). The incubation solution was mixed for 1 h, and then hydrolyzed by
enzymes to obtain the present data. Data are presented as the average value with standard deviation (n = 3). More data are available for longer incubation times in Figs. S4 and S5.
as long as 96 h in this study (Fig. S5c, d). Thus it is likely that only a
small proportion of DNA in the prepared solution could form complexes
with HA or a combination of HA and metal ions. In a previous study,
maximal adsorption of DNA by HA was found to be approximately
0.23 μmol P/mg HA at pH 3.0 or 4.0 (Crecchio and Stotzky, 1998; Saeki
et al., 2011). This indicates that only a small portion of DNA was
adsorbed by HA in this study (10 μmol P/mg HA in the prepared solution). Metal ions, including Al3+, Ca2+, Fe3+ and Fe2+ cannot protect
DNA hydrolyzed by enzymes, but it is possible to preserve DNA by the
formation of HA–Mn+–DNA complexes (Fig. 6c). DNA was relatively
stable and few phosphates were released in the absence of enzymes
(Fig. S5c, d). However, there were notable proportions of DNA, characterized by 31P NMR, that could not be hydrolyzed by enzymes in sediments from Lake Tai, especially in sediments from the macrophytedominated region (Fig. 4). This might be because the total amounts of
DNA-P were small in the NaOH–EDTA extractants from sediments,
and then a relatively large proportion of DNA was combined with HA
or metal ions. Furthermore, only 27% to 54% DNA could be hydrolyzed
by enzymatic hydrolysis in the water from lakes with different trophic
status (Siuda and Chrόst, 2000). Therefore, more mechanisms for DNA
being resistant to enzymatic hydrolysis in lakes need to be identified
in the further works.
Enzymatic hydrolysis of IHP6, a representative phytate-like P, was
obviously inhibited by the presence of HA and metal ions (Al3+, Fe3+
and Fe2+), and partially inhibited by Ca2+ (Fig. 6d), though the activity
of phytase ensured full hydrolysis of p-nitrophenylphosphate (pNPP) in
the presence of HA, metals or a combination of HA and metals (Fig. S6).
Free IHP6 was stable and resistant to chemical hydrolysis, except for
enzymatic hydrolysis (Figs. 6d and S4c, d). Similar to the HA–PP
complex, the HA–IHP6 complex couldn't be hydrolyzed by phytase,
which is likely due to the formation of a HA–Mn+–IHP6 complex with
a trace amount of metals in the purified HA solution (Table S1). The
large molecular size of HA–Mn+–IHP6 could result in it being inaccessible to phytase. For the interaction of IHP6 with HA and Ca2 +, it was
obvious that much more IHP6 was hydrolyzed by phytase for the
Ca2 +–IHP6 complex than other Mn +–IHP6 complexes (Fig. 6d). This
result of a large proportion of Ca2 +–IHP6 hydrolysis by phytase was
similar to that observed in previous studies (Maenz et al., 1999). The
stability of the complex is an important factor for resistance to enzymatic hydrolysis. Ca2+–IHP6 is less stable than other Mn+–IHP6 complexes,
which might result in hydrolysis of Ca2 +–IHP6 (Maenz et al., 1999;
Angel et al., 2002). However, the HA–Ca2 +–IHP6 complex was more
resistant to enzymatic hydrolysis than the Ca2 +–IHP6 complex. The
presence of trace amounts of metals (Al, Fe) with HA would enhance
the formation of the other HA–Mn+–IHP6 complexes. It has been
shown previously that concentrations of myo-IHP6 (characterized by
31
P NMR) were significantly and positively correlated with total Al in
the surface sediments from lakes (Jørgensen et al., 2011). It is possible
that the Al–IHP6 complex protects IHP6 from enzymatic hydrolysis.
HA, as a model for natural, polyphenolic organic matter, and HA combined with metal ions were found to be important in the sequestration
of phytate-like P in sediments, which results in inositol phosphates
being the predominant constituent of Po in sediments of most lakes
(De Groot and Golterman, 1993; Reitzel et al., 2007; Jørgensen et al.,
2011). Inositol phosphates are even thought to be stable and could be
used as a paleo-indicator of P in sediments (Turner and Weckström,
2009). Therefore, a large proportion of phytate-like P may be resistant
to enzymatic and chemical hydrolysis in sediments, especially in the
macrophyte-dominated region of Lake Tai (Figs. 4 and 5).
The results of this study demonstrate that some Po characterized by
31
P NMR (including condensed P) cannot be hydrolyzed by phosphatase, and is not bioavailable when combined with HA with metal
bridging or metal ions to form complex Po compounds. This is probably
a mechanism for the preservation of Po, especially inositol phosphates,
Y. Zhu et al. / Chemical Geology 397 (2015) 51–60
in sediments. Enzymatic hydrolysis and ultraviolet irradiation have
shown that approximately 50% of P associated with humic and fulvic
acids is labile; this percentage was even lower (10–17%) for P after humification of environmental samples (He et al., 2006a, 2009b, 2011).
Based on the results of this study, we propose that free Po is transformed
to more recalcitrant P during early stages of diagenesis, which can sequester Po in sediments of eutrophic lakes for long periods. For example,
a 23 year half-life was reached for monoester P in sediments (extracted
by NaOH–EDTA) in Lake Erken, Sweden (Ahlgren et al., 2005) and a
27 year half-life was reported for Lake Tai, China (Ding et al., 2013).
4. Conclusions
• Accumulation of NOM increased the content of enzymatically hydrolysable Po, and thus bioavailable Po, in lake sediments. Bioavailable
Po is an important internal source of P in the sediments, which
might play a significant role in maintaining the eutrophic status of
lakes.
• Of H2O-Po and NaOH–EDTA Po, 10.6% to 54.5% (average, 31.0%) and
28.7% to 69.6% (average, 42.3%) were unidentified Po by enzymatic
hydrolysis. A comprehensive analysis of NaOH–EDTA Po by 31P NMR
and enzymatic hydrolysis suggests that some Po would be combined
with NOM to form Po–NOM complexes that are resistant to enzymatic
hydrolysis, thus unavailable. Enzymatic hydrolysis combined with 31P
NMR is a valuable tool to analyze interactions between Po and NOM in
environmental samples.
• Enzymatic hydrolysis of model P compounds, especially phytate-like
P, was inhibited in the presence of HA and metal ions; the formation
of Po–metal–HA or Po–metal complexes might be the mechanism responsible for these processes. These results suggest that incorporation
of some Po compounds into NOM, such as humic matrixes, is likely to
be an important mechanism for Po preservation in lake sediments.
Acknowledgments
This research was jointly supported by the National Natural Science
Foundation of China (Nos. 41130743, 41403094, 41261140337).
Appendix A. Supplementary data
Supplementary data to this article can be found online at http://dx.
doi.org/10.1016/j.chemgeo.2015.01.006.
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Supplementary data
Manuscript title: Influence of natural organic matter on the bioavailability and preservation of
organic phosphorus in lake sediments
Authors: Yuanrong Zhu, Fengchang Wu*, Zhongqi He, John P. Giesy, Weiying Feng, Yunsong
Mu, Chenglian Feng, Xiaoli Zhao, Haiqing Liao, Zhi Tang
Contents
pages
Supplementary Tables (S1-S6)
S2-S4
Supplementary Figures (S1-S6)
S5-S9
Submitted to: Chemical Geology
S1
Table S1. Concentrations (μM) of Al, Ca, Fe, Mn and P in the prepared HA solution a (200 mg C/L)
Element
Al
Ca
Fe
Mn
P
Concentration
26.4±10.1 b
-
10.1±5.9
-
2.3±0.8
a
determined by ICP-MS
b
average value (triple) ± S.D
Table S2. Concentrations (mM) of Al, Ca, Fe, Mn and the ratio of P to Fe and Mn concentration (w/v) in the
NaOH-EDTA extracts from sediments of Lake Tai
Location
Al
Ca
Fe
Mn
P/(Fe+Mn)
T1
1.508
4.089
0.034
0.074
3.243
T2
1.101
5.406
0.029
0.068
1.989
T3
1.556
4.788
0.034
0.077
2.200
T4
1.253
5.104
0.043
0.086
5.252
T5
1.025
6.624
0.089
0.162
1.492
Table S3. Composition of water-soluble P of the sediments characterized by enzymatic hydrolysis
TP
Pi
Po
Pi
Po
Labile monoester P
Diester P
Phytate-like P
Unidentified Po
Location
mg kg-1
% of H2O-Pt
% of H2O-Po
T1
2.4
0.7
1.7
28.1
71.9
32.9
5.2
7.3
54.5
T2
2.0
1.2
0.8
59.3
40.7
44.0
45.4
0.0
10.6
T3
2.1
0.6
1.5
29.4
70.6
30.2
13.4
45.3
11.2
T4
2.4
1.1
1.3
47.0
53.0
27.1
0.0
22.0
53.1
T5
2.8
0.8
2.0
27.2
72.8
8.2
0.0
68.7
25.8
Average
2.4
0.9
1.5
38.2
61.8
28.5
12.8
28.7
31.0
SD
0.3
0.3
0.5
14.4
14.4
13.0
19.0
28.3
21.7
S2
Table S4. Phosphorus extracted by HCl and NaOH-EDTA
HCl-TP
Location
HCl-Pi
HCl-Po
HCl-TP
mg kg -1
HCl-TP+NaOH-EDTA-TP a
% of TP in the sediments
T1
72.0
67.3
4.7
12.1
77.2
T2
44.7
44.8
-b
11.5
66.1
T3
65.0
63.9
1.1
14.9
76.7
T4
249.2
250.5
-
21.3
85.5
T5
109.5
108.2
1.3
17.7
84.8
a
HCl-TP + NaOH-EDTA-TP was the sum of total P extracted by HCl and NaOH-EDTA.
b
negative value, cannot be detectable
Table S5. Composition of NaOH-EDTA extractable P of the sediments characterized by enzymatic hydrolysis
Sampling
TP
Pi
Po
Labile monoester
Diester
Phytate-like
Unidentified
P
P
P
Po
TP
sites
mg kg-1
Recovery (%)
% of NaOH-EDTA Po
T1
387.4
327.3
59.4
65.1
30.4
7.6
23.4
38.6
T2
213.3
201.7
11.6
54.7
18.8
0.0
11.6
69.6
T3
270.1
205.8
64.7
61.8
13.2
6.6
51.5
28.7
T4
749.7
680.9
72.4
64.2
27.7
0.0
41.6
30.7
T5
414.6
273.9
141.1
67.1
35.0
6.9
14.0
44.1
Average
407.0
337.9
69.9
62.6
25.0
4.2
28.4
42.3
SD
208.7
198.6
46.4
4.8
8.9
3.9
17.5
16.5
S3
Table S6. Influence of metals (Al3+, Ca2+, Fe3+, Fe2+) and humic acid (HA) on the determination of phosphate
(PO43-) by molybdenum blue method
Samples a/time
0h
3h
7h
18h
24h
48h
96h
PO43-
196.9±1.1b
193.6±0.2
194.9±4.3
199.3±3.8
194.8±4.0
192.3±2.2
195.3±3.3
PO43-+ HA
190.9±4.5
193.3±3.3
195.8±3.3
197.6±1.6
189.1±4.4
193.7±0.2
192.0±0.2
PO43-+ Al3+
191.2±0.3
196.9±0.5
196.1±0.5
194.8±6.1
197.7±0.4
201.0±1.0
190.1±2.9
PO43-+ HA + Al3+
196.7±5.4
199.9±1.7
196.1±1.1
201.1±1.3
193.9±2.0
197.6±0.9
190.8±3.5
PO43-+ Ca2+
191.6±6.0
191.3±8.5
194.0±2.5
192.1±2.2
189.0±0.0
195.6±0.5
190.4±3.3
PO43-+ HA + Ca2+
194.5±5.7
194.2±1.9
195.9±0.4
198.1±3.9
189.5±2.0
197.2±14.4
191.9±6.9
PO43-+ Fe3+
195.7±7.9
198.0±3.5
193.4±3.8
191.4±3.4
191.7±6.3
193.0±4.0
192.4±7.5
PO43-+ HA + Fe3+
191.9±0.6
199.2±4.2
195.6±4.5
190.3±1.7
190.6±0.9
195.2±0.6
192.3±5.8
PO43-+ Fe2+
197.8±5.9
197.0±4.1
195.2±2.1
193.2±0.1
189.2±0.7
194.5±1.8
192.4±6.4
PO43-+ HA + Fe2+
193.6±2.2
197.3±1.9
194.4±1.7
194.8±3.4
189.0±6.9
197.3±3.0
190.4±0.9
a
Samples preparation described in Figure 3.
b
Mean (μM) ± standard deviation (n = 2).
S4
Total hydrolyzable Po (mg kg-1)
-1
Labile monoester P (mg kg )
60
50
y = 17.912x - 4.9599
40
(R2 = 0.977, p=0.002)
30
20
10
(a)
0
0.0
0.5
1.0
1.5
2.0
2.5
3.0
100
80
60
y = 24.990x + 8.710
(R2 = 0.848, p=0.026)
40
20
0
(b)
0.0
3.5
0.5
1.0
TOC(%)
25
20
15
5
y = 3.742x + 14.552
(R2 = 0.085, p=0.635)
0
(c)
0.5
1.0
1.5
2.0
2.5
3.0
3.5
Phytate-like P+Unknown Po (mg kg-1)
-1
Phytate-like P (mg kg )
30
0.0
2.0
2.5
3.0
3.5
TOC(%)
35
10
1.5
TOC(%)
100
y = 24.209x + 13.220
80
2
(R = 0.843, p=0.028)
60
40
20
(d)
0
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
TOC(%)
Figure S1. The liner regressions of bioavailable Po species (labile monoester P, phytate-like P, and total
hydrolyzable Po), the sum of phytate-like P and unknown Po (phytate-like P + unidentified Po) in NaOH-EDTA
extracts with TOC (%) in the sediments from Lake Tai
S5
b
c
a
e f
d
T1
T2
T3
T4
T5
20
15
10
5
0
-5
-10
-15
-20
Chemical shift (ppm)
Figure S2. 31P-NMR spectra of NaOH-EDTA extracts of sediments from Lake Tai (a, phosphonates; b,
orthophosphate; c, orthophosphate monoester; d, phospholipids; e, DNA-P; f, pyrophosphate)
25
60
40
20
0
0.0
(a)
0.5
1.0
1.5
2.0
20
-1
80
y = 0.0381x - 0.1529
(R2 = 0.948, p=0.05)
DNA-P (mg kg )
-1
Monoester-P (mg kg )
100
2.5
3.0
15
10
5
0
0.0
3.5
y = 0.1225x + 0.2111
(R2 = 0.987, p=0.001)
(b)
0.5
1.0
TOC (%)
3.0
3.5
100
-1
3.5
NMR-Po (mg kg )
-1
2.5
120
4.0
Pyro-P (mg kg )
2.0
TOC (%)
4.5
3.0
y = 1.299x + 0.2482
2
(R = 0.711, p=0.073)
2.5
2.0
1.5
0.5
1.0
1.5
2.0
2.5
3.0
80
60
y = 34.225x + 7.1673
2
(R = 0.912, p=0.011)
40
20
(c)
1.0
0.5
0.0
1.5
0
0.0
3.5
(d)
0.5
1.0
1.5
2.0
2.5
3.0
3.5
TOC (%)
TOC (%)
Figure S3. The relationships between organic P species (monoester-P, diester-P, pyro-P) characterized by NMR,
total Po determined by NMR (NMR-Po) in NaOH-EDTA Po and TOC (%) of sediments from Lake Tai
S6
% of PP hydrolyzed in buffer control % of PP hydrolyzed by phosphotase
120
(a)
100
80
60
40
20
0
100
1h
24h
80
60
40
20
0
PP
% of IHP6 hydrolyzed by phosphotase
% of IHP6 hydrolyzed in buffer control
(b)
PP
A
+H
PP
+
+
+
+
2+
3+
2+
l3 +
Fe2
Fe3
Al
Fe2
Fe3
Ca
Ca
+A
P+
P+
P+
A+
A+
A+
A+
P
P
P
H
H
H
H
+
+
+
+
PP
PP
PP
PP
120
(c)
100
80
60
40
20
0
20
1h
24h
(d)
15
10
5
0
+
+
A
2+
2+
3+
3+
P6
2+
3+
Al
Fe2
Fe3 +Fe
Al
IH P 6 + H
Ca
Ca 6+Fe
6
6+ HA+
6+ HA+
A+
A+
P
P
P
P
H
H
IH
IH P 6 +
IH P 6 +
IH P 6 +
IH P6+
IH
IH
IH
IH
Figure S4. Effect of humic acid (HA) and mental (M) on enzymatic hydrolysis of organic P. Solution prepared
including Po, mixture of Po and HA (Po + HA), and mixture of Po, HA and M ((Po + HA + M). The solution was
mixed 1h and 24h, and then hydrolyzed by enzymes here. Data are presented as the average value with standard
deviation (n=3). (a), (b), PP was pyrophosphate; (c), (d), IHP6 was inositol hexakisphosphate.
S7
% of G6P hydroyzed in buffer control % of G6P hydroyzed by phosphotase
120
(a)
100
80
60
40
20
0
100
1h
24h
80
60
40
20
0
% of DNA hydrolyzed by phosphotase
P
G6
% of DNA hydrolyzed by phosphotase
(b)
48h
96h
P
G6
A
+H
P
G6
l3 +
l3 +
a2+
e3+
e2+
a2+
e3+
e2+
+A
+F
+F
+A
+F
+F
+C
+C
6P
6P
6P
HA
HA
HA
HA
G
G
G
+
+
+
+
P
P
P
P
G6
G6
G6
G6
120
(c)
100
80
60
40
20
0
(d)
20
1h
24h
48h
96h
15
10
5
0
A
DN
l3 +
l3 +
HA
e3+
a2+
e2+
e3+
a2+
e2+
+A
+F
+F
+A
+F
+F
A+
+C
+C
A
A
A
A
A
A
N
A
A
H
H
H
D
H
DN
DN
DN
DN
A+
A+
A+
A+
DN
DN
DN
DN
Figure S5. Effect of humic acid (HA) and mental (M) on enzymatic hydrolysis of organic P. Solution prepared
including Po, mixture of Po and HA (Po + HA), and mixture of Po, HA and M ((Po + HA + M). The solution was
mixed 1h, 24h, 48h and 96h, and then hydrolyzed by enzymes here. Data are presented as the average value
with standard deviation (n=3). (a), (b), G6P was glucose-6-phosphate; (c), (d), DNA was deoxyribonucleic acid.
S8
120
Phytase Hydrolysis
Buffer Control
% of pNPP hydrolyzed
100
80
60
40
20
pNPP+HA+Fe2+
pNPP+Fe2+
pNPP+HA+Fe3+
pNPP+Fe3+
pNPP+HA+Ca2+
pNPP+Ca2+
3+
pNPP+HA+Al
pNPP+Al3+
pNPP+HA
pNPP
0
Figure S6. Influence of humic acid (HA) and metals (Al3+, Ca2+, Fe3+, Fe2+) on the activity of phytase prepared.
Solution prepared including pNPP, mixture of Po and HA (Po + HA), and mixture of Po, HA and M ((Po + HA +
M). The solution was mixed 1h, and then hydrolyzed by phytase here. Test was conducted by the substrate of
p-nitrophenylphosphate (pNPP) at pH 5.15, and 37 ºC. Data are presented as the average value with standard
deviation (n=3)
S9