Chemical Geology 397 (2015) 51–60

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Chemical Geology

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Influence of natural organic matter on the bioavailability andpreservation 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, Chinab College of Water Sciences, Beijing Normal University, Beijing 100875, Chinac USDA-ARS Southern Regional Research Center, 1100 Robert E Lee Blvd, New Orleans, LA 70124, USAd Department of Biomedical Veterinary Biosciences and Toxicology Centre, University of Saskatchewan, Saskatoon, Saskatchewan, Canadae Department of Biology and Chemistry, City University of Hong Kong, Kowloon, Hong Kong, Chinaf State Key Laboratory for Marine Pollution, City University of Hong Kong, Kowloon, Hong Kong, China

⁎ Corresponding author. Tel.: +86 10 84915312; fax: +E-mail address: wufengchang@vip.skleg.cn (F. Wu).

http://dx.doi.org/10.1016/j.chemgeo.2015.01.0060009-2541/© 2015 Elsevier B.V. All rights reserved.

a b s t r a c t

a r t i c l e i n f o

Article history:Received 25 August 2014Received in revised form 12 January 2015Accepted 13 January 2015Available online 21 January 2015

Editor: Carla M Koretsky

Keywords:Enzymatic hydrolysis31P NMROrganic phosphorusBioavailabilityPreservationNatural organic matter

Information about the bioavailability and sequestration of organic phosphorus (Po) in sediments is fundamentalto understanding biogeochemical cycling of phosphorus (P) in eutrophic lakes. However, the processesgoverning 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 appliedto 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% respectivelywere hydrolyzed by phosphatase, and therefore considered to be biolog-ically available. Of NaOH–EDTA Po, 28.7–69.6% could not be hydrolyzed by phosphatase; this portion wascharacterized by 31P NMR as monoester P and/or diester P. Simulation experiments of hydrolysis of model Pocompounds 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 monoesterP 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 insediments 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. Formationof 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 nutri-ents due to activities of humans, which then stimulates excessivegrowth of plants, has become a worldwide concern and its causesand strategies to control it are areas of active research (Smith andSchindler, 2009; McMahon and Read, 2013). Continuous allochthonousinputs and internal recycling of nutrients are both significant factorscontributing to eutrophication of lakes. Phosphorus (P) is the primarycontrollable, limiting nutrient, especially with respect to long-term con-trol of blooms of nuisance algae in most lakes (McMahon and Read,2013). As external inputs of P have gradually been reduced over thelast few decades, biogeochemical cycling of internal P that has accumu-lated in sediments has become the primary factor maintaining the

86 10 84931804.

trophic status of lakes (Søndergaard et al., 2003; Zhu et al., 2013a,b).Organic P (Po), which can constitute a substantial pool of internal Pfrom 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 importantrole in maintaining eutrophic status for lakes, especially after externalsources of P have been controlled (Zhang et al., 2008; Zhu et al.,2013a). Thus, knowledge of the composition, bioavailability, and preser-vation of Po in sediments is necessary to understand P dynamics ineutrophic lakes.

Phosphorus nuclear magnetic resonance (31P NMR) spectroscopyhas become a preferred technique that is widely used to characterizeforms of P present in sediments that has significantly advanced knowl-edge of Po in sediment from lakes (Baldwin, 2013). This techniquedistinguishes P compounds, including orthophosphate, pyrophos-phates, polyphosphate, phosphate monoester, diester phosphate, andphosphonates (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 abasic solution of NaOH–EDTA, and preconcentration methods such aslyophilization, are needed for solution 31P NMR spectroscopy analysis,and these result in some degradation of labile Po compounds (Turneret al., 2003; Cade-Menun et al., 2006). Enzymatic hydrolysis, a biochem-ical analysis procedure, provides a relatively mild approach for charac-terizing the lability of Po in environmental samples (He et al., 2006c,2008;Monbet et al., 2007; Zhu et al., 2013a). Organic P has been charac-terized previously by a combination of enzymatic hydrolysis and 31PNMR (He et al., 2007, 2008). Both methods have been found to be suit-able for characterization of Po compounds from environmental samples.With solution 31PNMR,most P compounds that have different function-al groups can be identified and quantified (Turner et al., 2003;Cade-Menun, 2005). Though a well designed procedure for enzymatichydrolysis cannot identify all Po contained in samples, this techniquecan provide an estimate of hydrolyzable, and thus bioavailable Posuch as labile monoester P, diester P, and phytate-like P in sediments(Bünemann, 2008; Zhu et al., 2003a). Since both enzymatic hydrolysisand 31P NMR have advantages and disadvantages, characterization ofPo using both methods concurrently allows for a more comprehensivecharacterization of P in sediments.

Humic acids (HA) have been used as representatives of naturalorganic matter (NOM) in studies of interactions of Po with NOMand metals (He et al., 2006a, 2009a). Most naturally occurring Pocompounds are present in either the mono- or diester form, whichleaves one or two non-ester hydroxyl groups to bind with NOM ormetal bridging to form complexes (Laarkamp, 2000; He et al., 2006b;Zhu et al., 2013a), HA as a model for NOM, such as Po combinedwith HA (Po–HA) (Brannon and Sommers, 1985a) and Po combinedwith HA with metal bridging (Po–metal–HA) (Laarkamp, 2000;Benitez-Nelson et al., 2004; Monbet et al., 2007) have been widelydiscussed and investigated. However, solution 31P NMR spectroscopycannot distinguish whether monoester P or diester P is linked to a car-bon chain like HA or metal ions in complex compounds. For example,the complexes of HA–DNA would be detected as DNA only in solution31P NMR spectroscopy. Compared with 31P NMR, enzymatic hydrolysisis a potential tool to assess the effect of NOM or metals on the reactivityof Po, because results of previous studies have shown that hydrolysis ofPo by enzymes is likely to be influenced byNOMormetal ions present inlake sediments (De Groot and Golterman, 1993; Zhu et al., 2013a). Re-sults of investigations of the stability of phosphate esters, includingphosphoserine and phosphoethanolamine, incorporated into modelhumic polymers, indicate that Po–humic materials are resistant toboth chemical and enzymatic hydrolysis (Brannon and Sommers,1985b). For diester P, DNA-P bound to HAwas protected more stronglyagainst degradation by DNase than free DNA (Crecchio and Stotzky,1998). Therefore, enzymatic hydrolysis alone (or combined with 31PNMR) is a valuable (or comprehensive) tool to investigate the interac-tions of Po with NOM or metals. Incorporation of Po into NOM and theassociation of Po with metal ions might both be mechanisms by whichPo 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 bythese interactions, which influences the biogeochemical cycle of P inlakes. Few studies have investigated mechanisms for preservation ofPo or quantified them in lake sediments (Carman et al., 2000; Reitzelet al., 2007, 2012; Ding et al., 2013), hence further and more detailedclarification on these processes was warranted.

In this study, enzymatic hydrolysis and 31P NMR were used tocharacterize Po in sediments from algae-dominated and macrophyte-dominated regions of Lake Tai. The influence of NOM on enzymatichydrolysis was also investigated. Finally, enzymatic hydrolysis ofmodel Po–HA, Po–metal, and Po–metal–HA complexes were analyzedto confirm observations about the influence of NOM on Po hydrolysis,which has implications for bioavailability and preservation of Po insediments.

2. Materials and methods

2.1. Study site and sampling

Lake Tai (Ch: Taihu), located in the Yangtze River delta, JiangsuProvince (Fig. 1), is the third largest freshwater lake in China. It's a typ-ical shallow lake with a surface area of 2338 km2, and an average depthof 1.9 m (Qin et al., 2007). With economic development and changes inland use of this area, large loads of pollutants and nutrients have beeninput to the lake. Currently, eutrophication is one of the main problemsin Lake Tai (Zhao et al., 2013). Algal blooms have occurredwith increas-ing frequency and intensity in the western and northern parts of thelake, especially in algal-dominated regions including Zhushan Bay andMeiliang Bay since the 1980s, and in recent years, blooms of phyto-plankton, including those of cyanobacteria have expanded to GonghuBay (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 eastand southeast regions of Lake Tai, including south of Gonghu and,Xukou Bays and the southeastern part of Lake Tai (called “East LakeTai”) (Zhao et al., 2013). These regions have better water quality.However, eutrophication has been accelerated by increasing nutrientconcentrations in the water and sediments of East Lake Tai (Qin et al.,2007). Especially, there were large amounts of organic matter accumu-lated in sediments from the macrophyte-dominated East Lake Tai.

In May 2009, surface sediments (top 3 cm) were collected by use ofa gravity core sampler from four regions of Lake Tai (Fig. 1). Of thesesites (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 re-gion dominated by algae. T3 (31°24′50.64″N, 120°21′5.86″E) is locatedin Gonghu Bay, which is a transitional region between macrophytesand algae. T5 (31°5′51.65″N, 120°32′54.97″E) is located in the EastLake Tai, which was in a macrophyte-dominated region. Sedimentswere transported to the laboratory in air-tight plastic bags and placedin cold storage on dry ice. Sediments were lyophilized and ground topowder 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 useof inductively coupled plasma optical-emission spectrometry (ICP-OES)after micro-acid (HNO3–HCl–HF) wet digestion of sediments. Chinesestandard reference samples of sediment (GSD-12) were analyzedsimultaneously in order to check the accuracy of results. General charac-teristics of forms of P were determined by methods that had beenharmonized and validated by use of the Standards, Measurements andTesting (SMT) program of the European Commission (Ruban et al.,1999, 2001). The operationally defined scheme was composed of fivesteps 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 Pextractable by HCl (Ca-P, P associated with Ca). For each fraction of P,concentrations were analyzed by the use of the molybdenum bluemethod (Murphy and Riley, 1962). Sediments were pretreated by anexcess of 1 mol/L HCl to remove carbonates, then analyzed for totalorganic carbon (TOC) and total nitrogen (TN) using an elementalanalyzer (Vario EL Ш, Elementar, Germany). Properties of sedimentsfrom 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 shak-ing with 60 mL of deionized water (2 h) in the first step to characterizewater-soluble Po (H2O-Po). The residue was then pretreated with 0.1 M

Fig. 1. Map of sampling sites in Lake Tai (Ch: Taihu).

53Y. Zhu et al. / Chemical Geology 397 (2015) 51–60

HCl by shaking for 1 h at room temperature to remove cations and thusreduce interferences in NMR spectroscopy of Fe and Mn (Turner et al.,2005). Before enzymatic hydrolysis, it was important to obtain a con-centrated fraction of Po by removing as much Pi as possible. The residuewas then extractedwith a solution of 0.25MNaOH–25mMEDTA (16 h)to obtain NaOH–EDTA-Po for enzymatic hydrolysis and 31P NMR analy-sis. Though 0.25 M NaOH–50 mM EDTA has been widely used forextraction of Po from sediments and soils (Cade-Menun, 2005; Turneret al., 2005), this relatively large concentration of EDTA could inhibit en-zyme activity, such as alkaline phosphatase (Chen et al., 1996) andphosphodiesterase (Wang et al., 2001), during enzymatic hydrolysis.Therefore, 0.25MNaOH–25mMEDTAwas used. This solution exhibitedefficiencies of extraction of Po that were consistent with those obtainedwith 0.25 M NaOH–50 mM EDTA (Xu et al., 2012). H2O-Po was looselyadsorbed 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 Po,H2O-Po, a generally small but mobile Po fraction in sediments, wasalso analyzed. Therefore, H2O-Po and NaOH–EDTA Po were obtainedthrough this procedure. Dissolved reactive phosphorus (DRP) in ex-tracts was analyzed by the molybdenum blue method (Murphy andRiley, 1962). TP in extracts was determined after digestion with potas-sium persulfate (K2S2O8) in an autoclave at 121 °C for 30 min. Organic

Table 1Physical and chemical properties of sediments.

Location Al Ca Fe Mn TP Pi

g kg−1 d.w. mg kg−1 d.w.

T1 50.5 7.2 28.7 1.0 595.4 411.8T2 50.5 14.0 29.7 0.7 390.2 188.4T3 23.3 3.7 22.5 0.4 437.0 266.6T4 50.3 11.2 28.0 1.2 1168.5 929.0T5 38.8 5.4 18.3 0.5 618.3 366.1

P in extracts was then determined by calculation of the differencebetween TP and DRP. An aliquot was taken for the measurement of Al,Ca, Fe and Mn by ICP-OES. The remaining NaOH–EDTA extracts wereused 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 fromSigma. H2O-Po and NaOH–EDTA Po were analyzed by enzymatic hydro-lysis, 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 waspurified to remove DRP. Aliquots of 200 mg of enzyme were dissolvedin 30 mL of an 80% saturated (NH4)2SO4 solution, then purified byprecipitation at 4 °C overnight. The precipitate was collected by centri-fugation (10,000 g) for 20 min at 4 °C. The precipitate was dissolvedin 10 mL of 10 mM NaAc–HAc buffer (pH 5.15) and dialyzed sixtimes using a Spectra/Por Float-Lyzer (MWCO: 3500–5000, SpectrumLaboratories, Inc.) for 16 hwith 2 L buffer. The dialyzed enzyme solutionwas then centrifuged (10,000 g, 4 °C) for 20 min. The purified phytasewas prepared in a NaAc–HAc buffer (0.1M, pH 5.15) or a Tris–HCl buffer(0.1 M, pH 7.0) to obtain a concentration of approximately 0.1 unit/mL.

Po Fe/Al-P Ca-P TOC TN TOC/TN

% d.w. Molar ratio

183.5 170.1 257.9 1.10 0.21 6.1201.8 126.2 104.2 0.36 0.10 4.2170.4 85.9 209.3 1.00 0.16 7.1239.6 703.5 278.9 1.36 0.21 7.6252.2 161.2 263.9 3.04 0.43 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 forthe APase, PDEase and phytase mixtures) to a final volume of 8 mL ina 10-mL tube, then incubated at 37 °C for 16 h. Samples containing anenzyme-free buffer were simultaneously incubated to monitor andcorrect for any non-enzymatic hydrolysis and effects of the matrixthrough the use of a blank. Each extract was analyzed in triplicate andmeans were reported. DRP concentrations were quantified by the mo-lybdenum blue/ascorbic acid method. Standards for DRP quantificationwere analyzed simultaneously with each procedure to correct for inter-ferences induced by enzymes andmatrices (Bünemann, 2008). Throughthis procedure (Zhu et al., 2013a), Po was classified into four species:(1) labilemonoester P (hydrolyzed by APase), (2) diester P (hydrolyzedby APase + PDEase minus labile monoester P), (3) phytate-like P(hydrolyzed by APase + PDEase + phytase minus labile monoesterand diester P), (4) unidentified Po (the portion of Po that was not hydro-lyzed by APase, PDEase and phytase).

Freeze-dried untreated NaOH–EDTA extracts were redissolved in2 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 ofD2Owas added for use as a signal lock. 31P NMR spectra weremeasuredat 161.98 MHz by a Bruker AV 400 MHz spectrometer equipped with a5 mm broadband observe probe. The conditions used to collect spectraincluded a 12 μs pulse (90°), 2-s pulse delay, and acquisition time of0.2 s,with approximately 24,000 transients. Temperaturewas regulatedat 20 °C. Based on the ratio of P to Fe andMn concentrations (w/v) in thefinal NMR sample, the total delay time used was adequate to obtainquantitative spectra of the extracts (McDowell et al., 2006). A 5 spulse delay, and an acquisition time of 0.5 swere tested again to analyzeT5 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 peakareas, and converted to concentrations of P relative to concentrationsof 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 andabsence of humic acids and metals

Four model P compounds representing a variety of molecular sizesand functional types were selected and purchased from Sigma-AldrichChemicals (Shanghai, China). They consisted of labile monoesterphosphate (glucose-6-phosphate, Glu6P), condensed-P compounds(tetra-sodium pyrophosphate, PP), diester phosphate (DNA), andphytate (inositol hexakisphosphate, IHP6). Solutions of model P com-pounds containing approximately 2 mM (P) were prepared and accu-rate concentrations were determined after digestion by H2SO4/K2S2O8.Commercially available HA (No. 53680) was also purchased fromSigma-Aldrich, then purified according to a modified IHSS purificationprocedure as described elsewhere (Hong and Elimelech, 1997). Thecharacteristics of HA here could be found in Fetsch and Havel (1998)and Rigol et al. (1998). The composition of the purified solid HA was51.3% 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 compoundswith HA, Mn+ and combinations of HA and Mn+ is described below. Avolume of 5 mL of individual model P compounds was added, then5 mL of a solution containing HA and/or Mn+ were added. The mixturewas diluted to 50 mL, and the final concentrations of metal ions werematched 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 removedand analyzed by enzymatic hydrolysis. Solutions containing Glu6P andPP respectivelywere hydrolyzed by APase at pH9.0, 37 °C; solutions con-taining DNAwere hydrolyzed by PDEase and APase at pH 9.0, 37 °C; andsolutions containing IHP6 were hydrolyzed by phytase at pH 5.15, 37 °C.To determine the effects of HA, Al3+, Ca2+, Fe3+ and Fe2+ on quantifica-tion of DRP, DRP (prepared byKH2PO4)was quantified aswell asmodel Pcompounds according to this scheme; DRP was then quantified by themolybdenum blue/ascorbic acid method. In this study, some Fe2+

could be oxidized to Fe3+ by O2. There was no detectable DRP releasedfrom 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 differentbetween regions of Lake Tai (Table 1). The greatest concentration of TPwas observed in sediments from Zhushan Bay (T4) (1168.5 mg kg−1),while the concentration was low in the sediments from outer MeiliangBay (T2). Inorganic P, including Fe/Al-P and Ca-P was the primaryform of TP in sediments from Lake Tai. Compared with TP, Fe/Al-P is agood indicator of sediment polluted by external P inputs (Zhu et al.,2013b). In sediments from Zhushan Bay, the concentration of Fe/Al-Pwas703.5mgkg−1,which accounted for 75.7% of Pi. This result indicatesa relatively large external input of P to this region. Fe/Al-P is a bioavail-able form of P in sediments, which could be an important source of P forassimilation by algae/bacteria during blooms in Meiliang Bay. At otherlocations, Ca-P, which is a relatively stable fraction of sedimentary Pand contributes to the permanent sequestration of P in sediments(Gonsiorczyk et al., 1998; Zhu et al., 2013c), was the primary form ofPi. Compared with bioavailable Fe/Al-P, internal cycling of Po is morelikely an important source of internal P in sediments, especially inMeiliang Bay, Gonghu Bay, and East Lake Tai (Table 1).

TOC content in the sediments ranged from0.36% to 3.04%. Content ofTOC was greatest in East Lake Tai, which is dominated by emergentmacrophytes, which is characterized by high amounts of organic matteraccumulation in sediments. The molar ratio of TOC/TN ranged from 4.2to 8.2, which is in the range of typical autochthonous sources, ratherthan allochthonous sources (Meyers and Ishiwatari, 1993). These find-ings indicate that organic matter in sediments of Lake Tai is derivedmainly fromdecomposition of aquaticmacrophytes, algae, and bacteria.The molar ratio of TOC/TN was greatest in sediment at T5 in East LakeTai. This is possibly because of the predominance of aquatic macro-phytes 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 to2.8 mg kg−1, 40.7% to 72.8% of which were H2O-Po (Table S3). Insediments, concentrations of H2O-Po increased as the TOC contentincreased, which is possibly due to greater decomposition of NOM asa result of greater microbial activity in spring and summer when tem-peratures are higher (De Vicente et al., 2003). Based on enzymatic hy-drolysis of H2O-Po (Fig. 2a), concentrations of individual forms of Poidentified in the H2O-Po were: labile monoester P, 0.2–0.6 mg kg−1; di-ester P, 0–0.4 mg kg−1; phytate-like P, 0–1.4 mg kg−1 and unidentifiedPo, 0.1–1.0 mg kg−1 (Fig. 2b).

Labile monoester P was an important constituent of H2O-Po, andaccounted for 8.2% to 44.0% (average, 28.5%) of H2O-Po (Fig. 2band Table S3). For Lake Dianchi, a highly eutrophic lake, the mean con-centration and proportion were 1.0 mg kg−1 and 36.7% respectively(Zhu et al., 2013a). Concentrations and proportions of labile monoesterPwere lesser in H2O-Po from Lake Tai. This indicates that eutrophicationhas enhanced the accumulation of labile monoester P of H2O-Po in sed-iments. Labile monoester P in H2O-Po was an important autochthonous

Fig. 2. Total and enzymatically released Po (a) and derived Po species (b) inwater extracts ofsediments from Lake Tai. Data are presented as the average value with standard deviation(n = 3).

Fig. 3. Total and enzymatically released Po (a) and derived Po species (b) in NaOH–EDTAextracts of sediments from Lake Tai. Data is presented as the average value with standarddeviation (n = 3).

55Y. Zhu et al. / Chemical Geology 397 (2015) 51–60

source of P within Lake Tai because of its lability and bioavailability. La-bile monoester P would be released and hydrolyzed by enzymes duringperiods when minimal DRP is available (Zhou et al., 2002; Zhu et al.,2013a). Diester P is derived mainly from algae, bacteria, and aquaticmacrophytes, which decompose faster than monoester P in Lake Tai(Ding et al., 2013). Concentrations and proportions (average, 12.8%) ofbioavailable diester P in H2O-Po were also lesser than those in H2O-Pofrom Lake Dianchi (Zhu et al., 2013a). Phytate-like P, which accountedfor 0 to 45.3% (average, 28.7%) (Table S3),was also an important constit-uent of H2O-Po in sediments from Lake Tai. Compared with algae-dominated regions (T1, T2, and T4), concentrations and proportions ofphytate-like P in the H2O-Po were greater from the macrophyte-algaetransitional regions (T3) and macrophyte-dominated regions (T5).Inositol phosphates and the P bonding in NOM that is similar to thechemical structure of inositol phosphates were likely the primaryconstituents of phytate-like P (He et al., 2011; Zhu et al., 2013a).Macrophytes in lakes and terrestrial inputs are potential sources ofinositol phosphates in sediments of lakes (Turner et al., 2002). Organicmatter accumulated in sediments from Lake Tai was derived mainlyfrom aquatic macrophytes, algae and bacteria. Therefore, aquatic mac-rophytes (Suzumura and Kamatani, 1995; Turner et al., 2002) are possi-bly a significant source of inositol phosphates in sediments frommacrophyte-algae transitional regions and macrophyte-dominatedregions. Phytate-like P,whichwould be hydrolyzedbyphytase in aquat-ic environments, was found to be an important form of bioavailable Poto the overlying water in lakes such as Lake Dianchi, a eutrophiclake in China (Zhu et al., 2013a). Certain forms of phytate-like P, suchas inositol phosphates, may also be assimilated by algae directly(Whitton et al., 1991). The portion of unidentified Po thatwas potential-ly unavailable to biota accounted for 10.6% to 54.5% (average, 31.0%) ofH2O-Po, while the enzymatically hydrolyzable, thus bioavailable Po ofH2O-Po, would be a readily available source of P for internal cyclingfrom sediments.

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% to67.1% of TP in sediments. In addition, 11.5% to 21.3% of TP, mainly Pi,

was removed by pretreatment with HCl (Table S4). Therefore, recover-ies 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 positivelycorrelated with TOC (%) in sediments from Lake Tai (R2 = 0.962,P b 0.01, n=5). Additionally, unextractable P could be inert Pi or refrac-tory 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 andTable 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 ofphytate-like P ranged from 1.3 to 33.3 mg kg−1, and accountedfor 11.6% to 51.5% of NaOH–EDTA Po. Concentrations of labilemonoesterP of NaOH–EDTA Po were directly proportional to TOC contents of sedi-ments from Lake Tai (Fig. S1a), which indicates that NOM accumulationcould increase the accumulation of labile monoester P. In general,greater phosphatase activity (e.g., alkaline phosphatase) correspondedto greater amounts of NOM and Po in the sediments (Zhou et al., 2002,2008). Therefore, the readily available, labile monoester P in the sedi-ments from regions with greater amounts of NOM would maintainand accelerate the internal cycling of P (Zhu et al., 2013a). Althoughconcentrations of total hydrolysable Po were positively correlated withTOC content (Fig. S1b), there were no relationships between concentra-tions of diester P, or phytate-like P, and TOC contents in sediments fromdifferent types of lakes (Fig. S1c). Also, phytate-like Pwas not correlatedwith TOC in NaOH extracts of sediments from Lake Dianchi (Zhu et al.,2013a). Additionally, a large portion of NaOH–EDTA Po (unidentifiedPo) could not be hydrolyzed by APase, PDEase, or phytase. Concentra-tions of unidentified Po ranged from 8.1 to 62.9 mg kg−1, and accountedfor 28.7% to 69.6% (average, 42.3%) of NaOH–EDTA Po. The proportion ofunidentified Po was similar to that of P associated with humic materialsin soils (33–73%) (He et al., 2009b, 2011), but theproportionwas greaterthan that of nonhydrolyzable P in NaOH–EDTA extracts of animalmanure (9–26%) (He et al., 2007). Itwas postulated that the unidentifiedPo was present in more complex Po forms, such as a phytate-like P asso-ciated with HA or metals (Celi et al., 1999; Dao, 2003; He et al., 2004;

56 Y. Zhu et al. / Chemical Geology 397 (2015) 51–60

Monbet et al., 2007). Both absolute concentrations and relative propor-tions of phytate-like Pwere lesser in the typical macrophyte-dominatedEast Lake Tai (Fig. 3 and Table S5). However, the concentration ofunidentified Po was 62.9 mg kg−1, which accounted for 44.1% ofNaOH–EDTA Po in sediments from East Lake Tai (T5). The sum of theconcentrations of phytate-like P and unidentified Po was positivelycorrelated with TOC content in sediments (Fig. S1d). Therefore, wefurther speculate that some phytate-like P would be a constituent ofunidentified Po in sediments.

3.4. Comparison of Po forms and bioavailability by 31P NMR and enzymatichydrolysis

Results of 31P NMR spectroscopy of NaOH–EDTA extracts from sedi-ments of Lake Tai are shown in Table 2 and Fig. S2. All extracts containedOrtho-P, monoester-P, lipid-P, DNA-P and pyro-P. Trace amounts ofphosphonates were found in extracts of sediments from Meiliang Bay(T1) and Zhushan Bay (T4). Ortho-P, which mainly exists in sedimentsin phosphate form (e.g., Fe- and Ca-bound Pi), ranged in concentrationfrom 202.8 to 682.5 mg kg−1. Ortho-P was the main constituent ofNaOH–EDTA TP, which accounted for 74.6% to 95.1%. Concentrationsof 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-Pincludes a range of Po compounds, such as mononucleotides, sugarphosphates, by-products of decomposition of phospholipids, and inosi-tol phosphates (Cade-Menun, 2005; Jørgensen et al., 2011). In thisstudy, it was not possible to identify specific Po compounds in themonoester P region by 1D 31P NMR spectroscopy, as shown in Fig. S2;these could possibly be distinguished and identified by the use of 2D1H–31P NMR spectroscopy (Vestergren et al., 2012). Lipid-P, whichwould be expected to quickly degrade to α-glycerophosphate andβ-glycerophosphate (Shinohara et al., 2012), and then be hydrolyzedby APase, occurred in trace amounts in sediments. Concentrations oflipid-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-Pranged from 1.8 to 22.9 mg kg−1, which accounted for 0.9% to 5.5% ofNaOH–EDTATP extracted from sediments (Table 2). DNA-P,which orig-inated primarily from bacterial DNA, decomposing phytoplankton andmacrophytes, could be an indicator of bacterial abundance in sediments(Zhang et al., 2009; Baldwin, 2013). Concentrations of DNA-Pwere pos-itively 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 DNAor microbial cells was closely related with NOM in sediments fromLake 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 fromsediments. Pyro-P, which can be synthesized by algae, bacteria, andfungi as a response to oxic conditions, is considered the most labile ofthe P compound groups, and is responsible for a significant portion of Precycling in sediments (Hupfer et al., 2004; Ahlgren et al., 2006). Pyro-Pcan be quickly hydrolyzed by APase (Zhu et al., 2013a). Poly-P was notfound in sediments from Lake Tai. Additionally, phosphonates are agroup of Po compounds containing a direct C\P bond that is resistantto chemical, thermal, and photolytic degradation (Benitez-Nelson et al.,2004). Phosphonates also could not be hydrolyzed by phosphatase,

Table 2Concentrations (mg kg−1) and percentage (%) of P characterized in the NAOH–EDTA extracts

Location Phosphonates Ortho-Pa Monoester-P

T1 0.7 (0.2) 347.8 (89.8)b 31.6 (8.2)T2 ND 202.8 (95.1) 7.9 (3.7)T3 ND 227.8 (84.3) 36.2 (13.4)T4 6.8 (0.9) 682.5 (91.0) 46.4 (6.2)T5 ND 309.4 (74.6) 78.3 (18.9)

a Ortho-P, orthophosphate; monoester-P, orthophosphate monoesters; lipid-P, phospholipidb Values in parentheses are percentages of total P in the extracts.c ND, not detected.

such as APase, PDEase and phytase (Monbet et al., 2007; Bünemann,2008; Zhu et al., 2013a). It has also suggested that phosphonates wouldbe an unrecognized source of P for certainmicroorganisms in the aquaticenvironment (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 consistentwith results of studies of other lakes in China (Zhang et al., 2009; Dinget al., 2010). Therefore, further work is needed to elucidate the bioavail-ability 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 Powere directly determinedby the molybdenum blue method, or in combination with digestion byK2S2O8. Concentrations of Ortho-P determined by 31PNMRwere greaterthan those determined by enzymatic hydrolysis (Fig. 4a), while the op-posite trend was observed for the concentrations of total Po (Fig. 4b).The differences are the result of experimental artifacts of both 31PNMR and enzymatic hydrolysis. First, Po was possibly hydrolyzedthrough lyophilization of NaOH–EDTA extracts prior to 31P NMRanalysis (Cade-Menun et al., 2006), and analyzed under highly alkalineconditions (pH N 13) over a relatively long duration (approx. 15 h):this is widely accepted as an unavoidable limitation to characterizationof P by solution 31P NMR spectroscopy (Turner et al., 2003; He et al.,2008). Second, concentrations of NaOH–EDTA Po would possibly beoverestimated by the use of molybdate colorimetry, because a portionof Ortho-P associated with NOM would be determined as Po in theextracts (Turner et al., 2006).

The sum of the concentrations of monoester P, pyro-P, and poly-Pdetermined by 31P NMR, which Po compounds, including pyro-P andpoly-P here, are likely to be hydrolyzed by APase and phytase (Zhuet al., 2013a), was compared with the sum of concentrations of labilemonoester P and phytate-like P characterized by enzymatic hydrolysis.It should be noted that someproportion of labilemonoester P character-ized by enzymatic hydrolysis, such as glucose phosphates, would bedegraded into Ortho-P when analyzed by solution 31P NMR withNaOH–EDTA pretreatment (Cade-Menun et al., 2006). Some diester P,such as RNA, would also be rapidly degraded to labile monoester Pwhen characterized by solution 31P NMR with NaOH–EDTA pretreat-ment (Turner et al., 2003). Though it would be imprecise to compareconcentrations of monoester/pyro/poly-P between these two methods,it was clear that large portions of monoester/pyro/poly-P characterizedby 31P NMR could not be hydrolyzed by enzymes in the sediments fromthemacrophyte-dominated East Lake Tai (Fig. 4c). In a previous study ofPo in environmental samples characterized by solution 31P NMR andenzymatic hydrolysis (He et al., 2007), it was found that concentrationsof phytate-like P and labile monoester P released by phosphatase werelower than the concentrations of inositol phosphates and other mono-ester P quantified by solution 31P NMR spectroscopy. These observa-tions suggest that the monoester P cannot be hydrolyzed totally byphosphatase, and therefore may not be totally bioavailable for microor-ganisms. The results of a comparison of DNA-P quantified by enzymatichydrolysis and solution 31P NMR have also shown that some DNA inextractants could not be totally hydrolyzed by phosphatase, and thusis possibly non-bioavailable (Fig. 4d). In general, Po determined in the

from sediments by solution 31P NMR spectroscopy.

Lipid-P DNA-P Pyro-P Poly-P

0.3 (0.1) 7.0 (1.8) 1.0 (0.3) NDc

0.4 (0.2) 1.8 (0.9) 0.8 (0.4) ND0.7 (0.3) 5.2 (1.9) 0.9 (0.3) ND4.7 (0.6) 10.6 (1.4) 3.4 (0.5) ND0.6 (0.1) 22.9 (5.5) 4.0 (1.0) ND

s; pyro-P, pyrophosphate; poly-P, polyphosphate.

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 byenzymatic hydrolysis was compared with monoester P, pyro-P, and poly-P determined by 31P NMR (c).

57Y. Zhu et al. / Chemical Geology 397 (2015) 51–60

extracts could not be totally hydrolyzed by phosphatase; this wasespecially true for samples from parts of the lake dominated by primaryproductivity ofmacrophytes (Fig. 5). Some Pomay be contained inmorecomplex 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 possiblyresist degradation in sediments (Reitzel et al., 2007).

3.5. Influence of humic acids andmetals on enzymatic hydrolysis ofmodel Pcompounds

There were no effects of HA or metals on the quantification ofphosphate in this study (Table S6), which indicates that the quantifica-tion of phosphate that was released from Po by enzymes in the presence

Fig. 5. Organic P (including condensed P) determined by K2S2O8 digestion/molybdatecolorimetry (a), 31P NMR (b), and enzymatic hydrolysis (c).

of HA and metals was accurate. Results of enzymatic hydrolysis ofmodel Po compounds in the presence of HA and/or metal ions showthat some Po could not be hydrolyzed by enzymes, though free Po com-pounds could be hydrolyzed totally by enzymes (Fig. 6). These resultssuggest that Po associated with HA and metals (Al3+, Ca2+, Fe3+,Fe2+) protect some Po, especially condensed P (e.g., PP) and phytate-like P (e.g., IHP6), from hydrolysis by phosphatase, thus leading tolower availability than free Po compounds. Enzymatic hydrolysis of PPwasmore affected byHA, Al3+, Fe3+, and Fe2+, while Glu6Pwas less in-fluenced by HA and metals. There were still trace amounts of metals,such as Al and Fe, in purified HA solutions (Table S1). Therefore, somePP could possibly be combined with HA in the presence of trace metals,and thus be rendered resistant to hydrolysis by APase. Pyrophosphateswere good complexing agents for metal ions, which were combinedstrongly 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, es-pecially for Ca2+. For metal ions including Ca2+, Fe3+, and Fe2+ alone,the HA–Mn+–PP complex was more resistant to enzymatic hydrolysisthan the Mn+–PP complex. These results indicate that the associationwith NOM and metals is a likely mechanism for preservation of somePP in surface sediments. Concentrations of PPwere positively correlatedwith concentrations of TOC in sediments (Fig. S3c). In a previous study,PP was found to be significantly correlated with content of the loss onignition (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 sedi-ments of lakes. Therefore, the significantly positive correlation betweenPP and NOM in the sediments would further indicate that NOM is re-sponsible 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 degradedin sediments of lakes (Ahlgren et al., 2005). ForGlu6P, a representative la-bile monoester P, the HA–Mn+–Glu6P model was hydrolyzed to a lesserextent by APase (Fig. 6b), which could protect a small proportion of labilemonoester P from enzymatic hydrolysis. Additionally, labile Glu6P waschemically hydrolyzed in the absence of enzymes (Fig. S5a, b).

Compared with free DNA, only a small portion of DNA could resistenzymatic hydrolysis in the presence of HA and/or metal ions(Fig. 6c). Even if the reaction times of HA, metal ions, and DNA were

Fig. 6. Effect of HA andmetal 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 byenzymes 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.

58 Y. Zhu et al. / Chemical Geology 397 (2015) 51–60

as long as 96 h in this study (Fig. S5c, d). Thus it is likely that only asmall proportion of DNA in the prepared solution could form complexeswith HA or a combination of HA and metal ions. In a previous study,maximal adsorption of DNA by HA was found to be approximately0.23 μmol P/mg HA at pH 3.0 or 4.0 (Crecchio and Stotzky, 1998; Saekiet al., 2011). This indicates that only a small portion of DNA wasadsorbed by HA in this study (10 μmol P/mg HA in the prepared solu-tion). Metal ions, including Al3+, Ca2+, Fe3+ and Fe2+ cannot protectDNA hydrolyzed by enzymes, but it is possible to preserve DNA by theformation of HA–Mn+–DNA complexes (Fig. 6c). DNA was relativelystable and few phosphates were released in the absence of enzymes(Fig. S5c, d). However, there were notable proportions of DNA, charac-terized by 31P NMR, that could not be hydrolyzed by enzymes in sedi-ments from Lake Tai, especially in sediments from the macrophyte-dominated region (Fig. 4). This might be because the total amounts ofDNA-P were small in the NaOH–EDTA extractants from sediments,and then a relatively large proportion of DNA was combined with HAor metal ions. Furthermore, only 27% to 54% DNA could be hydrolyzedby enzymatic hydrolysis in the water from lakes with different trophicstatus (Siuda and Chrόst, 2000). Therefore, more mechanisms for DNAbeing resistant to enzymatic hydrolysis in lakes need to be identifiedin the further works.

Enzymatic hydrolysis of IHP6, a representative phytate-like P, wasobviously inhibited by the presence of HA and metal ions (Al3+, Fe3+

and Fe2+), and partially inhibited by Ca2+ (Fig. 6d), though the activityof phytase ensured full hydrolysis of p-nitrophenylphosphate (pNPP) inthe presence of HA, metals or a combination of HA and metals (Fig. S6).Free IHP6 was stable and resistant to chemical hydrolysis, except forenzymatic hydrolysis (Figs. 6d and S4c, d). Similar to the HA–PPcomplex, the HA–IHP6 complex couldn't be hydrolyzed by phytase,which is likely due to the formation of a HA–Mn+–IHP6 complex witha trace amount of metals in the purified HA solution (Table S1). The

large molecular size of HA–Mn+–IHP6 could result in it being inaccessi-ble to phytase. For the interaction of IHP6 with HA and Ca2+, it wasobvious that much more IHP6 was hydrolyzed by phytase for theCa2+–IHP6 complex than other Mn+–IHP6 complexes (Fig. 6d). Thisresult of a large proportion of Ca2+–IHP6 hydrolysis by phytase wassimilar to that observed in previous studies (Maenz et al., 1999). Thestability of the complex is an important factor for resistance to enzymat-ic hydrolysis. Ca2+–IHP6 is less stable than otherMn+–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 moreresistant to enzymatic hydrolysis than the Ca2+–IHP6 complex. Thepresence of trace amounts of metals (Al, Fe) with HA would enhancethe formation of the other HA–Mn+–IHP6 complexes. It has beenshown previously that concentrations of myo-IHP6 (characterized by31P NMR) were significantly and positively correlated with total Al inthe surface sediments from lakes (Jørgensen et al., 2011). It is possiblethat the Al–IHP6 complex protects IHP6 from enzymatic hydrolysis.HA, as a model for natural, polyphenolic organic matter, and HA com-bined with metal ions were found to be important in the sequestrationof phytate-like P in sediments, which results in inositol phosphatesbeing 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 beused as a paleo-indicator of P in sediments (Turner and Weckström,2009). Therefore, a large proportion of phytate-like P may be resistantto enzymatic and chemical hydrolysis in sediments, especially in themacrophyte-dominated region of Lake Tai (Figs. 4 and 5).

The results of this study demonstrate that some Po characterized by31P NMR (including condensed P) cannot be hydrolyzed by phospha-tase, and is not bioavailable when combined with HA with metalbridging or metal ions to form complex Po compounds. This is probablya mechanism for the preservation of Po, especially inositol phosphates,

59Y. Zhu et al. / Chemical Geology 397 (2015) 51–60

in sediments. Enzymatic hydrolysis and ultraviolet irradiation haveshown that approximately 50% of P associated with humic and fulvicacids is labile; this percentage was even lower (10–17%) for P after hu-mification of environmental samples (He et al., 2006a, 2009b, 2011).Based on the results of this study,we propose that free Po is transformedto more recalcitrant P during early stages of diagenesis, which can se-quester Po in sediments of eutrophic lakes for long periods. For example,a 23 year half-life was reached for monoester P in sediments (extractedby NaOH–EDTA) in Lake Erken, Sweden (Ahlgren et al., 2005) and a27 year half-life was reported for Lake Tai, China (Ding et al., 2013).

4. Conclusions

• Accumulation of NOM increased the content of enzymatically hydro-lysable Po, and thus bioavailable Po, in lake sediments. BioavailablePo is an important internal source of P in the sediments, whichmight play a significant role in maintaining the eutrophic status oflakes.

• Of H2O-Po and NaOH–EDTA Po, 10.6% to 54.5% (average, 31.0%) and28.7% to 69.6% (average, 42.3%) were unidentified Po by enzymatichydrolysis. A comprehensive analysis of NaOH–EDTA Po by 31P NMRand enzymatic hydrolysis suggests that some Po would be combinedwith NOM to form Po–NOM complexes that are resistant to enzymatichydrolysis, thus unavailable. Enzymatic hydrolysis combinedwith 31PNMR is a valuable tool to analyze interactions between Po and NOM inenvironmental samples.

• Enzymatic hydrolysis of model P compounds, especially phytate-likeP, was inhibited in the presence of HA and metal ions; the formationof Po–metal–HA or Po–metal complexes might be the mechanism re-sponsible for these processes. These results suggest that incorporationof some Po compounds into NOM, such as humic matrixes, is likely tobe an important mechanism for Po preservation in lake sediments.

Acknowledgments

This research was jointly supported by the National Natural ScienceFoundation 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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S1

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

S2

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

Location TP Pi Po Pi Po Labile monoester P Diester P Phytate-like P Unidentified Po

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

S3

Table S4. Phosphorus extracted by HCl and NaOH-EDTA

Location HCl-TP HCl-Pi HCl-Po HCl-TP HCl-TP+NaOH-EDTA-TP

a

mg kg -1 % 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

sites

TP Pi Po TP Labile monoester

P

Diester

P

Phytate-like

P

Unidentified

Po

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

S4

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).

S5

y = 17.912x - 4.9599(R2 = 0.977, p=0.002)

TOC(%)

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5

Labi

le m

onoe

ster

P (m

g kg

-1)

0

10

20

30

40

50

60

y = 24.990x + 8.710(R2 = 0.848, p=0.026)

TOC(%)

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5

Tota

l hyd

roly

zabl

e P o

(mg

kg-1

)

0

20

40

60

80

100

y = 3.742x + 14.552(R2 = 0.085, p=0.635)

TOC(%)

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5

Phyt

ate-

like

P (m

g kg

-1)

05

101520253035

y = 24.209x + 13.220(R2 = 0.843, p=0.028)

TOC(%)

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5Phyt

ate-

like

P+U

nkno

wn

P o (m

g kg

-1)

0

20

40

60

80

100

(a) (b)

(c) (d)

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

S6

20 15 10 5 0 -5 -10 -15 -20

c feb

a

T5

T4

T2

T1

Chemical shift (ppm)

T3

d

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)

y = 0.0381x - 0.1529(R2 = 0.948, p=0.05)

TOC (%)

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5

Mon

oest

er-P

(mg

kg-1

)

0

20

40

60

80

100

y = 0.1225x + 0.2111(R2 = 0.987, p=0.001)

TOC (%)

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5

DN

A-P

(mg

kg-1

)

0

5

10

15

20

25

(a) (b)

y = 1.299x + 0.2482(R2 = 0.711, p=0.073)

TOC (%)

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5

Pyro

-P (m

g kg

-1)

0.51.01.52.02.53.03.54.04.5

(c)

y = 34.225x + 7.1673(R2 = 0.912, p=0.011)

TOC (%)

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5

NM

R-P

o (m

g kg

-1)

0

20

40

60

80

100

120

(d)

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

S7

% o

f PP

hydr

olyz

ed b

y ph

osph

otas

e

0

20

40

60

80

100

120

PPPP+HA

PP+Al3+

PP+HA+Al3+

PP+Ca2+

PP+HA+Ca2+PP+Fe3+

PP+HA+Fe3+PP+Fe2+

PP+HA+Fe2+

% o

f PP

hydr

olyz

ed in

buf

fer c

ontro

l

0

20

40

60

80

1001h24h

% o

f IH

P6 h

ydro

lyze

d by

pho

spho

tase

0

20

40

60

80

100

120

IHP6

IHP6+HA

IHP6+Al3+

IHP6+HA+Al3+

IHP6+Ca2+

IHP6+HA+Ca2+

IHP6+Fe3+

IHP6+HA+Fe3+

IHP6+Fe2+

IHP6+HA+Fe2+

% o

f IH

P6 h

ydro

lyze

d in

buf

fer c

ontro

l

0

5

10

15

20 1h24h

(a)

(b)

(c)

(d)

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.

S8

% o

f G6P

hyd

royz

ed b

y ph

osph

otas

e

0

20

40

60

80

100

120

G6PG6P+HA

G6P+Al3+

G6P+HA+Al3+

G6P+Ca2+

G6P+HA+Ca2+

G6P+Fe3+

G6P+HA+Fe3+

G6P+Fe2+

G6P+HA+Fe2+

% o

f G6P

hyd

royz

ed in

buf

fer c

ontro

l

0

20

40

60

80

1001h24h

48h96h

% o

f DN

A h

ydro

lyze

d by

pho

spho

tase

0

20

40

60

80

100

120

DNA

DNA+HA

DNA+Al3+

DNA+HA+Al3+

DNA+Ca2+

DNA+HA+Ca2+

DNA+Fe3+

DNA+HA+Fe3+

DNA+Fe2+

DNA+HA+Fe2+

% o

f DN

A h

ydro

lyze

d by

pho

spho

tase

0

5

10

15

201h 24h48h96h

(a)

(b)

(c)

(d)

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.

S9

% o

f pN

PP h

ydro

lyze

d

0

20

40

60

80

100

120

pNPP

pNPP

+HA

pNPP

+Al3+

pNPP

+HA

+Al3+

pNPP

+Ca2+

pNPP

+HA

+Ca2+

pNPP

+Fe3+

pNPP

+HA

+Fe3+

pNPP

+Fe2+

pNPP

+HA

+Fe2+

Phytase Hydrolysis Buffer Control

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)