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Water dynamics and its role in structural hysteresis of dissolved organic matter Pellegrino Conte, and Jiri Ku#erík Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.5b04639 • Publication Date (Web): 26 Jan 2016 Downloaded from http://pubs.acs.org on January 29, 2016
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Environmental Science & Technology
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Water dynamics and its role in structural hysteresis of dissolved organic
2
matter
3 4
Pellegrino Conte1 and Jiri Kucerik2*
5
1
6
Scienze edificio 4, 90128, Palermo, Italy
7
2
8
Fortstrasse 7, 768 29, Landau, Germany
Dipartimento di Scienze Agrarie e Forestali, Università degli Studi di Palermo, v.le delle
Institute for Environmental Sciences, University of Koblenz-Landau, Campus Landau,
9 10 11 12 13 14 15 16 17
* Corresponding author: Jiri Kucerik, e-mail [email protected], tel. +49 6341 280 31582
18
fax +49 6341 280 31576
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Abstract
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Knowledge of structural dynamics of dissolved organic matter (DOM) is of paramount importance
21
for understanding DOM stability and role in the fate of solubilized organic and inorganic
22
compounds (e.g. nutrients and pollutants) either in soils or aquatic systems. In this study, Fast
23
Field Cycling (FFC) 1H NMR relaxometry was applied to elucidate structural dynamics of terrestrial
24
DOM, represented by two structurally contrasting DOM models such as the Suwanee river (SRFA)
25
and the Pahokee peat (PPFA) fulvic acids purchased by the International Humic Substance Society.
26
Measurement of NMR relaxation rate of water protons in heating-cooling cycles revealed
27
structural hysteresis in both fulvic acids. In particular, structural hysteresis was related to the
28
delay in re-establishing water network around fulvic molecules as a result of temperature
29
fluctuations. The experiments revealed that the structural temperature dependency and
30
hysteresis were more pronounced in SRFA than in PPFA. This was attributed to the larger content
31
of hydrogel-like structure in SRFA stabilized, at a larger extent, by H-bonds between carboxylic
32
and phenolic groups. Moreover, results supported the view that terrestrial DOM consist of a
33
hydrophobic rigid core surrounded by progressively assembling amphiphilic and polar molecules,
34
which form an elastic structure that can mediate reactivity of the whole DOM.
35 36 37 38
Key words: dissolved organic matter, water hysteresis, Fast Filed Cycling NMR, correlation time,
39
relaxation, structural hysteresis, temperature variation
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Introduction
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Dissolved organic matter (DOM) represents an important part of natural organic matter (NOM).
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In terrestrial eco-systems, DOM mediates a myriad of biogeochemical mechanisms and responses
43
to changes in ecological processes.1 In particular, terrestrial DOM is involved in the solubilization
44
and transport of both inorganic2,3,4 and organic molecules and colloids,3, 2, 9
10
rock weathering, pedogenesis of topsoils and subsoils,
6, 11
5-8
micronutrient
soil water repellency12
45
availability,
46
and soil texture.13
47
Up to now, conformational arrangement of terrestrial DOM has been described by many, often
48
contradicting, models including the linear macromolecular polyelectrolyte hypothesis,14, 15 the
49
supramolecular assemblies of molecules stabilized by weak interactions,16, 17 the heterogeneous
50
Donnan gel phases,18 or, the mixture of supra and macromolecules.19 Although there is still not a
51
general consensus about DOM primary composition, DOM dynamics is attributed to its structural
52
complexity and flexibility.20, 21
53
To explain DOM ability to enhance solubility of hydrophic organic compounds (HOC),22 or its
54
capacity to decrease water surface tension,20 DOM has been assumed to form micelles akin to
55
classical surfactants.20, 23-25 The formation of micelle, i.e. the critical micelle concentration (CMC),
56
is reported in the range from 5 to 10 g L-1.26 However, in some cases, HOC solubilization was also
57
observed at concentrations far below the aforementioned CMC values.17 As an example, Kopinke
58
et al.,8 showed that for very hydrophobic compounds, dissolved humic acids show a significant
59
sorption effect at the concentration of 10 mg L-1. In addition, DOM ability to solubilize either
60
1,1,1-trichloro-2,2-bis(p-chlorophenyl) ethane or DDT appeared to decrease with increasing DOM
61
concentration.27 This anomaly cannot be explained by a classical micellar concept, but it can be
62
explained by the formation of premicellar aggregates, i.e. assemblies containing partially
63
hydrophobic regions formed below the critical micelle concentration.28-31 In addition, Drastik et
64
al.32 hypothesized that enhanced HOC solubility at low DOM concentration can be caused by a co-
65
solute effect (i.e. hydrotrophy) of the dissolved polar amphiphilic and hydrophilic molecules.
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Conte and Berns33 showed that humic and fulvic acids, considered as representatives of
67
NOM, consist of condensed aromatic hydrophobic cores surrounded by more hydrophilic
68
molecules. Recent ultrasonic velocimetry study of DOM supported this view and showed
69
that hydration of such a hydrophilic envelope is concentration dependent.32 At low
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concentration, small hydrophobic assemblies are stabilized by dissolved hydrophilic and
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amphiphilic molecules. As concentration increases, these molecules progressively form a 3 ACS Paragon Plus Environment
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physical hydrogel. Around concentration of 1 g L-1, an abrupt change in hydration
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characteristics appears. This can be explained as a change in domination hydration
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mechanisms.32, 34 The same concentration range was observed also by analyzing adiabatic
75
compressibility of humic acids sols.35
76
Similar model was also suggested by Baigorri et al.,19 who concluded that humic substances
77
in solution consist of a macromolecular fraction with stable aggregates and supramolecular
78
associations of low molecular weight. Chilom and Rice26 demonstrated that the least
79
abundant fraction of humic acids consisting of amphiphilic molecules facilitates aggregation
80
of other aromatic and more abundant fractions. The authors also showed that only
81
approximately one-third of their components displayed amphiphilic characteristics and
82
supported the idea of Guetzloff and Rice20 that not all molecules participate in micelle
83
formation. Lam et al.,36 showed that carboxyl-rich alicyclic molecules play a more prominent
84
role in DOM aggregation than material derived from linear terpenoids, lignin and
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carbohydrates.
86
Apart from concentration, pH and salinity,37 DOM structure and reactivity are also affected
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by temperature 3,36. Upon heating and cooling, Jia et al.38 observed a hysteresis in sorption
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and desorption of perfluorooctane sulfonate (PFOS) in dissolved humic substances. Palmer
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and von Wandruszka39 studied the influence of temperature on the size of DOM aggregates.
90
They observed an increase in DOM aggregate sizes akin to surfactant clouding at around
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40°C. On the contrary, another study32 reported two strong transitions at around 20 and
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40°C in DOM systems up to concentration of around 4 g L-1. The transitions were attributed
93
to the disruption of H-bonds stabilizing the hydrogel formed by polar and amphiphilic DOM
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moieties. At all investigated concentrations (from 0.05 up to 10 g L-1), DOM exhibited
95
structural hysteresis as temperature was varied, which was explained as a difference
96
between the rate of structural collapse and H-bond regeneration.32 In past, hysteresis in
97
ultrasonic velocities has already been observed also for pure water and its mixtures with
98
other liquids.40 This implies that DOM structural hysteresis might be caused primarily by
99
water cluster dynamics. However, this hypothesis requires additional experimental
100
verifications.
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It is worth noting that the self-assembling of molecules in aqueous systems is primarily due
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to the dynamics and arrangement of surrounding water-cluster-network (i.e. water
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structure).41, 42 Therefore, it is beneficial to assess the dynamics of dissolved and aggregated 4 ACS Paragon Plus Environment
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molecules by studying the dynamics of surrounding water molecules (e.g. refs43, 44). Indeed,
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the role of water dynamics is frequently studied for biomolecules such as proteins and DNA,
106
45
107
assembling are still scarce.
108
In order to verify the view reported by Drastik et al.32 we tested the hypotheses that (i) DOM
109
with larger content of polar and amphiphilic molecules is more vulnerable to structural
110
transitions and (ii) water dynamics plays an important role in DOM temperature-induced
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hysteresis. For this reason, we analyzed water behavior in two water soluble fulvic acids
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from the International Humic Substances Society (IHSS) by high performance size exclusion
113
chromatography (HPSEC) and fast field cycling (FFC) NMR relaxometry.47-51 Results showed
114
that DOM structural dynamics is largely governed by H-bonds, which stabilize the hydrogel
115
formed by polar and amphiphilic molecules surrounding a hydrophobic core which is, in turn,
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made predominantly by aromatic molecules held together by van der Waals forces. The
117
experimental data also suggest that temperature induced hysteresis in DOM structure is
118
caused primarily by hysteresis in water clustering, which, then, feedbacks in H-bonds
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rearrangement delay.
synthetic polymers,43 or cations and anions,46 but relevant studies on its role in DOM self-
120 121
Materials and methods
122
Samples
123
Suwanee River (SRFA) and Pahokee peat (PPFA) fulvic acids (FA) were purchased from the
124
International Humic Substances Society (IHSS) and analyzed as is. Table 1 lists the selected
125
results
126
(http://www.humicsubstances.org) and in Thorn et al.52
of
the
elemental
and
structural
analysis
reported
by
the
provider
127 128
High performance size exclusion chromatography (HPSEC)
129
HPSEC experiments were performed by using a Ultimate 3000 Standard Chromatography
130
Station (Dionex Inc., USA), equipped with a 600 × 7.8 mm BioSep S2000 column
131
thermostated at 25°C, and a BioSep Guard pre-column with a 0.2-µm stainless steel inlet
132
filter (Phenomenex Inc., USA). The UV detector set at 280 nm was used for the detection.
133
The eluent was a 50 mmol L–1 water (MilliQ) solution of NaH2PO4.H2O adjusted to pH 7 by
134
using 1 mol L-1 NaOH solution. The flow rate was set at 0.5 mL min–1, the concentration of
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SRFA and PPFA solutions used for the analysis were 0.6 mg mL–1, injected volume was 100 5 ACS Paragon Plus Environment
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µL. The calibration of the column was carried out using poly(styrenesulphonates) standards
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of 194.2, 145, 32.9, 14.9, 6.53 and 0.91 kDa mass (Polymer Standards Service Ltd., Germany).
138
The weight (MW) and number (MN) averaged molecular weights and polydispersity index
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(PDI= MW/MN) were calculated according to the ref.53 based on the semi-logarithmic
140
calibration curve of retention time and molecular weight of eluted standards. According to
141
Sutton and Sposito,16 the determined values can be considered only as apparent values, but
142
their comparison provides a valuable insight into aggregation of FAs.
143 144
Fast field cycling nuclear magnetic resonance (FFC NMR)
145
1
146
Larmor frequencies) were acquired on a Stelar Spinmaster FFC2000 Relaxometer (Stelars.r.l.;
147
Mede, PV, Italy) at several temperatures from 25 to 52°C. The details of the technique for
148
environmental applications are reported in ref.51 Briefly, proton spins were polarized at a
149
polarization field (BPOL) corresponding to a proton Larmor frequency (ωL) of 24 MHz for a
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period of polarization (TPOL) corresponding to about five times the T1 estimated at this
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frequency. After each BPOL application, the magnetic field intensity (abbreviated as BRLX) was
152
systematically changed in the proton Larmor frequency (ωL) comprised in the range 0.01–
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30.0 MHz. The period τ, during which BRLX was applied, has been varied on 20 logarithmic
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spaced time sets, each of them adjusted at every relaxation field to optimize the sampling of
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the decay/recovery curves. Free induction decays (FID) were recorded following a single 1H
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90° pulse applied at an acquisition field (BACQ) corresponding to the proton Larmor frequency
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of 16 MHz. A time domain of 100 µs sampled with 512 points was applied. Field switching
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time was 3 ms, whereas spectrometer dead time was 15 µs. For all experiments a recycle
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delay of 5 times T1 was used.
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Both FA were prepared by their dissolution in Milli-Q grade water and homogenization using
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a Vortex Mixer Cole Palmer for 1 minute in order to homogenize the system and to remove
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paramagnetic oxygen, which may affect the relaxometry experiments. The concentration of
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both solutions was chosen 40 g L-1 in order to suppress as much as possible the
164
conformation changes in FA and thus measure only changes induced by water properties.32
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Furthermore, this concertation is far above the critical micelle concentration reported by
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other authors e.g.20 thus, the molecules were self-assembled in thermodynamically stable
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aggregates. The pH of both sols were around 4. The obtained sols were measured under
H nuclear magnetic resonance dispersion profiles (i.e. relaxation rates R1 or 1/T1 vs. proton
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isothermal conditions in hermetically sealed cuvettes. The measurement was started exactly
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after 30 minutes after reaching the requested temperature by the NMR system. This
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protocol was applied for both heating and cooling cycles. Furthermore, the results in
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previous work showed that the changes in FA are reversible up to 90°C,32 therefore, only
172
physical structure of FA was affected under conditions used in this work.
173 174
FFC NMR data processing
175
The NMRD profiles were fitted in Origin Pro 7.5 SR6 with a Lorentzian function of the
176
type49,51:
Aiτ C ,i
n
R1 = ∑
177
1
1 + (ω Lτ C ,i )
2
(1)
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This equation is needed in order to account for the stretched shape of the dispersion
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curves.51 In equation (1), R1 is the longitudinal relaxation rate, ωL is the proton Larmor
180
frequency, Ai is a constant containing the proton quantum-spin number, the proton
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magnetogyric ratio, the Planck constant, and the electron-nuclear hyperfine coupling
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constant describing interactions between resonant protons and unpaired electrons. As a
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matter of fact, the larger this constant, the faster is the longitudinal relaxation rate due to
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stronger electron-nucleus interactions. τ C ,i is the correlation time of the i-th relaxing
185
component measuring the time needed for molecular re-orientation. It is a typical
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parameter for spectral density which, in turn, describes random molecular motions.49 The
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number n of Lorentzians that can be included in eq. (1) without unreasonably increasing the
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number of parameters were determined by means of the Merit function analysis.54 For the
189
present paper, n=4 was used for the mathematical fit of the NMRD profiles. It must be
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stated that the fitting parameters achieved in equation (1) do not have physical
191
significance.50 They can only be used to provide a weight average correlation time according
192
to equation (2):51 n
∑ Aτ
i i
193
τC =
1
(2)
n
∑A
i
1
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The activation energy and frequency factor of the proton relaxation was calculated
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according to the equation derived from the Arrhenius equation55 7 ACS Paragon Plus Environment
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R1 = A exp(−
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EA ) RT
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(3)
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Where R1 is the proton relaxation rate, A is the frequency factor, Ea is the activation energy,
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R is the gas constant, and T is the thermodynamic temperature.
199 200
Results and Discussion
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HPSEC evaluation of PPFA and SRFA characteristics
202
Figure 1 reports the HPSEC profiles of PPFA and SRFA. To illustrate the distribution of
203
molecular weights, the retention time was recalculated into molecular weight scale. In
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particular, PPFA revealed a more intense band in the high molecular weight range (∼ 9 kg
205
mol-1) and weaker bands in the low molecular weight interval (∼4 kg mol-1 and ∼2 kg mol-1)
206
(Figure 1).
207
Thorn et al.52 reported a comprehensive study on the chemical properties of both FAs
208
investigated in this work. The results showed the larger aromatic carbon content and C/H
209
ratio (Table 1) in PPFA, which suggest that its aggregates are stabilized to a larger extent by
210
van der Waals forces. Larger content of aromatic carbon systems implies that the amount of
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π-electrons in PPFA is bigger than in SRFA. π-clouds are responsible for the hyperchromic
212
effect,56 which explains the large HPSEC band observed at ∼ 9 kg mol-1 (Figure 1) and causes
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the larger PPFA nominal weight average molecular weight (Mw). In addition, PPFA was also
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shown to have a lower amount of phenolic groups, and a larger content of carboxylic groups
215
as compared to SRFA.52 Under the experimental conditions applied here, most of the
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carboxylic groups are deprotonated and thus negatively charged. For this reason they repel
217
each other and cause the lower tendency of PPFA (as compared to SRFA) to form inter- and
218
intra-molecular hydrogen bonds,57 thereby supporting the statement about the
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predominant stabilization by van der Waals forces. On the contrary, SRFA shows lower
220
aromatic carbon content and larger content of phenolic groups.52 Under the experimental
221
conditions used here (i.e. acidic pH), the phenolic groups are protonized and may form H-
222
bonds with the deprotonized carboxylic groups.58 For this reason, we suggest that the H-
223
bonds play more important role in SRFA aggregates stabilization as compared to PPFA.
224
By accepting hypotheses of Drastik et al.32 that at high concentration DOM mainly consists of
225
hydrophobic (i.e. aromatic) core surrounded by hydrophilic moiety formed by progressive
226
aggregation of amphiphilic and polar molecules, we can argue that PPFA consists of large 8 ACS Paragon Plus Environment
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aggregates containing condensed aromatic hydrophobic systems covered by polar, mostly
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deprotonated carboxylic groups. They allow good solvation and make PPFA more thermally
229
stable and incompressible even upon dilution as also indicated in ref.32 Conversely, the SRFA
230
samples has more hydrogel-like character that allows a better penetration of water
231
molecules and larger structural compressibility, which makes the system more vulnerable to
232
heat-induced transitions.32
233 234
Fast field cycling 1H nuclear magnetic resonance (FFC 1H NMR) relaxometry results
235
The contrasting structural features of both FAs as described above are important for
236
understanding the relaxation of water protons in DOM.
237
FFC NMR experiments revealed a decrease of the R1 values in the whole range of the applied
238
magnetic field (BRLX) for both fulvic acids as temperature was changed from 25 to 52 °C
239
(Figure 2). In particular, all the relaxation rates were faster for the PPFA than for SRFA
240
(compare the y-axes in Figure 2a and 2b).
241
The T1 relaxation mechanism refers to the energy transfer between water and dissolved
242
molecules.55 Two motion regimes, each reflecting different proton longitudinal relaxation
243
mechanisms can occur, as water is located in the close proximity of FAs’ surfaces. The outer-
244
sphere relaxation mechanism (which causes the slow motion regime), refers to water
245
diffusing in rigid structures. As a consequence, the rate of relaxation is reciprocally
246
proportional to the solvent diffusion coefficient.59 The second relaxation mechanism, i.e. the
247
inner sphere one (causing the fast motion regime), is less affected by diffusion, while the
248
chemical exchange between water and the solute (i.e. the fulvic acids) plays the dominant
249
role. Figure 3 shows that the fast motion regime occurs in both FAs.60 However, difference in
250
relaxation rates in both FAs (Figure 2) implies that relaxation of water protons in the
251
hydrogel-like SRFA is slightly decelerated by diffusion. On the contrary, due to its rigidity,
252
PPFA structure is less penetrable and thus diffusion influences relaxation at a lesser extent.
253
To verify this conclusion, the relaxation rates obtained at different temperatures were used
254
to determine activation energies and frequency factors from the slope and y-intercept of the
255
linearized form of the Arrhenius equation (3) (i.e. ln(R1) vs 1000/T), respectively. These
256
parameters were calculated for relaxation rates obtained at Larmor frequencies of 0.08 and
257
30 MHz (Figure 3, and Table 2).
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As reported in Table 2, the activation energies obtained by using R1 values are negative,
259
which resulted from decreasing relaxation rate with increasing temperature. As a rule,
260
negative values of activation energy indicate that the investigated interaction is complex and
261
does not proceed in one step or that the processes proceed along attractive potential energy
262
surfaces without energy barriers on which slower collisions are more reactive than faster
263
ones.61 Therefore, the obtained activation energies should not be simply interpreted in
264
terms of height of the potential (energy) barrier separating two minima. Instead, they are
265
interpreted as values reflecting a complex mechanism of interactions between water and
266
fulvic acids.
267
The second parameter retrieved from the Arrhenius equation is the frequency factor (Table
268
2). This parameter is the factor reflecting the frequency at which the collisions between
269
water and fulvic acids take place regardless their energy and for this reason it is less
270
influenced by interaction complexity. Consequently, the lower frequency factor for SRFA
271
indicates a lower count of interactions between water and SRFA molecules caused by the
272
restricted diffusion of water in SRFA.
273
The activation energy and the frequency factor measured at the proton Larmor frequencies
274
of 30 and 0.08 MHz (Table 2) resulted in different values for SRFA, but similar for PPFA.
275
Measurements at short Larmor frequencies are more sensitive to slowly relaxing pool of
276
water protons, such as the weakly bound or freely moving proton relaxation pools.
277
Conversely, as proton Larmor frequency increases, measurements provide information on
278
the quickly relaxing proton pools, which are due to strong interactions.49 Therefore, the
279
larger span activation energies can be interpreted with respect to FA structure as being
280
related to its heterogeneity. SRFA, due to its hydrogel-like character, contains assumingly
281
more heterogeneous environment for water proton relaxation in its structure than PPFA,
282
where the relaxation between water and polar groups takes place mainly on the surface. For
283
this reason, the values of activation energies are similar at high and low frequencies in PPFA
284
and different in SRFA. This statement accords with the polydispersity index (Table 1), which
285
is a measure of FA heterogeneity, thereby resulting higher for SRFA.
286
The results above explain the peculiarity that although water relaxes faster in PPFA (Figure
287
2), its correlation time (τc) is lower (Figure 4). In fact, correlation time represents a time
288
necessary for rotation of 1 rad of a water molecule.55 In light of above discussion, water
289
rotation is faster in the PPFA than in the SRFA structure (Table 2). Furthermore, Figure 4 10 ACS Paragon Plus Environment
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confirms the larger structural rigidity of PPFA caused by its aromatic structure. In fact, Figure
291
4 shows that the correlation times of PPFA surrounding water do not change significantly at
292
different temperatures. In contrast, the τc values of the water system in SRFA decreases with
293
increasing temperature. This means that temperature increment causes an increase in water
294
motion in SRFA due to the weakening of the FA structure. Accordingly, the physical structure
295
of SRFA is less thermally stable as in PPFA, which can be attributed to larger number of H-
296
bond stabilizing SRFA structure. Shaffer and von Wandruszka62 concluded that two
297
counteracting processes are effective at elevated temperatures both thermal agitation,
298
which tends to disrupt the aggregates, and dehydration, which promotes aggregate
299
formation. Therefore, in relatively rigid and aromatic PPFA, the temperature increase causes
300
a disruption of polar assemblies and simultaneously increases hydrophobic effect stabilizing
301
most van der Waals forces63 in hydrophobic assemblies. This leads to the increasing
302
hydrophobicity of hydrophobic domains in diluted humic acids solution upon heating as
303
observed by Shaffer and von Wandruszka62 and indicates that these domains, composed of
304
hydrophobic molecules, are porous, which enables their interaction with hydrophobic
305
compounds. It is also noteworthy that the results in Figure 4 are in contrast to observations
306
using ultrasonic velocimetry, which indicated absence of structural changes with increasing
307
temperature.64
308
Kucerik et al.65 showed that humic and fulvic acids from different sources (IHSS standards)
309
start aggregating already at concentrations around 0.01 g L-1. Zheng and Price66 investigated
310
diffusion coefficients of hydrophilic and aliphatic moieties of DOM and observed their
311
aggregation only at higher concentrations. Similarly, Smejkalova and Piccolo25 reported
312
micelle-like behavior only for two out of nine investigated humic acids and concluded that in
313
the rest of investigated samples, the length of amphiphilic molecules is either too short or
314
their abundance is too low to form micelles. Based on these results, Drastik et al32 concluded
315
that hydrophobic molecules are aggregated already at very low concentration and
316
successively involved in gluing hydrogels as concentration increases. In this work, we showed
317
that structural dynamics of DOM is largely influenced by hydrogel-like structure composed of
318
either amphiphilic or polar molecules stabilized by H-bonds, which is in accordance with
319
results of Lam et al.,36 who observed the importance of carboxyl-rich alicyclic molecules in
320
aggregation of SRFA. This DOM part responses to thermal fluctuation (and other factors such
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321
as pH and ionic strength) and presumably modulates the reactivity of hydrophobic
322
aggregates trapped in the gel structure.
323 324
Structural hysteresis of DOM
325
The relaxation rates R1 values retrieved from the heating and cooling cycles revealed the
326
hysteresis in R1 values for water as well as for both fulvic acids (Figure 5 reports exemplary
327
results only for SRFA). In other words, the rate of water protons relaxation differs as the
328
system is cooled down or heated up during the heating/cooling cycle.
329
Liquid water is a molecular 3D network stabilized by H-bonds and van der Waals forces. The
330
formation of H-bonds brings about energetic changes in enthalpy and entropy of the whole
331
network. Under equilibrium, the enthalpy and the entropy factors are mutually
332
compensated,67 but their response to temperature variation is different. We hypothesize
333
that this difference can cause the delay in re-establishing of water structure. This conclusion
334
is in accordance with the longer relaxation time during water cooling (Figure 5). The slower
335
relaxation rate indicates a change in arrangement of water structure and a decrease in
336
strength of H-bonds. The decreased strength of H-bonds in water influences the self-
337
assembling of dissolved FA molecules and consequently decreases the correlation time of
338
water in FAs upon heating and cooling (Table 2). The correlation times in Table 2 also
339
indicate that the structural hysteresis is more pronounced in hydrogel-like SRFA, i.e. in
340
flexible system containing more H-bonds than in rigid, aromatic PPFA. The results in Figure 5
341
suggest that delay in re-conformation of 3D water network is primary reason of re-
342
establishing of H-bonds in FA structure. This is in accordance with recent conclusions that
343
break of H-bonds in diluted FA and humic acids solutions are the main reasons of phase
344
transitions at 20 and 40°C and in shift of their temperatures upon heating and cooling.32
345
The variation of temperature in nature is a rule. Investigation of water properties in two
346
structurally contrasting FA showed that the temperature variation changes the properties of
347
water structure, thereby changing the properties of self-assembled colloidal systems. The
348
character of DOM self-assemblies influences their reactivity and mobility, which indicates
349
that partitioning of DOM with pollutants and nutrients and their transport depend on DOM
350
thermal history. Future research is needed in order to reveal the factors influencing the
351
DOM hysteresis. In addition, a new question arises: how relevant is DOM hysteresis in
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ecological processes such as the non-reversible sorption of and the fate of engineered
353
nanoparticles, nutrients and contaminants.
354 355
References
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1. Bolan, N. S. et al. Dissolved Organic Matter: Biogeochemistry, Dynamics, and Environmental Significance in Soils. In Advances in Agronomy, Vol 110, 2011; Vol. 110, pp 1-75. 2. Blaser, P., The role of natural organic matter in the dynamics of metals in forest soils. In Humic Substances in the Global Environment and Implications on Human Health, Senesi, N.; Miano, T., Eds. Elsevier: Amsterdam, 1994; pp 943– 960. 3. Philippe, A.; Schaumann, G. E., Interactions of dissolved organic matter with artificial inorganic colloids: a review. Environ. Sci. Technol. 2014, 48, (16), 8946–8962. 4. Zsolnay, A., Dissolved organic matter: artefacts, definitions, and functions. Geoderma 2003, 113, (3-4), 187-209. 5. Chiou, C. T.; Malcolm, R. L.; Brinton, T. I.; Kile, D. E., Water Solubility Enhancement of Some Organic Pollutants and Pesticides by Dissolved Humic and Fulvic-Acids. Environ. Sci. Technol. 1986, 20, (5), 502-508. 6. Kalbitz, K.; Solinger, S.; Park, J. H.; Michalzik, B.; Matzner, E., Controls on the dynamics of dissolved organic matter in soils: A review. Soil Sci. 2000, 165, (4), 277-304. 7. Kopinke, F. D.; Georgi, A.; MacKenzie, K., Sorption of pyrene to dissolved humic substances and related model polymers. 1. Structure-property correlation. Environ. Sci. Technol. 2001, 35, (12), 25362542. 8. Kopinke, F. D.; Georgi, A.; Mackenzie, K., Sorption and chemical reactions of PAHs with dissolved humic substances and related model polymers. Acta Hydrochim. Hydrobiol. 2001, 28, (7), 385-399. 9. Kaiser, K.; Guggenberger, G.; Zech, W., Organically bound nutrients in dissolved organic matter fractions in seepage and pore water of weakly developed forest soils. Acta Hydrochim. Hydrobiol. 2001, 28, 411-419. 10. Raulund-Rasmussen, K.; Borggaard, O. K.; Hansen, H. C. B.; Olsson, M., Effect of natural organic soil solutes on weathering rates of soil minerals. Eur. J. Soil Sci. 1998, 49, 397-406. 11. Lundström, U. S.; van Breemen, N.; Iongmans, A. G., Evidence for microbial decomposition of organic acids during podsolization. Eur. J. Soil Sci. 1995, 46, 489-496. 12. Diehl, D.; Bayer, J. V.; Woche, S. K.; Bryant, R.; Doerr, S. H.; Schaumann, G. E., Reaction of soil water repellency on artificially induced changes in soil pH. Geoderma 2010, 158, (3-4), 375-384. 13. Mavi, M. S.; Marschner, P.; Chittleborough, D. J.; Cox, J. W.; Sanderman, J., Salinity and sodicity affect soil respiration and dissolved organic matter dynamics differentially in soils varying in texture. Soil Biol. Biochem. 2012, 45, 8-13. 14. Ghosh, K.; Schnitzer, M., Macromolecular structure of humic substances. Soil Sci. 1980, 129, 266276. 15. Tombacz, E., Colloidal properties of humic acids and spontaneous changes of their colloidal state under variable solution conditions. Soil Sci. 1999, 164, (11), 814-824. 16. Sutton, R.; Sposito, G., Molecular structure in soil humic substances: The new view. Environ. Sci. Technol. 2005, 39, (23), 9009-9015. 17. Wershaw, R. L., Molecular aggregation of humic substances. Soil Sci. 1999, 164, (11), 803-813. 18. Benedetti, M. F.; vanRiemsdik, W. H.; Koopal, L. K., Humic substances considered as a heterogeneous donnan gel phase. Environ. Sci. Technol. 1996, 30, (6), 1805-1813. 19. Baigorri, R.; Fuentes, M.; Gonzalez-Gaitano, G.; Garcia-Mina, J. M., Analysis of molecular aggregation in humic substances in solution. Colloid Surface A. 2007, 302, (1-3), 301-306. 20. Guetzloff, T. F.; Rice, J. A., Does Humic-Acid Form a Micelle. Sci. Total Environ. 1994, 152, (1), 3135.
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21. MacCarthy, P.; Rice, J. A., An ecological rationale for the heterogeneous nature of humic substances. In Scientists on Gaia, Schneider, S.; Boston, P. J., Eds. MIT Press, Cambridge, MA: 1991; pp 339-345. 22. Kopinke, F. D.; Georgi, A.; Mackenzie, K., Water solubility enhancement of pyrene in the presence of humic substances, by S. Tanaka et al.: comments. Anal. Chim. Acta. 1997, 355, (2-3), 101-103. 23. Hayase, K.; Tsubota, H., Sedimentary humic acid and fulvic acid as surface active substances. Geochim. Cosmochim. Acta. 1983, 47, 947-952. 24. Simpson, A. J., Determining the molecular weight, aggregation, structures and interactions of natural organic matter using diffusion ordered spectroscopy. Magn. Reson. Chem. 2002, 40, S72-S82. 25. Smejkalova, D.; Piccolo, A., Aggregation and Disaggregation of Humic Supramolecular Assemblies by NMR Diffusion Ordered Spectroscopy (DOSY-NMR). Environ. Sci. Technol. 2008, 42, 699-706. 26. Chilom, G.; Bruns, A. S.; Rice, J. A., Aggregation of humic acid in solution: Contributions of different fractions. Org. Geochem. 2009, 40, (4), 455-460. 27. Carter, C. W.; Suffet, I. H., Binding of DDT to dissolved humic materials. Environ. Sci. Technol. 1982, 16, 735-740. 28. von Wandruszka, R., The micellar model of humic acid: Evidence from pyrene fluorescence measurements. Soil Sci. 1998, 163, (12), 921-930. 29. Engebretson, R. R.; van Wandruszka, R., Microorganization in dissolved Humic Acids. Environ. Sci. Technol. 1994, 28, 1934-1941. 30. von Wandruszka, R.; Ragle, C.; Engebretson, R., The role of selected cations in the formation of pseudomicelles in aqueous humic acid. Talanta 1997, 44, (5), 805-809. 31. Yates, L. M.; Engebretson, R. R.; Haakenson, T. J.; von Wandruszka, R., Immobilization of aqueous pyrene by dissolved humic acid. Anal. Chim. Acta. 1997, 356, (2-3), 295-300. 32. Drastik, M.; Novak, F.; Kucerík, J., Origin of heat - induced structural changes in dissolved organic matter. Chemosphere 2013, 90, 789-795. 33. Conte, P.; Berns, A. E., Dynamics of Cross Polarization in Solid State Nuclear Magnetic Resonance Experiments of Amorphous and Heterogeneous Natural Organic Substances. Anal. Sci. 2008, 24, 1183-1188. 34. Kucerik, J.; Cechlovska, H.; Bursakova, P.; Pekar, M., Lignite humic acids aggregates studied by high resolution ultrasonic spetroscopy: Thermodynamic stability and molecular feature. J. Therm. Anal. Calorim. 2009, 96, (2), 637-643. 35. Klucakova, M.; Kargerova, A.; Novackova, K., Conformational changes in humic acids in aqueous solutions. Chem. Pap. 2012, 66, (9), 875-880. 36. Lam, B.; Simpson, A. J., Investigating Aggregation in Suwannee River, USA, Dissolved Organic Matter Using Diffusion-Ordered Nuclear Magnetic Resonance Spectroscopy. Environ. Toxicol. Chem. 2009, 28, (5), 931-939. 37. Tombacz, E.; Rice, J. A., Changes of colloidal state in aqueous systems of humic acids. In Understanding Humic Substances: Advanced Methods, Properties and Applications, Ghabbour, E. A.; Davies, G., Eds. 1999; pp 69-78. 38. Jia, C.; You, C.; Pan, G., Effect of temperature on the sorption and desorption of perfluorooctane sulfonate on humic acid. J. Environ. Sci. 2010, 22, (3), 355-361. 39. Palmer, N.; von Wandruszka, R., Dynamic light scattering measurements of particle size development in aqueous humic materials. Fresen. J. Anal. Chem. 2001, 371, (7), 951-954. 40. Koc, A. B.; Vatandas, M., Ultrasonic velocity measurements on some liquids under thermal cycle: Ultrasonic velocity hysteresis Food Research International 2006, 39, 1076-1083. 41. Israelachvili, J. N., Intermolecular and surface forces. 3rd ed.; Elsevier: Amsterdam, 2011. 42. Chaplin, M., Water structuring at colloidal surfaces. Springer: Dordrecht, 2006; Vol. 228, p 1-10. 43. McBrierty, V. J.; Martin, S. J.; Karasz, F. E., Understanding hydrated polymers: the perspective of NMR. J. Mol. Liq. 1999, 80, (2-3), 179-205. 44. Prusova, A.; Conte, P.; Kucerik, J.; Alonzo, G., Dynamics of hyaluronan aqueous solutions as assessed by fast field cycling NMR relaxometry. Anal. Bioanal. Chem. 2010, 397, (7), 3023-3028. 14 ACS Paragon Plus Environment
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45. Halle, B., Cross-relaxation between macromolecular and solvent spins: The role of long-range dipole couplings. J. Chem. Phys. 2003, 119, (23), 12372-12385. 46. Conte, P., Effects of ions on water structure: a low-field 1H T 1 NMR relaxometry approach. Magn. Reson. Chem. 2015, 53, (9), 711-718. 47. Korb, J. P., Surface dynamics of liquids in porous media. Magn. Reson. Imag. 2001, 19, (363-368). 48. Korb, J. P.; Bryant, R. G., Magnetic relaxation dispersion in porous and dynamically heterogeneous materials Adv. Inorg. Chem. 2006, 57, 293-326. 49. Kimmich, R.; Anoardo, E., Field-cycling NMR relaxometry. Prog. Nucl. Mag. Res. Sp. 2004, 44, (34), 257-320. 50. Laudicina, V. A.; De Pasquale, C.; Conte, P.; Badalucco, L.; Alonzo, G.; Palazzolo, E., Effects of afforestation with four unmixed plant species on the soil-water interactions in a semiarid Mediterranean region (Sicily, Italy). J. Soils Sediments 2012, 12, (8), 1222-1230. 51. Conte, P.; Alonzo, G., Environmental NMR: Fast-field-cycling Relaxometry. eMagRes 2013, 2, 389398. 52. Thorn, K. A.; Folan, D. W.; MacCarthy, P. Characterization of the International Humic Substances Society Standard and Reference Fulvic and Humic Acids by Solution State Carbon-13 (13C) and Hydrogen-1 (1H) Nuclear Magnetic Resonance Spectrometry; U.S. Geological Survey: Denver, CO, 1989; p 93. 53. Mori, S.; Barth, H. G., Size Exclusion Chromatography. Springer-Verlag: Heidelberg, Germany, 1999. 54. Halle, B.; Jóhannesson, H.; Venu, K., Model-Free Analysis of Stretched Relaxation Dispersions. J J. Magn. Res. 1998, 135, (1), 1-13. 55. Bakhmutov, V. I., Practical NMR relaxation for chemists. Wiley: 2004; p 202. 56. Conte, P.; Piccolo, A., Conformational Arrangement of Dissolved Humic Substances. Influence of Solution Composition on Association of Humic Molecules. . Environ. Sci. Technol. 1999, 33, (10), 1682-1690. 57. Alvarez-Puebla, R. A.; Valenzuela-Calahorro, C.; Garrido, J. J., Theoretical study on fulvic acid structure, conformation and aggregation - A molecular modelling approach. Sci. Total Environ. 2006, 358, (1-3), 243-254. 58. Saab, S. d. C.; Carvalho, E. R.; Bernardes Filho, R.; de Moura, M. R.; Martin-Neto, L.; Mattoso, L. H. C., pH Effect in Aquatic Fulvic Acid from a Brazilian River. J.Braz. Chem. Soc. 2010, 21, (8), 1490-1496. 59. Hwang, L. P.; Freed, J. H., Dynamic effects of pair correlation functions on spin relaxation by translational diffusion in liquids. J. Chem. Phys. 1975, 63, 4017-4025. 60. Conte, P.; Marsala, V.; De Pasquale, C.; Bubici, S.; Valagussa, M.; Pozzi, A.; Alonzo, G., Nature of water-biochar interface interactions. GCB Bioenergy 2013, 5, 116-121. 61. Revell, L. E.; Williamson, B. E., Why Are Some Reactions Slower at Higher Temperatures? J. Chem. Educ. 2013, 90, (8), 1024-1027. 62. Shaffer, L.; von Wandruszka, R., Temperature Induced Aggregation and Clouding in Humic Acid Solutions. Adv. Environ. Chem. 2015, 2015, Article ID 543614, 6 pages. 63. Parsegian, V. A.; Ninham, B. W., Temperature-dependent van der Waals Forces. Biophys. J. 1970, 10, 664-674. 64. Drastik, M.; Ctvrtnickova, A.; Zmeskal, O.; Kucerik, J., Aggregation of humic an fulvic acids in diluted solutions. In Energy, Environment, Ecosystems, Development and Landscape Architecture, Mastorakis, N.; Helmis, C.; Papageorgiou, C. D.; Bulucea, C. A.; Panagopoulos, T., Eds. 2009; pp 163168. 65. Kučerík, J.; Drastík, M.; Zmeškal, O.; Čtvrtníčková, A., Ultrasonic spectroscopy and fractal analysis in the study on progressive aggregation of humic substances in diluted solutions. WSEAS Trans. Environ. Develop. 2009, 5, 705-715. 66. Zheng, G.; Price, W. S., Direct Hydrodynamic Radius Measurement on Dissolved Organic Matter in Natural Waters Using Diffusion NMR. Environ. Sci. Technol. 2012, 46, (3), 1675-1680. 67. Chaplin, M. F., Water’s hydrogen bond strength. In Water and Life, Lynden-Bell, R. M.; Conway Morris, S.; Barrow, J. D.; Finney, J. L.; Harper, C. L. J., Eds. CRC Press, Boca Raton: 2010; pp 69-86. 15 ACS Paragon Plus Environment
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503 504 505
Captions to Figures
506
(dotted line) fulvic acids.
507
Figure 2. Nuclear magnetic resonance dispersion (NMRD) profiles of the Pahokee peat (a)
508
and Suwannee river (b) fulvic acids. The continuous lines are obtained by the fitting
509
procedure described in the text.
510
Figure 3. Relationship between the longitudinal relaxation times measured at 0.08 and 30
511
MHz for the Pahokee peat (a) and Suwannee river (b) fulvic acids.
512
Figure 4. Variation of the water correlation time (tC) during the heating cycle of the solutions
513
containing Suwannee river (black line) and Pahokee peat fulvic acids (red line).
514
Figure 5. Hysteresis in longitudinal relaxation time during the heating and cooling cycle of
515
demineralized water (a) and Suwannee river fulvic acid (b).
Figure 1. HPSEC chromatograms of the Suwannee River (continous line) and Pahokee Peat
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Table 1. Selected properties of IHSS fulvic acids standards obtained from http://www.humicsubstances.org/ and Thorn et al.52 C H N O C/H C/O Carboxylic Phenolic C/(O+N) Suwannee river 53.0 4.4 0.8 43.9 12.2 1.20 1.19 12.2 3.11 Pahokee peat 52.1 3.2 2.4 43.9 16.1 1.18 1.12 15.2 1.78
Table 2. Parameters determined from HPSEC and FFC NMR measurements Averaged molecular weight Activation energy (kDa) (kJ/mol) Weight Number PDI** 30 MHz* 0.08 MHz* (Mw) (MN) SRFA 9.03 7.20 1.25 18.1±1.1 25.9±1.2 PPFA 9.53 8.29 1.14 11.0±0.8 10.6±0.4 *Larmor frequency, ** polydispersity index
Frequency factor (s-1) 30 MHz*
0.08 MHz*
(2.9±0.2)×10-4 (9.8±0.2)×10-3
(1.3±0.1)×10-6 (1.5±0.1)×10-2
Correlation time (ns) Before After heating heating 15 2 0.28 0.19
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Figure 1. HPSEC chromatograms of the Suwannee River (continous line) and Pahokee Peat (dotted line) fulvic acids. 271x192mm (300 x 300 DPI)
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Figure 2a. Nuclear magnetic resonance dispersion (NMRD) profiles of the Pahokee peat (a) and Suwannee river (b) fulvic acids. The continuous lines are obtained by the fitting procedure described in the text. 272x221mm (300 x 300 DPI)
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Figure 2b. Nuclear magnetic resonance dispersion (NMRD) profiles of the Pahokee peat (a) and Suwannee river (b) fulvic acids. The continuous lines are obtained by the fitting procedure described in the text. 271x220mm (300 x 300 DPI)
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Figure 3. Relationship between the longitudinal relaxation times measured at 0.08 and 30 MHz for the Pahokee peat (a) and Suwannee river (b) fulvic acids. 366x276mm (237 x 237 DPI)
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Figure 4. Variation of the water correlation time (tC) during the heating cycle of the solutions containing Suwannee river (black line) and Pahokee peat fulvic acids (red line). 387x266mm (226 x 226 DPI)
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Figure 5. Hysteresis in longitudinal relaxation time during the heating and cooling cycle of demineralized water (a) and Suwannee river fulvic acid (b). 361x465mm (300 x 300 DPI)
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graphical abstract 40x27mm (300 x 300 DPI)
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Article
Water dynamics and its role in structural hysteresis of dissolved organic matter Pellegrino Conte, and Jiri Ku#erík Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.5b04639 • Publication Date (Web): 26 Jan 2016 Downloaded from http://pubs.acs.org on January 29, 2016
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Water dynamics and its role in structural hysteresis of dissolved organic
2
matter
3 4
Pellegrino Conte1 and Jiri Kucerik2*
5
1
6
Scienze edificio 4, 90128, Palermo, Italy
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2
8
Fortstrasse 7, 768 29, Landau, Germany
Dipartimento di Scienze Agrarie e Forestali, Università degli Studi di Palermo, v.le delle
Institute for Environmental Sciences, University of Koblenz-Landau, Campus Landau,
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* Corresponding author: Jiri Kucerik, e-mail [email protected], tel. +49 6341 280 31582
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fax +49 6341 280 31576
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19
Abstract
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Knowledge of structural dynamics of dissolved organic matter (DOM) is of paramount importance
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for understanding DOM stability and role in the fate of solubilized organic and inorganic
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compounds (e.g. nutrients and pollutants) either in soils or aquatic systems. In this study, Fast
23
Field Cycling (FFC) 1H NMR relaxometry was applied to elucidate structural dynamics of terrestrial
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DOM, represented by two structurally contrasting DOM models such as the Suwanee river (SRFA)
25
and the Pahokee peat (PPFA) fulvic acids purchased by the International Humic Substance Society.
26
Measurement of NMR relaxation rate of water protons in heating-cooling cycles revealed
27
structural hysteresis in both fulvic acids. In particular, structural hysteresis was related to the
28
delay in re-establishing water network around fulvic molecules as a result of temperature
29
fluctuations. The experiments revealed that the structural temperature dependency and
30
hysteresis were more pronounced in SRFA than in PPFA. This was attributed to the larger content
31
of hydrogel-like structure in SRFA stabilized, at a larger extent, by H-bonds between carboxylic
32
and phenolic groups. Moreover, results supported the view that terrestrial DOM consist of a
33
hydrophobic rigid core surrounded by progressively assembling amphiphilic and polar molecules,
34
which form an elastic structure that can mediate reactivity of the whole DOM.
35 36 37 38
Key words: dissolved organic matter, water hysteresis, Fast Filed Cycling NMR, correlation time,
39
relaxation, structural hysteresis, temperature variation
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40
Introduction
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Dissolved organic matter (DOM) represents an important part of natural organic matter (NOM).
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In terrestrial eco-systems, DOM mediates a myriad of biogeochemical mechanisms and responses
43
to changes in ecological processes.1 In particular, terrestrial DOM is involved in the solubilization
44
and transport of both inorganic2,3,4 and organic molecules and colloids,3, 2, 9
10
rock weathering, pedogenesis of topsoils and subsoils,
6, 11
5-8
micronutrient
soil water repellency12
45
availability,
46
and soil texture.13
47
Up to now, conformational arrangement of terrestrial DOM has been described by many, often
48
contradicting, models including the linear macromolecular polyelectrolyte hypothesis,14, 15 the
49
supramolecular assemblies of molecules stabilized by weak interactions,16, 17 the heterogeneous
50
Donnan gel phases,18 or, the mixture of supra and macromolecules.19 Although there is still not a
51
general consensus about DOM primary composition, DOM dynamics is attributed to its structural
52
complexity and flexibility.20, 21
53
To explain DOM ability to enhance solubility of hydrophic organic compounds (HOC),22 or its
54
capacity to decrease water surface tension,20 DOM has been assumed to form micelles akin to
55
classical surfactants.20, 23-25 The formation of micelle, i.e. the critical micelle concentration (CMC),
56
is reported in the range from 5 to 10 g L-1.26 However, in some cases, HOC solubilization was also
57
observed at concentrations far below the aforementioned CMC values.17 As an example, Kopinke
58
et al.,8 showed that for very hydrophobic compounds, dissolved humic acids show a significant
59
sorption effect at the concentration of 10 mg L-1. In addition, DOM ability to solubilize either
60
1,1,1-trichloro-2,2-bis(p-chlorophenyl) ethane or DDT appeared to decrease with increasing DOM
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concentration.27 This anomaly cannot be explained by a classical micellar concept, but it can be
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explained by the formation of premicellar aggregates, i.e. assemblies containing partially
63
hydrophobic regions formed below the critical micelle concentration.28-31 In addition, Drastik et
64
al.32 hypothesized that enhanced HOC solubility at low DOM concentration can be caused by a co-
65
solute effect (i.e. hydrotrophy) of the dissolved polar amphiphilic and hydrophilic molecules.
66
Conte and Berns33 showed that humic and fulvic acids, considered as representatives of
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NOM, consist of condensed aromatic hydrophobic cores surrounded by more hydrophilic
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molecules. Recent ultrasonic velocimetry study of DOM supported this view and showed
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that hydration of such a hydrophilic envelope is concentration dependent.32 At low
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concentration, small hydrophobic assemblies are stabilized by dissolved hydrophilic and
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amphiphilic molecules. As concentration increases, these molecules progressively form a 3 ACS Paragon Plus Environment
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physical hydrogel. Around concentration of 1 g L-1, an abrupt change in hydration
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characteristics appears. This can be explained as a change in domination hydration
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mechanisms.32, 34 The same concentration range was observed also by analyzing adiabatic
75
compressibility of humic acids sols.35
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Similar model was also suggested by Baigorri et al.,19 who concluded that humic substances
77
in solution consist of a macromolecular fraction with stable aggregates and supramolecular
78
associations of low molecular weight. Chilom and Rice26 demonstrated that the least
79
abundant fraction of humic acids consisting of amphiphilic molecules facilitates aggregation
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of other aromatic and more abundant fractions. The authors also showed that only
81
approximately one-third of their components displayed amphiphilic characteristics and
82
supported the idea of Guetzloff and Rice20 that not all molecules participate in micelle
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formation. Lam et al.,36 showed that carboxyl-rich alicyclic molecules play a more prominent
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role in DOM aggregation than material derived from linear terpenoids, lignin and
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carbohydrates.
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Apart from concentration, pH and salinity,37 DOM structure and reactivity are also affected
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by temperature 3,36. Upon heating and cooling, Jia et al.38 observed a hysteresis in sorption
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and desorption of perfluorooctane sulfonate (PFOS) in dissolved humic substances. Palmer
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and von Wandruszka39 studied the influence of temperature on the size of DOM aggregates.
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They observed an increase in DOM aggregate sizes akin to surfactant clouding at around
91
40°C. On the contrary, another study32 reported two strong transitions at around 20 and
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40°C in DOM systems up to concentration of around 4 g L-1. The transitions were attributed
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to the disruption of H-bonds stabilizing the hydrogel formed by polar and amphiphilic DOM
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moieties. At all investigated concentrations (from 0.05 up to 10 g L-1), DOM exhibited
95
structural hysteresis as temperature was varied, which was explained as a difference
96
between the rate of structural collapse and H-bond regeneration.32 In past, hysteresis in
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ultrasonic velocities has already been observed also for pure water and its mixtures with
98
other liquids.40 This implies that DOM structural hysteresis might be caused primarily by
99
water cluster dynamics. However, this hypothesis requires additional experimental
100
verifications.
101
It is worth noting that the self-assembling of molecules in aqueous systems is primarily due
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to the dynamics and arrangement of surrounding water-cluster-network (i.e. water
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structure).41, 42 Therefore, it is beneficial to assess the dynamics of dissolved and aggregated 4 ACS Paragon Plus Environment
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molecules by studying the dynamics of surrounding water molecules (e.g. refs43, 44). Indeed,
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the role of water dynamics is frequently studied for biomolecules such as proteins and DNA,
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45
107
assembling are still scarce.
108
In order to verify the view reported by Drastik et al.32 we tested the hypotheses that (i) DOM
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with larger content of polar and amphiphilic molecules is more vulnerable to structural
110
transitions and (ii) water dynamics plays an important role in DOM temperature-induced
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hysteresis. For this reason, we analyzed water behavior in two water soluble fulvic acids
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from the International Humic Substances Society (IHSS) by high performance size exclusion
113
chromatography (HPSEC) and fast field cycling (FFC) NMR relaxometry.47-51 Results showed
114
that DOM structural dynamics is largely governed by H-bonds, which stabilize the hydrogel
115
formed by polar and amphiphilic molecules surrounding a hydrophobic core which is, in turn,
116
made predominantly by aromatic molecules held together by van der Waals forces. The
117
experimental data also suggest that temperature induced hysteresis in DOM structure is
118
caused primarily by hysteresis in water clustering, which, then, feedbacks in H-bonds
119
rearrangement delay.
synthetic polymers,43 or cations and anions,46 but relevant studies on its role in DOM self-
120 121
Materials and methods
122
Samples
123
Suwanee River (SRFA) and Pahokee peat (PPFA) fulvic acids (FA) were purchased from the
124
International Humic Substances Society (IHSS) and analyzed as is. Table 1 lists the selected
125
results
126
(http://www.humicsubstances.org) and in Thorn et al.52
of
the
elemental
and
structural
analysis
reported
by
the
provider
127 128
High performance size exclusion chromatography (HPSEC)
129
HPSEC experiments were performed by using a Ultimate 3000 Standard Chromatography
130
Station (Dionex Inc., USA), equipped with a 600 × 7.8 mm BioSep S2000 column
131
thermostated at 25°C, and a BioSep Guard pre-column with a 0.2-µm stainless steel inlet
132
filter (Phenomenex Inc., USA). The UV detector set at 280 nm was used for the detection.
133
The eluent was a 50 mmol L–1 water (MilliQ) solution of NaH2PO4.H2O adjusted to pH 7 by
134
using 1 mol L-1 NaOH solution. The flow rate was set at 0.5 mL min–1, the concentration of
135
SRFA and PPFA solutions used for the analysis were 0.6 mg mL–1, injected volume was 100 5 ACS Paragon Plus Environment
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136
µL. The calibration of the column was carried out using poly(styrenesulphonates) standards
137
of 194.2, 145, 32.9, 14.9, 6.53 and 0.91 kDa mass (Polymer Standards Service Ltd., Germany).
138
The weight (MW) and number (MN) averaged molecular weights and polydispersity index
139
(PDI= MW/MN) were calculated according to the ref.53 based on the semi-logarithmic
140
calibration curve of retention time and molecular weight of eluted standards. According to
141
Sutton and Sposito,16 the determined values can be considered only as apparent values, but
142
their comparison provides a valuable insight into aggregation of FAs.
143 144
Fast field cycling nuclear magnetic resonance (FFC NMR)
145
1
146
Larmor frequencies) were acquired on a Stelar Spinmaster FFC2000 Relaxometer (Stelars.r.l.;
147
Mede, PV, Italy) at several temperatures from 25 to 52°C. The details of the technique for
148
environmental applications are reported in ref.51 Briefly, proton spins were polarized at a
149
polarization field (BPOL) corresponding to a proton Larmor frequency (ωL) of 24 MHz for a
150
period of polarization (TPOL) corresponding to about five times the T1 estimated at this
151
frequency. After each BPOL application, the magnetic field intensity (abbreviated as BRLX) was
152
systematically changed in the proton Larmor frequency (ωL) comprised in the range 0.01–
153
30.0 MHz. The period τ, during which BRLX was applied, has been varied on 20 logarithmic
154
spaced time sets, each of them adjusted at every relaxation field to optimize the sampling of
155
the decay/recovery curves. Free induction decays (FID) were recorded following a single 1H
156
90° pulse applied at an acquisition field (BACQ) corresponding to the proton Larmor frequency
157
of 16 MHz. A time domain of 100 µs sampled with 512 points was applied. Field switching
158
time was 3 ms, whereas spectrometer dead time was 15 µs. For all experiments a recycle
159
delay of 5 times T1 was used.
160
Both FA were prepared by their dissolution in Milli-Q grade water and homogenization using
161
a Vortex Mixer Cole Palmer for 1 minute in order to homogenize the system and to remove
162
paramagnetic oxygen, which may affect the relaxometry experiments. The concentration of
163
both solutions was chosen 40 g L-1 in order to suppress as much as possible the
164
conformation changes in FA and thus measure only changes induced by water properties.32
165
Furthermore, this concertation is far above the critical micelle concentration reported by
166
other authors e.g.20 thus, the molecules were self-assembled in thermodynamically stable
167
aggregates. The pH of both sols were around 4. The obtained sols were measured under
H nuclear magnetic resonance dispersion profiles (i.e. relaxation rates R1 or 1/T1 vs. proton
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isothermal conditions in hermetically sealed cuvettes. The measurement was started exactly
169
after 30 minutes after reaching the requested temperature by the NMR system. This
170
protocol was applied for both heating and cooling cycles. Furthermore, the results in
171
previous work showed that the changes in FA are reversible up to 90°C,32 therefore, only
172
physical structure of FA was affected under conditions used in this work.
173 174
FFC NMR data processing
175
The NMRD profiles were fitted in Origin Pro 7.5 SR6 with a Lorentzian function of the
176
type49,51:
Aiτ C ,i
n
R1 = ∑
177
1
1 + (ω Lτ C ,i )
2
(1)
178
This equation is needed in order to account for the stretched shape of the dispersion
179
curves.51 In equation (1), R1 is the longitudinal relaxation rate, ωL is the proton Larmor
180
frequency, Ai is a constant containing the proton quantum-spin number, the proton
181
magnetogyric ratio, the Planck constant, and the electron-nuclear hyperfine coupling
182
constant describing interactions between resonant protons and unpaired electrons. As a
183
matter of fact, the larger this constant, the faster is the longitudinal relaxation rate due to
184
stronger electron-nucleus interactions. τ C ,i is the correlation time of the i-th relaxing
185
component measuring the time needed for molecular re-orientation. It is a typical
186
parameter for spectral density which, in turn, describes random molecular motions.49 The
187
number n of Lorentzians that can be included in eq. (1) without unreasonably increasing the
188
number of parameters were determined by means of the Merit function analysis.54 For the
189
present paper, n=4 was used for the mathematical fit of the NMRD profiles. It must be
190
stated that the fitting parameters achieved in equation (1) do not have physical
191
significance.50 They can only be used to provide a weight average correlation time according
192
to equation (2):51 n
∑ Aτ
i i
193
τC =
1
(2)
n
∑A
i
1
194
The activation energy and frequency factor of the proton relaxation was calculated
195
according to the equation derived from the Arrhenius equation55 7 ACS Paragon Plus Environment
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R1 = A exp(−
196
EA ) RT
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(3)
197
Where R1 is the proton relaxation rate, A is the frequency factor, Ea is the activation energy,
198
R is the gas constant, and T is the thermodynamic temperature.
199 200
Results and Discussion
201
HPSEC evaluation of PPFA and SRFA characteristics
202
Figure 1 reports the HPSEC profiles of PPFA and SRFA. To illustrate the distribution of
203
molecular weights, the retention time was recalculated into molecular weight scale. In
204
particular, PPFA revealed a more intense band in the high molecular weight range (∼ 9 kg
205
mol-1) and weaker bands in the low molecular weight interval (∼4 kg mol-1 and ∼2 kg mol-1)
206
(Figure 1).
207
Thorn et al.52 reported a comprehensive study on the chemical properties of both FAs
208
investigated in this work. The results showed the larger aromatic carbon content and C/H
209
ratio (Table 1) in PPFA, which suggest that its aggregates are stabilized to a larger extent by
210
van der Waals forces. Larger content of aromatic carbon systems implies that the amount of
211
π-electrons in PPFA is bigger than in SRFA. π-clouds are responsible for the hyperchromic
212
effect,56 which explains the large HPSEC band observed at ∼ 9 kg mol-1 (Figure 1) and causes
213
the larger PPFA nominal weight average molecular weight (Mw). In addition, PPFA was also
214
shown to have a lower amount of phenolic groups, and a larger content of carboxylic groups
215
as compared to SRFA.52 Under the experimental conditions applied here, most of the
216
carboxylic groups are deprotonated and thus negatively charged. For this reason they repel
217
each other and cause the lower tendency of PPFA (as compared to SRFA) to form inter- and
218
intra-molecular hydrogen bonds,57 thereby supporting the statement about the
219
predominant stabilization by van der Waals forces. On the contrary, SRFA shows lower
220
aromatic carbon content and larger content of phenolic groups.52 Under the experimental
221
conditions used here (i.e. acidic pH), the phenolic groups are protonized and may form H-
222
bonds with the deprotonized carboxylic groups.58 For this reason, we suggest that the H-
223
bonds play more important role in SRFA aggregates stabilization as compared to PPFA.
224
By accepting hypotheses of Drastik et al.32 that at high concentration DOM mainly consists of
225
hydrophobic (i.e. aromatic) core surrounded by hydrophilic moiety formed by progressive
226
aggregation of amphiphilic and polar molecules, we can argue that PPFA consists of large 8 ACS Paragon Plus Environment
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aggregates containing condensed aromatic hydrophobic systems covered by polar, mostly
228
deprotonated carboxylic groups. They allow good solvation and make PPFA more thermally
229
stable and incompressible even upon dilution as also indicated in ref.32 Conversely, the SRFA
230
samples has more hydrogel-like character that allows a better penetration of water
231
molecules and larger structural compressibility, which makes the system more vulnerable to
232
heat-induced transitions.32
233 234
Fast field cycling 1H nuclear magnetic resonance (FFC 1H NMR) relaxometry results
235
The contrasting structural features of both FAs as described above are important for
236
understanding the relaxation of water protons in DOM.
237
FFC NMR experiments revealed a decrease of the R1 values in the whole range of the applied
238
magnetic field (BRLX) for both fulvic acids as temperature was changed from 25 to 52 °C
239
(Figure 2). In particular, all the relaxation rates were faster for the PPFA than for SRFA
240
(compare the y-axes in Figure 2a and 2b).
241
The T1 relaxation mechanism refers to the energy transfer between water and dissolved
242
molecules.55 Two motion regimes, each reflecting different proton longitudinal relaxation
243
mechanisms can occur, as water is located in the close proximity of FAs’ surfaces. The outer-
244
sphere relaxation mechanism (which causes the slow motion regime), refers to water
245
diffusing in rigid structures. As a consequence, the rate of relaxation is reciprocally
246
proportional to the solvent diffusion coefficient.59 The second relaxation mechanism, i.e. the
247
inner sphere one (causing the fast motion regime), is less affected by diffusion, while the
248
chemical exchange between water and the solute (i.e. the fulvic acids) plays the dominant
249
role. Figure 3 shows that the fast motion regime occurs in both FAs.60 However, difference in
250
relaxation rates in both FAs (Figure 2) implies that relaxation of water protons in the
251
hydrogel-like SRFA is slightly decelerated by diffusion. On the contrary, due to its rigidity,
252
PPFA structure is less penetrable and thus diffusion influences relaxation at a lesser extent.
253
To verify this conclusion, the relaxation rates obtained at different temperatures were used
254
to determine activation energies and frequency factors from the slope and y-intercept of the
255
linearized form of the Arrhenius equation (3) (i.e. ln(R1) vs 1000/T), respectively. These
256
parameters were calculated for relaxation rates obtained at Larmor frequencies of 0.08 and
257
30 MHz (Figure 3, and Table 2).
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258
As reported in Table 2, the activation energies obtained by using R1 values are negative,
259
which resulted from decreasing relaxation rate with increasing temperature. As a rule,
260
negative values of activation energy indicate that the investigated interaction is complex and
261
does not proceed in one step or that the processes proceed along attractive potential energy
262
surfaces without energy barriers on which slower collisions are more reactive than faster
263
ones.61 Therefore, the obtained activation energies should not be simply interpreted in
264
terms of height of the potential (energy) barrier separating two minima. Instead, they are
265
interpreted as values reflecting a complex mechanism of interactions between water and
266
fulvic acids.
267
The second parameter retrieved from the Arrhenius equation is the frequency factor (Table
268
2). This parameter is the factor reflecting the frequency at which the collisions between
269
water and fulvic acids take place regardless their energy and for this reason it is less
270
influenced by interaction complexity. Consequently, the lower frequency factor for SRFA
271
indicates a lower count of interactions between water and SRFA molecules caused by the
272
restricted diffusion of water in SRFA.
273
The activation energy and the frequency factor measured at the proton Larmor frequencies
274
of 30 and 0.08 MHz (Table 2) resulted in different values for SRFA, but similar for PPFA.
275
Measurements at short Larmor frequencies are more sensitive to slowly relaxing pool of
276
water protons, such as the weakly bound or freely moving proton relaxation pools.
277
Conversely, as proton Larmor frequency increases, measurements provide information on
278
the quickly relaxing proton pools, which are due to strong interactions.49 Therefore, the
279
larger span activation energies can be interpreted with respect to FA structure as being
280
related to its heterogeneity. SRFA, due to its hydrogel-like character, contains assumingly
281
more heterogeneous environment for water proton relaxation in its structure than PPFA,
282
where the relaxation between water and polar groups takes place mainly on the surface. For
283
this reason, the values of activation energies are similar at high and low frequencies in PPFA
284
and different in SRFA. This statement accords with the polydispersity index (Table 1), which
285
is a measure of FA heterogeneity, thereby resulting higher for SRFA.
286
The results above explain the peculiarity that although water relaxes faster in PPFA (Figure
287
2), its correlation time (τc) is lower (Figure 4). In fact, correlation time represents a time
288
necessary for rotation of 1 rad of a water molecule.55 In light of above discussion, water
289
rotation is faster in the PPFA than in the SRFA structure (Table 2). Furthermore, Figure 4 10 ACS Paragon Plus Environment
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confirms the larger structural rigidity of PPFA caused by its aromatic structure. In fact, Figure
291
4 shows that the correlation times of PPFA surrounding water do not change significantly at
292
different temperatures. In contrast, the τc values of the water system in SRFA decreases with
293
increasing temperature. This means that temperature increment causes an increase in water
294
motion in SRFA due to the weakening of the FA structure. Accordingly, the physical structure
295
of SRFA is less thermally stable as in PPFA, which can be attributed to larger number of H-
296
bond stabilizing SRFA structure. Shaffer and von Wandruszka62 concluded that two
297
counteracting processes are effective at elevated temperatures both thermal agitation,
298
which tends to disrupt the aggregates, and dehydration, which promotes aggregate
299
formation. Therefore, in relatively rigid and aromatic PPFA, the temperature increase causes
300
a disruption of polar assemblies and simultaneously increases hydrophobic effect stabilizing
301
most van der Waals forces63 in hydrophobic assemblies. This leads to the increasing
302
hydrophobicity of hydrophobic domains in diluted humic acids solution upon heating as
303
observed by Shaffer and von Wandruszka62 and indicates that these domains, composed of
304
hydrophobic molecules, are porous, which enables their interaction with hydrophobic
305
compounds. It is also noteworthy that the results in Figure 4 are in contrast to observations
306
using ultrasonic velocimetry, which indicated absence of structural changes with increasing
307
temperature.64
308
Kucerik et al.65 showed that humic and fulvic acids from different sources (IHSS standards)
309
start aggregating already at concentrations around 0.01 g L-1. Zheng and Price66 investigated
310
diffusion coefficients of hydrophilic and aliphatic moieties of DOM and observed their
311
aggregation only at higher concentrations. Similarly, Smejkalova and Piccolo25 reported
312
micelle-like behavior only for two out of nine investigated humic acids and concluded that in
313
the rest of investigated samples, the length of amphiphilic molecules is either too short or
314
their abundance is too low to form micelles. Based on these results, Drastik et al32 concluded
315
that hydrophobic molecules are aggregated already at very low concentration and
316
successively involved in gluing hydrogels as concentration increases. In this work, we showed
317
that structural dynamics of DOM is largely influenced by hydrogel-like structure composed of
318
either amphiphilic or polar molecules stabilized by H-bonds, which is in accordance with
319
results of Lam et al.,36 who observed the importance of carboxyl-rich alicyclic molecules in
320
aggregation of SRFA. This DOM part responses to thermal fluctuation (and other factors such
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321
as pH and ionic strength) and presumably modulates the reactivity of hydrophobic
322
aggregates trapped in the gel structure.
323 324
Structural hysteresis of DOM
325
The relaxation rates R1 values retrieved from the heating and cooling cycles revealed the
326
hysteresis in R1 values for water as well as for both fulvic acids (Figure 5 reports exemplary
327
results only for SRFA). In other words, the rate of water protons relaxation differs as the
328
system is cooled down or heated up during the heating/cooling cycle.
329
Liquid water is a molecular 3D network stabilized by H-bonds and van der Waals forces. The
330
formation of H-bonds brings about energetic changes in enthalpy and entropy of the whole
331
network. Under equilibrium, the enthalpy and the entropy factors are mutually
332
compensated,67 but their response to temperature variation is different. We hypothesize
333
that this difference can cause the delay in re-establishing of water structure. This conclusion
334
is in accordance with the longer relaxation time during water cooling (Figure 5). The slower
335
relaxation rate indicates a change in arrangement of water structure and a decrease in
336
strength of H-bonds. The decreased strength of H-bonds in water influences the self-
337
assembling of dissolved FA molecules and consequently decreases the correlation time of
338
water in FAs upon heating and cooling (Table 2). The correlation times in Table 2 also
339
indicate that the structural hysteresis is more pronounced in hydrogel-like SRFA, i.e. in
340
flexible system containing more H-bonds than in rigid, aromatic PPFA. The results in Figure 5
341
suggest that delay in re-conformation of 3D water network is primary reason of re-
342
establishing of H-bonds in FA structure. This is in accordance with recent conclusions that
343
break of H-bonds in diluted FA and humic acids solutions are the main reasons of phase
344
transitions at 20 and 40°C and in shift of their temperatures upon heating and cooling.32
345
The variation of temperature in nature is a rule. Investigation of water properties in two
346
structurally contrasting FA showed that the temperature variation changes the properties of
347
water structure, thereby changing the properties of self-assembled colloidal systems. The
348
character of DOM self-assemblies influences their reactivity and mobility, which indicates
349
that partitioning of DOM with pollutants and nutrients and their transport depend on DOM
350
thermal history. Future research is needed in order to reveal the factors influencing the
351
DOM hysteresis. In addition, a new question arises: how relevant is DOM hysteresis in
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ecological processes such as the non-reversible sorption of and the fate of engineered
353
nanoparticles, nutrients and contaminants.
354 355
References
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45. Halle, B., Cross-relaxation between macromolecular and solvent spins: The role of long-range dipole couplings. J. Chem. Phys. 2003, 119, (23), 12372-12385. 46. Conte, P., Effects of ions on water structure: a low-field 1H T 1 NMR relaxometry approach. Magn. Reson. Chem. 2015, 53, (9), 711-718. 47. Korb, J. P., Surface dynamics of liquids in porous media. Magn. Reson. Imag. 2001, 19, (363-368). 48. Korb, J. P.; Bryant, R. G., Magnetic relaxation dispersion in porous and dynamically heterogeneous materials Adv. Inorg. Chem. 2006, 57, 293-326. 49. Kimmich, R.; Anoardo, E., Field-cycling NMR relaxometry. Prog. Nucl. Mag. Res. Sp. 2004, 44, (34), 257-320. 50. Laudicina, V. A.; De Pasquale, C.; Conte, P.; Badalucco, L.; Alonzo, G.; Palazzolo, E., Effects of afforestation with four unmixed plant species on the soil-water interactions in a semiarid Mediterranean region (Sicily, Italy). J. Soils Sediments 2012, 12, (8), 1222-1230. 51. Conte, P.; Alonzo, G., Environmental NMR: Fast-field-cycling Relaxometry. eMagRes 2013, 2, 389398. 52. Thorn, K. A.; Folan, D. W.; MacCarthy, P. Characterization of the International Humic Substances Society Standard and Reference Fulvic and Humic Acids by Solution State Carbon-13 (13C) and Hydrogen-1 (1H) Nuclear Magnetic Resonance Spectrometry; U.S. Geological Survey: Denver, CO, 1989; p 93. 53. Mori, S.; Barth, H. G., Size Exclusion Chromatography. Springer-Verlag: Heidelberg, Germany, 1999. 54. Halle, B.; Jóhannesson, H.; Venu, K., Model-Free Analysis of Stretched Relaxation Dispersions. J J. Magn. Res. 1998, 135, (1), 1-13. 55. Bakhmutov, V. I., Practical NMR relaxation for chemists. Wiley: 2004; p 202. 56. Conte, P.; Piccolo, A., Conformational Arrangement of Dissolved Humic Substances. Influence of Solution Composition on Association of Humic Molecules. . Environ. Sci. Technol. 1999, 33, (10), 1682-1690. 57. Alvarez-Puebla, R. A.; Valenzuela-Calahorro, C.; Garrido, J. J., Theoretical study on fulvic acid structure, conformation and aggregation - A molecular modelling approach. Sci. Total Environ. 2006, 358, (1-3), 243-254. 58. Saab, S. d. C.; Carvalho, E. R.; Bernardes Filho, R.; de Moura, M. R.; Martin-Neto, L.; Mattoso, L. H. C., pH Effect in Aquatic Fulvic Acid from a Brazilian River. J.Braz. Chem. Soc. 2010, 21, (8), 1490-1496. 59. Hwang, L. P.; Freed, J. H., Dynamic effects of pair correlation functions on spin relaxation by translational diffusion in liquids. J. Chem. Phys. 1975, 63, 4017-4025. 60. Conte, P.; Marsala, V.; De Pasquale, C.; Bubici, S.; Valagussa, M.; Pozzi, A.; Alonzo, G., Nature of water-biochar interface interactions. GCB Bioenergy 2013, 5, 116-121. 61. Revell, L. E.; Williamson, B. E., Why Are Some Reactions Slower at Higher Temperatures? J. Chem. Educ. 2013, 90, (8), 1024-1027. 62. Shaffer, L.; von Wandruszka, R., Temperature Induced Aggregation and Clouding in Humic Acid Solutions. Adv. Environ. Chem. 2015, 2015, Article ID 543614, 6 pages. 63. Parsegian, V. A.; Ninham, B. W., Temperature-dependent van der Waals Forces. Biophys. J. 1970, 10, 664-674. 64. Drastik, M.; Ctvrtnickova, A.; Zmeskal, O.; Kucerik, J., Aggregation of humic an fulvic acids in diluted solutions. In Energy, Environment, Ecosystems, Development and Landscape Architecture, Mastorakis, N.; Helmis, C.; Papageorgiou, C. D.; Bulucea, C. A.; Panagopoulos, T., Eds. 2009; pp 163168. 65. Kučerík, J.; Drastík, M.; Zmeškal, O.; Čtvrtníčková, A., Ultrasonic spectroscopy and fractal analysis in the study on progressive aggregation of humic substances in diluted solutions. WSEAS Trans. Environ. Develop. 2009, 5, 705-715. 66. Zheng, G.; Price, W. S., Direct Hydrodynamic Radius Measurement on Dissolved Organic Matter in Natural Waters Using Diffusion NMR. Environ. Sci. Technol. 2012, 46, (3), 1675-1680. 67. Chaplin, M. F., Water’s hydrogen bond strength. In Water and Life, Lynden-Bell, R. M.; Conway Morris, S.; Barrow, J. D.; Finney, J. L.; Harper, C. L. J., Eds. CRC Press, Boca Raton: 2010; pp 69-86. 15 ACS Paragon Plus Environment
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Captions to Figures
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(dotted line) fulvic acids.
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Figure 2. Nuclear magnetic resonance dispersion (NMRD) profiles of the Pahokee peat (a)
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and Suwannee river (b) fulvic acids. The continuous lines are obtained by the fitting
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procedure described in the text.
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Figure 3. Relationship between the longitudinal relaxation times measured at 0.08 and 30
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MHz for the Pahokee peat (a) and Suwannee river (b) fulvic acids.
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Figure 4. Variation of the water correlation time (tC) during the heating cycle of the solutions
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containing Suwannee river (black line) and Pahokee peat fulvic acids (red line).
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Figure 5. Hysteresis in longitudinal relaxation time during the heating and cooling cycle of
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demineralized water (a) and Suwannee river fulvic acid (b).
Figure 1. HPSEC chromatograms of the Suwannee River (continous line) and Pahokee Peat
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Table 1. Selected properties of IHSS fulvic acids standards obtained from http://www.humicsubstances.org/ and Thorn et al.52 C H N O C/H C/O Carboxylic Phenolic C/(O+N) Suwannee river 53.0 4.4 0.8 43.9 12.2 1.20 1.19 12.2 3.11 Pahokee peat 52.1 3.2 2.4 43.9 16.1 1.18 1.12 15.2 1.78
Table 2. Parameters determined from HPSEC and FFC NMR measurements Averaged molecular weight Activation energy (kDa) (kJ/mol) Weight Number PDI** 30 MHz* 0.08 MHz* (Mw) (MN) SRFA 9.03 7.20 1.25 18.1±1.1 25.9±1.2 PPFA 9.53 8.29 1.14 11.0±0.8 10.6±0.4 *Larmor frequency, ** polydispersity index
Frequency factor (s-1) 30 MHz*
0.08 MHz*
(2.9±0.2)×10-4 (9.8±0.2)×10-3
(1.3±0.1)×10-6 (1.5±0.1)×10-2
Correlation time (ns) Before After heating heating 15 2 0.28 0.19
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Figure 1. HPSEC chromatograms of the Suwannee River (continous line) and Pahokee Peat (dotted line) fulvic acids. 271x192mm (300 x 300 DPI)
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Figure 2a. Nuclear magnetic resonance dispersion (NMRD) profiles of the Pahokee peat (a) and Suwannee river (b) fulvic acids. The continuous lines are obtained by the fitting procedure described in the text. 272x221mm (300 x 300 DPI)
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Figure 2b. Nuclear magnetic resonance dispersion (NMRD) profiles of the Pahokee peat (a) and Suwannee river (b) fulvic acids. The continuous lines are obtained by the fitting procedure described in the text. 271x220mm (300 x 300 DPI)
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Figure 3. Relationship between the longitudinal relaxation times measured at 0.08 and 30 MHz for the Pahokee peat (a) and Suwannee river (b) fulvic acids. 366x276mm (237 x 237 DPI)
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Figure 4. Variation of the water correlation time (tC) during the heating cycle of the solutions containing Suwannee river (black line) and Pahokee peat fulvic acids (red line). 387x266mm (226 x 226 DPI)
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Figure 5. Hysteresis in longitudinal relaxation time during the heating and cooling cycle of demineralized water (a) and Suwannee river fulvic acid (b). 361x465mm (300 x 300 DPI)
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graphical abstract 40x27mm (300 x 300 DPI)
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