Selective extraction of humic acids from an anthropogenic

Biol Fertil Soils (2014) 50:1223–1232
DOI 10.1007/s00374-014-0940-9
SPECIAL ISSUE
Selective extraction of humic acids from an anthropogenic
Amazonian dark earth and from a chemically oxidized charcoal
Joyce R. Araujo & Braulio S. Archanjo & Katia R. de Souza & Witold Kwapinski &
Newton P. S. Falcão & Etelvino H. Novotny & Carlos A. Achete
Received: 10 February 2014 / Revised: 30 May 2014 / Accepted: 19 June 2014 / Published online: 1 July 2014
# Springer-Verlag Berlin Heidelberg 2014
Abstract Spectroscopic techniques including X-ray photoelectron spectroscopy (XPS) can identify particular chemical
groups of humic acids (HA) from “Terra Preta de Índios”
(TPI) or Amazonian dark earth, the highly fertile anthropogenic soil found in the Amazonian region. The high fertility
and resilience of these soils cannot be explained by their
chemically inert pyrogenic C content alone, but the natural
aging of this C generates reactive carboxyl functional groups
attached directly to the recalcitrant polycondensed aromatic
backbone. Through spectroscopic techniques used in this
work, the HA fraction (the alkaline-soluble organic matter
that precipitates at low pH) of the TPI soil was compared with
humic and fulvic acids, obtained by oxidizing activated charcoal with sodium hypochlorite. The yields recovery of HAlike substances was 12 and 28 wt% by using 10 and
20 cmol L−1 of oxidizing agent, respectively. X-ray photoelectron spectroscopy, energy dispersive X-ray, and solid-state
13
C nuclear magnetic resonance (13C NMR) spectroscopies
J. R. Araujo (*) : B. S. Archanjo : K. R. de Souza : C. A. Achete
Instituto Nacional de Metrologia, Qualidade e Tecnologia, Av. Nossa
Sra. das Graças, 50, Duque de Caxias, RJ 25250-020, Brazil
e-mail: [email protected]
W. Kwapinski
Carbolea Research Group, Department of Chemical and
Environmental Science, University of Limerick, Limerick, Ireland
N. P. S. Falcão
Departamento de Ciências Agronômicas, Instituto Nacional de
Pesquisas da Amazônia, Manaus, AM 69011-970, Brazil
E. H. Novotny
Embrapa Solos, Jardim Botânico, Rio de Janeiro, RJ 22460-000,
Brazil
C. A. Achete
Departamento de Engenharia Metalúrgica e de Materiais,
Universidade Federal do Rio de Janeiro, Rio de Janeiro,
RJ 21941-972, Brazil
were used to evaluate the elements and structures present in all
samples. XPS C 1 s spectra of HA extracted from TPI soil and
from prepared HA showed aromatic structures (C=C and
π–π* shake-up satellite peak) bounded to carboxyl groups
(COOH). The morphology and polycondensation level of
aromatic C were evaluated by scanning electron microscopy
(SEM). The similarities of the spectra indicated that the used
method was efficient to obtain an organic amendment similar
to TPI soil organic matter.
Keywords Oxidized charcoal . Amazonian dark earth . Soil
organic matter . Humic acids . X-ray photoelectron
spectroscopy . Pyrogenic C
Introduction
Soil organic matter (SOM) was found to play a fundamental
role, and significant attention is given to the most stable SOM
types, such as humic substances, and to carbonaceous materials, as black C (pyrogenic C), pyrolyzed biomass, and, more
recently, biochar (Brady and Weil 1999; Jorio et al. 2012;
Lehmann 2007; Lehmann et al. 2008; Marris 2006). This
interest primarily arises from the discovery of Terra Preta de
Índios (TPI) soils, also known as Amazonian dark earths,
anthropogenic dark earths, Indian black earths, or
archeological dark earths. These soils, found in the Amazon
Basin, were modified and formed by the agricultural and
household activities of the indigenous people during the preColumbian era (Jorio et al. 2012). In addition to having higher
fertility than the adjacent non-anthropogenic soils, these soils
exhibit a peculiar resilience to intense degradative use
(Novotny et al. 2009). These properties can be explained by
the organic matter of these soils—pyrogenic C (Cpy) with
peripheral aromatic units that partially oxidize to yield acidic
(carboxyl) substituents (Glaser et al. 2001; Kramer et al. 2004;
1224
Masiello 2004; Novotny et al. 2009)—and by the relatively
high total acidity, which contributes to soil fertility and sustainability (Glaser et al. 2001).
Incorporation of charred biomass maybe more efficient
than incorporating plant residues for organic C storage in soil
(Jorio et al. 2012; Lehmann 2007; Lehmann et al. 2008;
Marris 2006; Schmidt et al. 2011) because Cpy is more stable
and increases the mean SOM residence time. Furthermore,
Cpy can increase plant yield (Lehmann 2007; Novotny et al.
2009; Schmidt et al. 2011), which further can increase Cpy
production. However, the turnover rate and extended residence time of C py in the soil remain under debate
(Brodowski et al. 2006; Oades 1988; Schimel et al. 1994;
Schmidt et al. 2011; Six et al. 2002; Torn et al. 1997).
Researchers agree that the partial oxidation of the Cpy aromatic rings (Brodowski et al. 2006; Oades 1988; Schimel et al.
1994; Schmidt and Noack 2000; Schmidt et al. 2011; Six et al.
2002) and Cpy’s interaction with soil minerals (Brodowski
et al. 2006; Kogel-Knabner et al. 2008; Oades 1988;
Schimel et al. 1994; Schmidt et al. 2011; Six et al. 2002;
Torn et al. 1997) are fundamental to Cpy stability.
Detailed investigations have yielded an efficient model for
the peculiar organic matter in TPI (Novotny et al. 2007, 2009;
Linhares et al. 2012). Humic acid (HA) from TPI can be
satisfactorily modeled as a binary mixture composed of ordinary HA and polycondensed aromatic structures with carboxyl groups attached directly to the aromatic C backbone
(Novotny et al. 2009). At the typical pH of TPI soil (4–6),
carboxylic acids are deprotonated generating carboxylate anions which contribute to soil cation exchange capacity (CEC);
these peculiar HA structures are not only extremely recalcitrant, which is important for C sequestration, but also reactive,
with a high CEC, which is important to soil fertility. On the
other hand, the HA of the adjacent soils contain mainly labile
compounds such as carbohydrates, amino acids, and lignin
residues (Novotny et al. 2009), which explains the low residence time of organic matter under cultivation. Based on this
proposed model, an oxidized material was obtained which
share several chemical properties and resilience with the
polycondensed aromatic structures, containing carboxylic
functionality, present in TPI; however, after an ecotoxicological test using Daphnia similes, this product was classified as
moderately toxic, likely due to the production of aryl chloride
during oxidation as inferred from the infrared spectroscopic
analysis (Linhares et al. 2012).
Recently, advanced spectroscopic techniques have been
used to study soil properties and determine the chemical
structures of SOM properties that are responsible for the
recalcitrance and reactivity of highly fertile soils such as TPI
(Jorio et al. 2012; Lehmann 2007; Lehmann et al. 2008;
Marris 2006; Schmidt et al. 2011). For example, chemical
characterization using X-ray photoelectron spectroscopy
(XPS) provided the C, O, and Cl core-electron binding
Biol Fertil Soils (2014) 50:1223–1232
energies of the laboratory-prepared fractions to allow a detailed study of the humic acid-like materials (HALM) and
fulvic acid-like materials (FALM).
To our knowledge, this is the first report where a comparison of HA extracted from TPI soils with laboratory-prepared
HALM from charcoal oxidation was performed. The use of
many coupled spectroscopic techniques is a powerful tool to
determine the chemical structure of target materials, due to the
different sensitivity of each technique; the type of sample to be
analyzed for each technique is also important, for example,
bulk vs. surface. Arenella et al. (2014) reported the effect of
soil-borne humic substances (HS) on the identification of
model proteins with different properties, such as myoglobin
(Mb), α-glucosidase (αG), and β-glucosidase (βG), by using
electrophoretic and ESI- and MALDI-mass spectrometry
(MS) methodologies. Results showed that the contact between
proteins and HS did not alter protein electrophoretic mobility
but led to protein modifications that affected protein identification by MS.
The use of mathematical tool (multivariate curve resolution—MCR) on 13C nuclear magnetic resonance (13C NMR)
spectra of HA from TPI and HA from control adjacent soils
facilitated the identification of a HA fraction peculiar from
TPI, i.e., polycondensed aromatic backbone heavily carboxylated (Novotny et al. 2009). It is important to prove the
existence of this fraction in TPI soils. Here, we have used a
modification, proposed by Hayes and Graham (2000), of the
traditional method of humic substance extraction suggested by
International Humic Substances Society (IHSS) to obtain,
preferentially, the peculiar HA from TPI substances estimated
by MCR (Novotny et al. 2009). In addition, we have compared the properties of this fraction with those of analogous
substances, obtained from charcoal oxidation, using XPS
analysis. This modification only selectively extracts the recalcitrant and reactive structures (polycondensed aromatic structures functionalized with carboxylic acids) characterizing TPI.
The novelty of this study is to replicate the HA of TPI through
oxidation of charcoal sample and compare the final product
with HA extracted from TPI soil in NaOH solutions of two
different pHs.
Materials and methods
HALM and FALM fractions obtained from charcoal
Activated charcoal (P.A., Vetec Química Fina Ltda., São
Paulo, Brazil) was chemically oxidized using sodium hypochlorite (NaOCl) at two concentrations (10 and 20 cmol L−1).
Activated charcoal was obtained from carbonaceous sources
as peat, wood, coir, lignite, coal, and petroleum pitch after
their incubation at 600–900 °C, under inert atmosphere with
argon or nitrogen. Five-gram-activated charcoal samples were
Biol Fertil Soils (2014) 50:1223–1232
placed in glass beakers with 200 mL of NaOCl of different
concentrations. Then, 2.4 g of sodium hydroxide (NaOH)
were added. The mixture was heated and stirred on a magnetic
stirrer-heater (60±2 °C) for 3 h and then filtered (filter paper
white ribbon, grade 389). The supernatant was acidified to pH
~ 2 to precipitate the HALM, while the FALM fraction
remained in the solution. In order to compare fractions isolated by exactly the same procedures, the precipitate was
redissolved in 0.1 mol L−1 KOH under a N2 atmosphere,
and KCl was added until the K+ concentration reached
0.3 mol L−1. The flocculated colloidal particles were centrifuged at 40,000g (Relative Centrifugal Force—RCF) for
20 min. The supernatant was acidified to pH~2 with concentrated HCl, and the precipitated HALM was separated from
the supernatant by centrifugation and dialysis. The unpurified
FALM was freeze-dried (Swift 1996) and then purified using
XAD-7 and Amberlite IR-120 resins. The purified HALM
and FALM were then freeze-dried (Table 1; Linhares et al.
2012).
Extracting humic acids (HA) from TPI
The 0- to 20-cm depth soil samples (Anthroposols—FAO
World Reference Base for Soil Resources 2007) were first
H+-exchanged by treating them with 0.1 M HCl, followed by
washing with distilled water until chloride free. The soils were
then repeatedly and exhaustively extracted (until the optical
density of the extract at 400 nm was below 0.1) with aqueous
solutions, with the soil/solution suspension pH sequentially
adjusted to 7 and then to 10.6 using 0.1 mol L−1 NaOH
(Simpson et al. 2007). The obtained suspensions were shaken
overnight under a N2 atmosphere. This procedure has been
described in detail by Hayes and Graham (2000). The supernatant was separated from the residue by centrifugation (5,000
g for 10 min) and filtration, and HCl was added to the filtrate
to reach pH~2 and precipitate the HA, which was separated
from the supernatant by centrifugation (5,000g for 10 min).
The residue was redissolved in 0.1 mol L−1 KOH under a N2
atmosphere, and KCl was added until the K+ concentration
reached 0.3 mol L−1. The flocculated colloidal particles were
separated by centrifugation at 40,000g for 20 min. The supernatant was acidified to pH~2 using concentrated HCl, and the
precipitated HA was separated from the supernatant by centrifugation. Afterwards, the HA was dialyzed to remove any
soluble salts and then it was freeze-dried.
Solid-state 13C nuclear magnetic resonance (NMR)
spectroscopy
Solid-state 13C NMR spectra were obtained using a Varian
INOVA (11.74 T) spectrometer with 13C and 1H frequencies
of 125.7 and 500.0 MHz, respectively. A variable-amplitude
cross-polarization pulse sequence was employed. The
1225
Table 1 Description of samples analyzed by XPS and EDS. The humic/
fulvic fractions were obtained using different concentrations of the oxidizing agent (NaOCl), whereas the TPI-humic fractions were exhaustively extracted using NaOH solutions at two different pHs (in parentheses)
Sample
NaOCl
(cmol L−1)
Maximum pH
of extraction
FALM-1a
20
–
FALM-2b
HALM-1a
HALM-2b
HA-TPI (7)c
HA-TPI (10)c
10
20
10
–
–
–
–
–
7
10
FALM fulvic acid-like material, HALM humic acid-like material, HA-TPI
humic acids extracted from TPI
a
20 cmol L−1 NaOCl
b
10 cmol L−1 NaOCl
c
Humic acids extracted at pH 7
d
Humic acids extracted at pH 10.6
experiments were performed using magic-angle spinning
(MAS) of 15 kHz, cross-polarization time of 1 ms, acquisition
time of 15 ms, recycle delay of 500 ms; and high-power twopulse phase-modulation (TPPM) proton decoupling of
70 kHz.
X-ray photoelectron spectroscopy (XPS)
X-ray photoelectron spectroscopy (XPS, Omicron
Nanotechnology) was used to study the chemical composition
and chemical state of the prepared fractions (HALM, FALM,
and HA-TPI fractions) after the acid-base fractionation. The
XPS analyses were performed under ultra-high vacuum
(10−10 mbar) using an Al, Kα=1,486.7 eV X-ray source
powered by an emission current of 16 mA and a voltage of
12.5 kV. High-resolution spectra were obtained for C and Cl
elements using analyzer pass energy of a 30- and 0.05-eV
step. The binding energies referenced the C 1 s level, which
was set at 284.6 eV.
Semi-quantitative chemical analyses were obtained by
XPS survey spectra considering the peak areas of elements
evaluated by the CasaXPS software and the sensitivity factor
of each atom. The typical uncertainty in quantitative XPS is
about 10 % due mainly to uncertainties in the experimental
optical data (Powell and Jablonski 2010). Gaussian/
Lorentzian (70/30) line shapes were used to fit the C 1 s peak
curves. The Shirley background and a least-squares routine
were used for peak fitting. During the start of the C 1 s fitting
procedure, the full width at half-maximum (FWHM) values
were limited to 1 eV for all peaks. The sp2 peak of the C 1 s
envelope centered at 284.3 eV had a FWHM of 1.0±0.2 eV. In
addition, near the sp2 component at 284.3 eV, we found at
least three broad components overlapping the C 1 s features.
1226
Fig. 1 13C NMR spectra of humic acids extracted from TPI soils at
pH 7—HA-TPI (7)—and at pH 10.6—HA-TPI (10), and the estimated
spectra by multivariate curve resolution analysis of 22 humic acids
samples (TPI and non-anthropogenic control soils)
The bands appearing in the higher energy region tended to be
much broader (FWHM≈1.7 eV) than that in the sp2 component. In particular, the FWHM of the components near the tail
of the C 1 s envelopes was approximately 2 eV. These features
agree with what is already reported (Bagri et al. 2010; Mattevi
et al. 2009; Stankovich et al. 2007; Yang et al. 2009).
High-resolution Cl peaks were not fitted due to the lower
resolution and lower intensity of their spectra. Some molecules containing O, such as O2, H2O, and CO2, may be
adsorbed on the surface of porous samples, and thus O 1 s
peak was not analyzed.
Scanning electron microscopy (SEM)
The SEM images were collected using an FEI Nova Nanolab
600 dual-beam microscope with a 5-keV accelerating energy
and a 98-pA current. All samples were coated with a thin gold
layer (100-nm thick) using an Electron Microscopy Sciences
sputter coater. The energy-dispersive X-ray spectroscopy
(EDS) measurements were performed using an FEI Quanta
200 with a tungsten filament operating in the environmental
mode (pressure≈1 Torr).
Biol Fertil Soils (2014) 50:1223–1232
2009). These chemical groups indicate the presence of
plant material at different stages of humification (fatty
acids, proteinaceous material, lignin, and cellulose). In
contrast, the other MCR component is characterized by
polycondensed aromatic structures and aromatic carboxylic groups (Fig. 1), which can be modeled as a pyrogenic
and partially oxidized C (polycondensed rings functionalized with carboxylic groups). The NMR spectra of the HA
extracted from TPI at pH 7 and 10.6 are similar to those
estimated by the MCR model (Fig. 1); thus, the mathematical methods estimate a real component of the TPI soil and
the proposed extraction method selectively extracts this
peculiar organic matter from the TPI. Similar results were
obtained by Mao et al. (2012) using different methods.
These authors analyzed the whole organic matter of TPI
samples collected from the sub-surface layers (0.4- to 0.7m deep), to prevent contamination with fresh material,
after demineralization with HF.
X-ray photoelectron spectroscopy (XPS)
XPS counts electrons ejected from a sample surface when
irradiated by X-rays. The underlying assumption when quantifying XPS spectra is that the number of electrons recorded is
proportional to the number of atoms in a given state. As a
result, the best way to compare XPS intensities is by the socalled percentage atomic concentrations, which is percentage
of intensity of the target atom corrected by its relative sensitivity factor (RSF; Powell and Jablonski 2010).
Unlike charcoal, which only contains C and O (Fig. 2), TPI
contains a variety of elements (Fig. 3). Like other productive
soils, TPI contains Ca, P, N, Al, Mg, Si, and Na, and low levels
of K, Ti, Cr, Mn, Fe, Cu, and Zn (Jorio et al. 2012). The HA
extracted from the TPI samples primarily contained C and O
Results and discussion
Solid-state 13C nuclear magnetic resonance spectroscopy
(NMR)
The NMR spectra for several Amazon soil HA were
modeled by MCR as a binary mixture with one component
exhibiting peaks typical of alkyl groups (such as n-alkyl),
methoxy groups, carbohydrates, aromatics, O-aromatics,
aliphatic carboxylic acids, and amides (Novotny et al.
Fig. 2 XPS spectra of charcoal samples containing C and O
Biol Fertil Soils (2014) 50:1223–1232
1227
Fig. 3 XPS spectra of humic
acids extracted from TPI soils at
pH 7—HA-TPI (7)—and at
pH 10.6—HA-TPI (10), a XPS
spectrum of HA-TPI (7), b C 1 s
high-resolution XPS spectrum of
HA-TPI (7), c XPS spectrum of
HA-TPI (10), and d C 1 s highresolution XPS spectrum of HATPI (10)
(Fig. 3), with O/C atomic ratios of 0.27 (pH 10.6) and 0.29
(pH 7).
Survey XPS spectra of the FALM and HALM prepared
through the oxidation of activated charcoal with two
NaOCl solutions (20 and 10 cmol L−1) are presented in
Fig. 4. The FALM exhibited O/C ratios of 0.37 and 0.33
Fig. 4 XPS spectra of the
fractions extracted from activated
charcoal oxidized with NaOCl: a
FALM-1 fulvic acid-like material
(10 cmol L−1 NaOCl), b FALM-2
fulvic acid-like material
(20 cmol L−1 NaOCl), c HALM-1
humic acid-like material
(10 cmol L−1 NaOCl), and d
HALM-2 humic acid-like material
(20 cmol L−1 NaOCl)
for FALM-1 and FALM-2, respectively, whereas the
HALM yielded lower O/C ratios, 0.28 and 0.29 for
HALM-1 and HALM-2, respectively (Table 2). The O/C
ratios of the HALM were similar to those of the TPI-HA
samples, indicating similar oxidation level in these samples. The O/C ratio of the charcoal samples was 0.07
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Biol Fertil Soils (2014) 50:1223–1232
Table 2 Atomic percentages calculated from the XPS survey spectra
(details are provided in “Materials and methods”) and O/C and Cl/C
atomic ratios for the FALM and HALM fractions, respectively
Element
FALM-1a
(at.%)
FALM-2b
(at.%)
HALM-1c
(at.%)
HALM-2d
(at.%)
C
O
N
K
Cl
Na
O/C ratio
Cl/C ratio
61
30
1
5
1
2
0.37
0.01
63
28
1
5
2
1
0.33
0.01
56
21
1
10
11
1
0.28
0.07
54
21
1
11
12
<1
0.29
0.08
a
fulvic acid-like material (10 cmol L−1 NaOCl)
b
fulvic acid-like material (20 cmol L−1 NaOCl)
c
humic acid-like material (10 cmol L−1 NaOCl)
d
humic acid-like material (20 cmol L−1 NaOCl)
(Fig. 2), which indicates that functional groups containing
oxygen were introduced into the charcoal sample after
partial oxidation with sodium hypochlorite. The difference between the HALM and FALM can be explained
by their differing solubilities: FALM are more soluble due
to their additional oxygenated functional groups (higher
hydrophilicity) and lower molecular size than HALM.
In addition, the XPS survey spectra of the laboratoryprepared fractions indicate that the HALM fractions contain
high quantities of Cl and K. After being oxidized with NaOCl,
the functionalized charcoal samples were treated with KCl in a
KOH solution to remove colloidal particles before acidifying
to pH~2. Thus, the HALM was contaminated with Cl and K
from the KCl salt, which was not removed by dialysis.
Organic chlorine likely remained in the FALM, while the
chloride salts remained in the HALM fraction. This assumption was later confirmed by high-resolution XPS analysis.
Seven components are required to fit the C 1 s peak of the
FALM and HALM fractions (Table 3). These seven components in the C 1 s spectrum (Fig. 5) contain the following
functional groups: sp2-hybridized C (C=C, 284.0 eV), sp3hybridized C (C–C/C–H, 284.8 eV), hydroxyl/phenols (C–
OH/C–Cl, 285.5 eV), epoxy/ether (C–O–C, 286.2 eV), carbonyl (C=O, 287.5 eV), carboxyl (HO-C=O, 289.3 eV), and
a shake-up satellite peak (π→π*, 291.3 eV) characteristic of
aromatic C structures (Filik et al. 2003; Han et al. 2013; Zhu
et al. 2011). A binding energy of 284.3 eV essentially corresponds to non-functionalized sp2 C (Papirer et al. 1995). Filik
et al. (2003) reported a binding energy of 284 eV in a graphite
sample and a full width at half-maximum (FWHM) value of
0.98. The HALM fractions contain more C–C and C=C bonds
than the FALM fractions; these C groups make up 58–61 % of
the FALM and 73-74 % of the HALM (Table 3). HALM-2
exhibited the highest (33 %) and FALM-2 the lowest C=C
content (28 %). The polycondensed aromatic HALM fraction
has a more recalcitrant structure than the FALM fraction,
which confirms the NMR data of Linhares et al. (2012), who
showed that the HALM has more polycondensed aromatic
structure than the FALM.
The peak at binding energy of 285.5 eV is essentially
attributed to sp2 C atoms linked to a chlorine atom (chlorinesubstituted sp2 C atoms at the periphery of the polyaromatic
structures) (Pérez-Cadenas et al. 2003). Hydroxyl and phenol
groups can also contribute to this binding energy (~285.8 eV;
Larciprete et al. 2012). The FALM fractions exhibited higher
quantities of this component (~13 %), which confirms both
the inferred attribution and the results of the IR vibration
bands, and it indicates that the FALM contains more aryl
chlorides than HALM, and explains its toxicity (Linhares
Table 3 Binding energies (eV) of the C 1 s components and the percentage of each species (in parentheses) of the fulvic (FALM) and humic acid-like
materials (HALM) and the humic acids (HA) extracted from TPI at pH 7 or 10.6
Assignment
FALM-1a
FALM-2b
HALM-1c
HALM-2d
HA-TPI (7)e
HA-TPI (10)f
C=C
C–C/C–H
C–OH/C–Cl
C–O–C
C=O
284.0 (35)
284.8 (23)
285.5 (13)
286.3 (12)
287.4 (4)
284.1 (28)
284.8 (33)
285.5 (13)
286.2 (8)
287.5 (9)
284.0 (31)
284.8 (42)
285.6 (9)
286.3 (6)
287.5 (8)
284.0 (33)
284.9 (41)
285.7 (12)
286.5 (5)
287.5 (4)
284.1 (36)
284.9 (32)
285.7 (5)
286.3 (13)
287.6 (7)
284.0 (30)
284.8 (36)
285.5 (6)
286.1 (14)
287.3 (6)
HO–C=O
288.7 (13)
288.5 (9)
288.5 (4)
288.3 (5)
288.9 (7)
288.7 (8)
The numbers in parentheses are the atomic percentages obtained from the relative contributions of each peak.
a
fulvic acid-like material (10 cmol L−1 NaOCl)
b
fulvic acid-like material (20 cmol L−1 NaOCl)
c
humic acid-like material (10 cmol L−1 NaOCl)
d
humic acid-like material (20 cmol L−1 NaOCl)
e
humic acids extracted from TPI in pH 7
f
humic acids extracted from TPI in pH 10
Biol Fertil Soils (2014) 50:1223–1232
1229
Fig. 5 High-resolution XPS
spectra of the C 1 s for a FALM-1
fulvic acid-like material
(10 cmol L−1 NaOCl), b FALM-2
fulvic acid-like material
(20 cmol L−1 NaOCl), c HALM-1
humic acid-like material
(10 cmol L−1 NaOCl), and d
HALM-2 humic acid-like material
(20 cmol L−1 NaOCl). The C 1 s
peak fitting procedure was
performed with the following
considerations: C=C (sp2), C–C
(sp3), C–OH (phenol), C–O–C
(ether/epoxide), C=O (carbonyl),
HO–C=O (carboxyl), and π–π*
shake-up (satellite peak of the C
sp2 component)
et al. 2012). Because the binding energy of sp2 C–Cl
(285.5 eV) overlaps with that of oxygen-containing C atoms,
the final evaluated percentage corresponds to the overall contribution of these two chemical sites. The Cl 2p peak must be
examined to determine the percentage of Cl-aryl bonds in the
humic/fulvic fractions.
A binding energy of ~286.3 eV corresponds to both the
sp3 C bound to Cl and, to a lesser extent, the sp2 and sp3 C
linked to a single O atom (epoxide or ether; Papirer et al.
1995). The XPS peaks at 287.5 and 288.4 eV correspond to
carbonyl and carboxyl groups, respectively. The greatest
difference observed in the C 1 s peaks for the FALM and
HALM fractions occurs in the carboxyl peak (COOH;
Fig. 5a–d). The percentage of carboxyls attached to aromatic C in the FALM-1 and 2 are 13 and 9 %, respectively,
and the HALM 4–5 % (Table 3, Fig. 5). The high carboxylic content and lower molecular size explain the high
solubility of the FALM samples across all pH values and
low condensation rate in the aromatic ring, which agrees
with the previous NMR results (Linhares et al. 2012).
Chlorine atoms easily bind to C surfaces, which increases their Lewis acidity but decreases their Brönsted
acidity due to resonance from the aromatic rings (PérezCadenas et al. 2003). A binding energy of ~200.5 eV is
indicative of chlorine atoms covalently bonded to sp2 C as
reported for organo chlorine compounds (Papirer et al.
1995). The high-resolution Cl 2p peak (Fig. 6) arises from
two major components: inorganic chlorine (binding energy
<199 eV), from a chloride salt with Na, K, Ca, or Fe, and
organic chlorine (binding energy >200 eV), from chlorine
bonded to aromatic sp2 C atoms or due to Cl–C=O bonds
(Fiedler and Herzschuc 1993; Wang et al. 2012). The
FALM contained more organic chloride, as aryl chloride
(Fig. 6), than the HALM, which mainly contained chloride
salts, as sodium hypochlorite (NaOCl). Activated charcoal
oxidized with various sodium hypochlorite concentrations
formed aryl chloride. Also, non-reacted sodium hypochlorite remained in FALM and HALM fractions. This
byproduct was partially removed during purification; however, ecotoxicological testing of the FALM indicated that
the chemically functionalized charcoal was moderately
toxic, and infrared analysis indicated the presence of aryl
chloride compounds that were likely responsible for this
Fig. 6 Comparison of the high-resolution XPS spectra in the Cl 2p
region for fulvic (FALM) and humic acid-like materials (HALM) both
obtained using NaOCl concentrations of 20 and 10 cmol L−1
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Biol Fertil Soils (2014) 50:1223–1232
Fig. 7 a EDS spectra of fulvic acid-like material obtained using NaOCl
concentrations of 10 and 20 cmol L−1 (FALM-1 and FALM-2, respectively) and humic acid-like material obtained with NaOCl concentrations
of 10 and 20 cmol L−1 (HALM-1 and HALM-2, respectively). b EDS
spectra of humic acids extracted from TPI at pH 7 and 10.6 (HA-TPI (7)
and HA-TPI (10), respectively)
toxicity (Linhares et al. 2012). The presence of aryl chloride was confirmed by XPS analysis.
fractions identified organic chlorides, as potentially toxic aryl
chloride. These results highlight the need for either purification
after synthesis or a change in the used oxidant. The EDS results
for the HA extracted from TPI (Fig. 7b) revealed an important
feature of the TPI samples: the region between 0.75 and 10 keV
showed elements characteristic of TPI soils, such as Si, P, S, K,
Ca, Ti, and Fe (Jorio et al. 2012), and small quantities of Na and
Cl residues remaining after extraction.
EDS
The qualitative elemental composition obtained from the EDS
analysis of the FALM and HALM is shown in Fig. 7a. Both
FALM and HALM fractions contained Cl, K, and Na from the
reagents (NaOCl, KCl, and KOH) used during the synthesis
and purification. The apparent presence of Al was due to Xrays interacting with Al parts of the microscope and the
sample holder. These results agreed with the XPS measurements, where the chlorine- and potassium-binding energies of
HALM fractions were mainly assigned to these elements in
salt form, while the chlorine binding energy in the FALM
Fig. 8 SEM images of the
following: a fulvic acid-like
material (FALM-1); b humic
acid-like material (HALM-1),
both fractions were obtained
using NaOCl concentrations of
10 cmol L−1; c humic acids
extracted from TPI at pH 7 (HATPI (7)); d humic acids extracted
from TPI at pH 10.6 (HA-TPI
(10))
Scanning electron microscopy (SEM)
Figure 8 provides the SEM images of the laboratory-prepared
fractions (FALM, HALM) and TPI extracts (pH 7 and 10.6).
The soluble fulvic acid fraction (Fig. 8a) forms distinguishable
small particles. This characteristic promotes a high metal
Biol Fertil Soils (2014) 50:1223–1232
adsorption (Xu et al. 2006). The metals in this fraction (Fe, K,
and Ca) can form bridges with carboxyl groups, interact by
chemisorption with oxidized C groups and form coordination
compounds (Pandey et al. 2000) with other functional groups
of humic substances, such as hydroxyl, phenol, and methoxy
functional groups.
Compacted microaggregates were found both in the HALM
and HA-TPI samples; however, the aggregate size of the
HALM (Fig. 8b) was greater than those of the HA-TPI fractions (Fig. 8c, d). Changlung et al. (2007) reported that the
aggregation phenomenon is important to the transport of heavy
metal ions in natural environments. Zhang et al. (2009) reported that humic substances in dilute solutions form thin threadand net-like structures that grow into larger rings and sheets
with increasing humic and cation concentrations. The HA
fraction is insoluble at low pH (pH~2) due to aggregation at
this pH value. This aggregation can be observed in SEM image
of HALM-1 fraction (Fig. 8b), confirming this hypothesis.
Conclusion
Aqueous NaOH solutions with pH values of 7 and 10.6 were
used to selectively extract the peculiar humic fraction of the TPI
sample. Substances similar to these peculiar HA were successfully prepared by oxidizing activated charcoal. The XPS and
SEM images showed a polycondensed structure in the TPI-HA
similar to that of the laboratory-prepared fractions. The use of
spectroscopic techniques to compare chemically produced materials, and humic fractions from fertile soils, may be used to
measure the chemical composition of these new produced
materials in order to recreate TPI soils. This expertise may be
important to indicate the need for a purification process or
ecotoxicological tests. Fulvic and humic fractions were isolated
through the precipitation of the HALM, which is insoluble at
acidic pH. The XPS analyses indicated that the FALM fractions
contained more carboxyl/carbonyl groups than the humic fractions, which contained more aromatic/aliphatic carbons. Aryl
chloride was formed in the FALM, and thus, a purification
process or change in the used oxidant is necessary before
incorporating this synthetic fraction into the soil.
Acknowledgments We thank Austim M. Pimenta and Carlos Senna for
the EDS measurements. Katia R. de Souza thanks Conselho Nacional de
Desenvolvimento Científico e Tecnológico (CNPq) for the research fellowship (384692/2012-5) supporting her research.
Conflict of interest
interest.
The authors declare no competing financial
References
Arenella M, Giagnoni J, Masciandaro G, Ceccanti B, Nannipieri P,
Renella G (2014) Interactions between proteins and humic
1231
substances affect protein identification by mass spectrometry. Biol
Fertil Soils 50:447–454
Bagri A, Mattevi C, Acik M, Chabal YJ, Chhowalla M, Shenoy VB
(2010) Structural evolution during the reduction of chemically derived graphene oxide. Nat Chem 2:581–587
Brady N, Weil R (1999) Soil organic matter. In: The nature and properties
of soils, Prentice-Hall, Inc, New Jersey
Brodowski S, John B, Flessa H, Amelung W (2006) Aggregate-occluded
black carbon in soil. Eur J Soil Sci 57:539–546
Changlung C, Wang X, Jiang H, Hu E (2007) Direct observation of
macromolecular structures of humic acid by AFM and SEM.
Colloids Surf A 302:121–125
FAO World Reference Base for Soil Resources (2007), First update.
Available at: http://www.fao.org/nr/land/soils/soil/wrb-documents/
en/. Accessed 18 April 2014
Fiedler R, Herzschuc R (1993) An XPS investigation of the effects of heat
treatment on the chlorine surface chemistry of some lignites. Fuel
72:1501–1505
Filik J, May PW, Pearce SRJ, Wild RK, Hallam HR (2003) XPS and laser
Raman analysis of hydrogenated amorphous carbon films. Diamond
Relat Mater 12:974–978
Glaser B, Haumaier L, Guggenberger G, Zech W (2001) The ‘Terra
Preta’ phenomenon: a model for sustainable agriculture in the humid
tropics. Naturwissenschaften 88:37–41
Han HS, You JM, Jeong H, Jeon S (2013) Synthesis of graphene oxide
grafted poly(lactic acid) with palladium nanoparticles and its application to serotonin sensing. Appl Surf Sci 284:438–445
Hayes MHB, Graham CL (2000) Procedures for the isolation and fractionation of humic substances. In: Ghabbour EA, Davies G (eds)
Humic substances: versatile components of plants, soil and water.
Royal Society of Chemistry, Cambridge, England, pp 91–109
Jorio A, Ribeiro-Soares J, Cançado L, Falcao NPS, Dos Santos HF,
Baptista DL, Martins Ferreira EH, Archanjo BS, Achete CA
(2012) Microscopy and spectroscopy analysis of carbon nanostructures in highly fertile Amazonian anthrosoils. Soil Tillage Res 122:
61–66
Kogel-Knabner I, Ekschmitt K, Flessa H, Guggenberger G, Matzner E,
Marschner B, von Luetzow M (2008) An integrative approach of
organic matter stabilization in temperate soils: linking chemistry,
physics, and biology. J Plant Nutr Soil Sci 171:5–13
Kramer RW, Kujawinski EB, Hatcher PG (2004) Identification of black
carbon derived structures in a volcanic ash soil humic acid by
Fourier transform ion cyclotron resonance mass spectrometry.
Environ Sci Technol 38:3387–3395
Larciprete R, Lacovig P, Gardonio S, Baraldi A, Lizzit S (2012) Atomic
oxygen on graphite: chemical characterization and thermal reduction. J Phys Chem 116:9900–9908
Lehmann J (2007) A handful of carbon. Nature 447:143–144
Lehmann J, Solomon D, Kinyangi J, Dathe L, Wirick S, Jacobsen C
(2008) Spatial complexity of soil organic matter forms at nanometre
scales. Nat Geosci 1:238–242
Linhares CR, Lemke J, Auccaise R, Duo DA, Ziolli RL, Kwapinski W,
Novotny EH (2012) Reproducing the organic matter model of
anthropogenic dark earth of Amazonia and testing the ecotoxicity
of functionalized charcoal compounds. Pesq Agropec Bras 47:693–
698
Mao JD, Johnson RL, Lehmann J, Olk DC, Neves EG, Thompson ML,
Schmidt-Rohr K (2012) Abundant and stable char residues in soils:
implications for soil fertility and carbon sequestration. Environ Sci
Technol 46:9571–9576
Marris E (2006) Black is the new green. Nature 442:626–628
Masiello CA (2004) New directions in black carbon organic geochemistry. Mar Chem 92:201–213
Mattevi C, Eda G, Agnoli S, Miller S, Mkhoyan KA, Celik O,
Mastrogiovanni D, Granozzi G, Garfunkel E, Chhowalla M (2009)
Evolution of electrical, chemical, and structural properties of
1232
transparent and conducting chemically derived graphene thin films.
Adv Func Mater 19:2577–2583
Novotny EH, Deazevedo ER, Bonagamba TJ, Cunha TJF, Madari BE,
Benites VD, Hayes MHB (2007) Studies of the compositions of
humic acids from Amazonian dark earth soils. Environ Sci Technol
41:400–405
Novotny EH, Hayes MHB, Madari BE, Bonagamba TJ, de Azevedo ER,
de Souza AA, Song G, Nogueira CM, Magrich AS (2009) Lessons
from the Terra Preta de Índios of the Amazon region for the
utilisation of charcoal for soil amendment. J Braz Chem Soc 20:
1003–1010
Oades JM (1988) The retention of organic matter in soils.
Biogeochemistry 5:35–70
Pandey AK, Pandey SD, Misra V (2000) Stability constants of metalhumic acid complexes and its role in environmental detoxification.
Ecotoxicol Environ Saf 47:195–200
Papirer E, Lacroix R, Donnet JB, Nansé G, Fioux P (1995) XPS study of
the halogenations of carbon black—part 2. Chlorination Carbon 33:
63–72
Pérez-Cadenas AF, Hódar FJM, Moreno-Castilla C (2003) On the nature
of surface acid sites of chlorinated activated carbons. Carbon 41:
473–478
Powell CJ, Jablonski A (2010) Progress in quantitative surface
analysis by X-ray photoelectron spectroscopy: current status
and perspectives. J Electron Spectrosc Relat Phenom 178–
179:331–346
Schimel DS, Braswell BH, Holland EA, McKeown R, Ojima DS, Painter
TH, Parton WJ, Townsend AR (1994) Climatic, edaphic, and biotic
controls over storage and turnover of carbon in soils. Global
Biogeochem Cycles 8:279–293
Schmidt MWI, Noack AG (2000) Black carbon in soils and sediments:
analysis, distribution, implications, and current challenges. Global
Biogeochem Cycles 14:777–794
Schmidt MWI, Torn MS, Abiven S, Dittmar T, Guggenberger G,
Janssens IA, Kleber M, Kogel-Knabner I, Lehmann J, Manning
DAC, Nannipieri P, Rasse DP, Weiner S, Trumbore SE (2011)
Biol Fertil Soils (2014) 50:1223–1232
Persistence of soil organic matter as an ecosystem property. Nature
478:49–56
Simpson AJ, Song G, Smith E, Lam B, Novotny EH, Hayes MHB (2007)
Unraveling the structural components of soil humin by use of
solution-state nuclear magnetic resonance spectroscopy. Environ
Sci Technol 41:876–883
Six J, Conant RT, Paul EA, Paustian K (2002) Stabilization mechanisms
of soil organic matter: implications for C-saturation of soils. Plant
Soil 241:155–176
Stankovich S, Dikin DA, Piner RD, Kohlhaas KA, Kleinhammes A, Jia
Y, Wu Y, Nguyen SBT, Ruoff RS (2007) Synthesis of graphenebased nanosheets via chemical reduction of exfoliated graphite
oxide. Carbon 45:1558–1565
Swift RS (1996) Organic matter characterization. In: Sparks DL (ed)
Methods of soil analysis, part 3, chemical methods. Soil Science
Society of America, Madison, WI, pp 1018–1020
Torn MS, Trumbore SE, Chadwick OA, Vitousek PM, Hendricks DM
(1997) Mineral control of soil organic carbon storage and turnover.
Nature 389:170–173
Wang DW, Wu KH, Gentle IR, Lu GQ (2012) Anodic chlorine/nitrogen
co-doping of reduced graphene oxide films at room temperature.
Carbon 50:3333–3341
Xu D, Zhu S, Chen H, Li F (2006) Structural characterization of humic
acids isolated from typical soils in China and their adsorption
characteristics to phenanthrene. Colloids Surf A 276:1–7
Yang D, Velamakanni A, Bozoklu G, Park S, Stoller M, Piner RD,
Stankovich S, Jung I, Field DA, Ventrice CA, Ruoff RS (2009)
Chemical analysis of graphene oxide films after heat and chemical
treatments by X-ray photoelectron and Micro-Raman spectroscopy.
Carbon 47:145–152
Zhang J, Dai J, Wang R, Li F, Wang W (2009) Adsorption and desorption
of divalent mercury (Hg2+) on humic acids and fulvic acids extracted
from typical soils in China. Colloids Surf A 335:194–201
Zhu Y, Tang J, Zhu W, Zhang M, Liu G, Liu Y, Zhang W, Jia M (2011)
Graphite oxide-supported CaO catalysts for transesterification of
soybean oil with methanol. Bioresour Technol 102:8939–8944