Journal of Structural Biology xxx (xxxx) xxx–xxx

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Journal of Structural Biology

journal homepage: www.elsevier.com/locate/yjsbi

Vaterite induced by Lysinibacillus sp. GW-2 strain and its stability

⁎
Jie-Jie Lva, Fang Maa, Fu-Chun Lia, , Chong-Hong Zhanga, Jia-Ni Chenb
a
College of Resources and Environmental Science, Nanjing Agricultural University, Nanjing 210095, China
b
State Key Laboratory for Mineral Deposits Research, Nanjing University, Nanjing 210046, China

A R T I C L E I N F O A B S T R A C T

Keywords: Studies on the formation and stability of vaterite by bacteria in experimental systems are of great importance for
Biomineralization understanding the mechanism by which microbes contribute to carbonate mineralization. In this study, mi-
Vaterite neralization experiments using Lysinibacillus sp. strain GW-2 were carried out for 72 h under shaking conditions
Stability and aging experiments using biotic and chemically synthesized vaterite were performed for 60 days in distilled
Aging
water and air. Our results indicate that Lysinibacillus sp. strain GW-2 can induce the formation of vaterite with
Lysinibacillus sp.
spherical morphology from an amorphous calcium carbonate precursor. Biogenic vaterite was more stable than
chemically synthesized vaterite in distilled water, perhaps due to organic matter secreted by bacteria that en-
wrapped the vaterite and prevented it from transforming into more stable phases. Infrared spectrophotometry of
biogenic and chemically synthesized vaterite confirmed the presence of organic matter in biogenic vaterite.

1. Introduction biomineralization, several independent groups demonstrated accumu-

Microbial induced carbonate precipitation is one of the most im-
portant processes in biogeochemistry. This process is an ideal method
for fixation of atmospheric CO2 (Millo et al., 2012; Pires et al., 2012),
repair of deteriorated ornamental stone in historical monuments, and
reinforcement of concrete buildings (Rodrigue-Navarro et al., 2003;
Kim et al., 2010). The co-precipitation of carbonate minerals and heavy
metals could also play an important role in the treatment of polluted
water (Rothenstein et al., 2012). In addition, bacterially induced car-
bonate precipitation may also provide important clues in the search for
extraterrestrial life owing to the special morphologies of the minerals
produced by this process (McKay et al., 1996; Morris et al., 2010). Fi-
nally, work using microbes has contributed to solving the “dolomite
problem” of the origin of dolomite (Vasconcelos et al., 1995; Wright
and Wacey, 2005; Sánchez-Román et al., 2011).
Vaterite formed in the absence of an integrated biological/chemical
system (i.e. a supersaturated solution) is rapidly converted from a
thermodynamically metastable phase to calcite or aragonite (Vecht and
Ireland, 2000). Thus, vaterite is relatively rare in nature and is usually
found as vaterite or calcite plus vaterite assemblages only in vivo or
experimental systems designed for microbial mineralization
(Lowenstam and Abbott, 1975; Rivadeneyra et al., 1991; Grasby, 2003;
Braissant et al., 2003; Lian et al., 2006; Bundeleva et al., 2012). The
reason for the stability of vaterite precipitated in biotic experiments is
not clearly understood. Many researchers believe that biofilms which
consist of exopolysaccharides and protein play a physiological role in
lation of calcium carbonate within bacterial biofilms (Yaara et al.,
2016).
The purpose of this study was to assess the formation process of
vaterite and to examine vaterite stability in distilled water and air. The
GW-2 strain of Lysinibacillus sp. isolated from soil has been experi-
mentally demonstrated to induce vaterite precipitation (Li and Guo,
2013). Therefore, incubation experiments with Lysinibacillus sp. strain
GW-2 were carried out for 72 h under shaking conditions to obtain
biotic vaterite. Aging experiments of biotic and chemically synthesized
vaterite were then performed for 60 days.
2. Materials and methods
2.1. Characteristics of the strain GW-2
B4 liquid medium containing 2.5 g calcium acetate, 4 g yeast ex-
tract, and 1 L distilled water was used for mineralization experiments.
The solution was adjusted to pH 8.0 using 1 mol/L NaOH and the pH
decreased to 7.37 after sterilization. B4 solid medium, prepared by
adding 2% agar to B4 liquid media, was used for selection, enrichment,
and purification of bacteria.
Bacteria were isolated from soil samples (pH 7.74) from Nanjing
Botanical Garden, China. After several purification steps, strain GW-2
was obtained and used in this study. The basic characteristics of strain
GW-2 are as follows: colonies on solid media are raised and transparent
with smooth surfaces and a diameter of approximately 1 mm. The

⁎
Corresponding author.
E-mail address: fchli@njau.edu.cn (F.-C. Li).

http://dx.doi.org/10.1016/j.jsb.2017.09.008
Received 3 August 2017; Received in revised form 21 September 2017; Accepted 23 September 2017
1047-8477/ © 2017 Elsevier Inc. All rights reserved.

Please cite this article as: Lv, J.J., Journal of Structural Biology (2017), http://dx.doi.org/10.1016/j.jsb.2017.09.008
J.-J. Lv et al.
9.0
8.5
8.0
pH value
7.5
7.0
0 10 20 30 40 50 60
Time(h)
Fig. 2. Temporal curve of pH value in the medium.
bacterial cells are short and rod-like in shape and contain spores in the
center ranging in size from 0.7–1.1 × 0.4–0.7 µm (Fig. 1). Gram
staining showed that strain GW-2 is Gram-positive. A comprehensive
phylogenetic analysis of 16S rRNA gene sequences showed that strain
GW-2 belonged to Lysinibacillus sp. (Li and Guo, 2013).
2.2. Method of biomineralization experiments
Four colonies growing on solid media were selected for inoculation
of 300 ml of liquid media (without calcium acetate to avoid CaCO3
precipitation) and a seed culture was obtained after incubation for 24 h
at 28 °C on a rotary shaker. The cell density of the seed culture was
1.03 × 107 cfu/ml. Erlenmeyer flasks were filled with 90 ml of B4 li-
quid media and 10 ml of seed culture and incubated at 28 °C on a rotary
shaker. Samples were taken after inoculation for less than 5 min, after
every hour for the first 18 h, and every 8 h between 18 and 72 h after
inoculation. The last sample was taken 72 h after inoculation. Abiotic
experiments were also performed under shaking conditions. Liquid and
solid phases of samples were separated by centrifugation.
2.3. Synthetic method of abiotic vaterite
A 250 ml conical bottle containing the composite solution, 10 g L-
Asparagine powder, and 1 g CaCl2 powder was placed on an ultrasonic
shaker for 20 min at 45 °C and 1 g Na2CO3 powder was slowly added.
The precipitated product was immediately centrifuged and placed in a
thermostatic oven at 35 °C to obtain abiotic vaterite.
2.4. Method of aging experiment
For aging experiments, a certain amount of vaterite induced by
Journal of Structural Biology xxx (xxxx) xxx–xxx
Fig. 1. a) Transmission electron microscopy and b) optical mi-
croscopy (1000×) images of Lysinibacillus sp. strain GW-2 cells
grown on B4 media for 48 h.
either the GW-2 strain or the chemical method was placed into an
Erlenmeyer flask with distilled water and incubated at 30 °C. Samples
were taken every 10 days for a total of 60 days. Aging tests were also
carried out in dry conditions on slide glass in air at 30 °C for 60 days.
GW-2 2.5. Observation and measurement
CK
The plate counting method was used to count the number of bac-
teria in each sample. An electronic balance accurate to 1/10,000 was
used to weigh the precipitate. pH was measured with a pH meter (pHS-
3C, Yoke Instrument, Shanghai, China). An X-ray diffractometer (D/
max-B III, Rigaku, Beijing, China) with Cu-Kα radiation was used to
identify the mineral composition of the precipitates with an error
of < 5%. Each sample was scanned continuously at 2° (2θ) min−1from
10° to 60° (2θ) at 35 kV and 20 mA in 0.01° (2θ) steps. Qualitative
70 80 identification of mineral phases was performed using Jade 5.0. The
precipitates were analyzed using a Tensor 27 Fourier transform infrared
spectrophotometer (FT-IR) (Bruker, Ettlingen, Germany). The spectro-
meter was set to scan at 1 cm−1 resolution with a scan range of
4000–400 cm−1. An EVO18 scanning electron microscope (Carl Zeiss,
Oberkochen, Germany) equipped with an energy dispersive detector
was used to image minerals at the Nanjing Institute of Geography and
Limnology, Chinese Academy of Sciences, China. The dried samples
were coated with 8 nm of gold before scanning electron microscopy
(SEM) imaging. Transmission electron microscopy (TEM) was per-
formed using a Tecnai G2 F20 electron microscope (FEI Co., Hillsboro,
OR, USA) equipped with an energy dispersive spectrometer to analyze
the chemical composition and microstructure of the minerals. Samples
were prepared by dispersing and settling the mineral powder with clear
alcohol on a Cu grid.
2.6. Characterization of organic matters in vaterite
Biogenic and abiotic vaterite were ground with an agate mortar. A
5 ml centrifuge tube filled with 30 mg vaterite powder and 2 ml of a
solution of dichloromethane and methanol at a ratio of 93:7 was placed
in an ultrasonic generator at 4 °C for 30 min. The clear fluid was then
collected by centrifugation. A small amount of the clear fluid was re-
moved with a capillary and dripped onto dried KBr powder. Once the
organic solvent was completely volatilized, the KBr powder was sub-
jected to compression treatment and analyzed using the FT-IR (Qian
et al., 2017).
3. Results
3.1. Changes of pH value
The pH of the culture decreased slightly during the initial growth
stage, after which pH gradually increased. The maximum and minimum
pH values were 8.81 and 6.98, respectively. In abiotic experiments, pH
remained unchanged at 7.40 (Fig. 2).
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J.-J. Lv et al. Journal of Structural Biology xxx (xxxx) xxx–xxx

(16–40 h), and decline(> 40 h) phases. Interestingly, the stationary

25 stage (8–18 h) appeared in the exponential phase. After this stage,
the bacteria began to use previously secreted organic matter
(polysaccharides, organic acids, etc.) as a carbon source and were
Bacterial density( 10 cfu/ml)

20
able to continue exponential growth (Zhu et al., 2014). The initial
density of bacterial cells was 1.03 × 107 cfu/ml and the density
7

15 after 8 h, when CaCO 3 began to precipitate, was 12.27 × 10 7 cfu/

ml. The maximum bacterial density during incubation was ap-
proximately 25-fold higher than the initial value.
10
3.3. Change of precipitate amount

5
As shown in Fig. 4, although CaCO 3 began to precipitate after
8 h, only a small amount of precipitate formed before 16 h of in-
0
cubation. Precipitation then progressed rapidly between 16 and
0 10 20 30 40 50 60 70 80 18 h, after which the amount of precipitate increased slowly to a
Time(h) maximum of 99 mg per 100 ml culture media. In contrast to bac-
terial experiments, no mineral precipitation occurred in abiotic
Fig. 3. Growth curve of GW-2 strain in B4 liquid medium.
experiments.

3.4. Mineral species and morphologies

100

In X-ray diffraction experiments, no obvious diffraction peaks

were observed before 16 h of incubation, whereas diffraction peaks
80
belonging to vaterite were observed between 16 and 72 h (Fig. 5).
FT-IR absorption peaks at 1083, 875, 1490–1420, and 746 cm−1
Precipitate amount(mg)

corresponding to vaterite ν 1, ν2 , ν3 , and ν 4 vibrations, respectively,

60
were also observed after 32 and 72 h of incubation, suggesting that
vaterite was the predominant precipitate formed. In contrast, FT-IR
absorption peaks after 16 h of incubation corresponded to amor-
40
phous calcium carbonate (ACC) vibrations (Fig. 6).
SEM experiments revealed that the minerals formed in this study
were spherical and always aggregated together around bacterial
20
extracellular secretions. TEM examination of spherical minerals
showed that the minerals formed after 16 h of incubation were ACC
and poorly crystallized vaterite, whereas the minerals formed after
0
0 10 20 30 40 50 60 70 80 32 and 72 h of incubation were predominantly fully crystallized
Time(h) vaterite (Fig. 7).

Fig. 4. Temporal curve of precipitate amount.

3.5. The stability of vaterite

3.2. 2Change of bacterial density As shown by X-ray diffraction, neither biogenic nor chemically
synthesized vaterite underwent any phase transformations in air
As shown in Fig. 3, after incubation, bacterial growth occurred after 60 days. Similarly, biogenic vaterite aged in distilled water for
throughout the lag (< 4 h), exponential (4–16 h), stationary 60 days did not transform (Fig. 8a) and its morphology remained

Fig. 5. X-ray diffraction patterns of three representative pre-

3500 V
V cipitate.

3000 V
V V
2500
16h V V V
Intensity (counts)

2000

1500
32h
1000

500
72h
0
10 20 30 40 50 60
2ș (°)

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J.-J. Lv et al. Journal of Structural Biology xxx (xxxx) xxx–xxx

100 16h

90
874.04
1082.35
80
1402.07
70

60

50
100 32h
1088.51 744.69
80

60
875.56

40 1463.39

20
100 72h
744.30
1087.94
90

80 874.20
1438.42

70

4000 3000 2000 1000

Wave number (cm-1)

Fig. 6. Fourier transforms infrared spectrophotometers of three representative precipitates.

spherical (Fig. 9a, b). However, almost all of the chemically syn-
thesized vaterite in distilled water was transformed into calcite
after 10 days (Fig. 8b) and its morphology changed from spherical
to rhombohedral with a round pit in the center (Fig. 9c, d).
4. Discussion
4.1. The increase of pH value is prerequisite condition for the precipitation
of vaterite
The pH of the culture decreased slightly during the initial growth
stage, after which pH gradually increased. The maximum and minimum
pH values were 8.81 and 6.98, respectively (Fig. 2). In abiotic experi-
ments, pH remained unchanged at 7.40. It is well known that an al-
kaline environment is a prerequisite for carbonate precipitation. In this
study, although pH decreased slightly during the initial stage of the
experiment from 8.0 to 6.98, the media was still alkaline enough for
carbonate precipitation. As the incubation time increased, the pH value
also gradually increased. After 16 h, vaterite began to precipitate at pH
7.77. Correlation analysis showed that there was a significant positive
correlation between the amount of precipitate and the pH value (cor-
relation coefficient 0.87, P < 0.01) (Fig. 10). These results confirm
that an increase in alkalinity is favorable for the formation of vaterite
(Rodrigue-Navarro et al., 2007; Fernández-Remolar et al., 2012).
In this experimental system, four processes may have affected the pH
value. First, bacteria secrete low-molecular-weight organic acids during the
process of metabolism. At the initial stage of the experiment in this study,
this process likely dominated, leading to a slight reduction in pH value. At
this stage, the lower alkalinity was not conducive to carbonate precipitation.
Also leading to a reduction in pH is the process by which CO2 produced by
bacterial respiration dissolves in water and then forms a precipitate
according to the following reactions: CaCO3:CO2(aq) + H2O →
HCO3− +
(aq) + H ; HCO3
−
(aq) → CO3(aq) + H ;CO3(aq) + Ca
2− + 2− 2+
→ CaCO3(s).
Alternatively, two processes result in an increase in pH value (Li et al.,
2013). As bacteria degrade organic matter into ammonia, NH4+ and
OH−are simultaneously produced according to the reactions R-
−
NH2 + H2O → NH3 + R-OH and NH3 + H2O → NH4+ (aq) + OH . Simi-
larly, when bacterial cells autolyse, nitrogen-containing molecules release
ammonia. As the bacteria in this study began to reproduce logarithmically,
these two processes likely dominated, resulting in an increase in pH.
Overall, the alkaline environment resulting from these processes allowed for
carbonate precipitation.
4.2. The rapid growth of bacteria ensured the precipitation and stabilization
of vaterite
Figs. 2 and 3 showed that when bacterial growth went into sta-
tionary phase (16 h), precipitants began to form. Further studies con-
firmed that the precipitants were vaterite which has the highest nu-
cleation barrier (ΔGN∗) among the three anhydrous polymorphs of
calcium carbonate. According to homogeneous/ heterogeneous nu-
cleation theory, energy higher than the nucleation barrier is one of the
necessary conditions for generation of a new nucleus. Here, the nu-
cleation barrier is determined by ΔGN∗ = 16π rSL2Ω3/3(kTInS)2, where
rSL is the interfacial energy (J/m2), Ω is the volume of the base unit, k is
the Boltzmann constant, T is the thermodynamic temperature, and S is
the supersaturation of the solution (Cui et al., 2007). It is obvious that
both reducing rSL and increasing S can reduce the ΔGN∗ value of the
nucleation of calcium carbonate. Previous results have shown that
bacterial cells (Costerton, 1995; Guo et al., 2013; Zhang et al., 2017)
and excreted EPS by bacteria are both likely to become effective mi-
neral nucleation sites (Schultze-Lam et al., 1996; Aloisi et al., 2006). As

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J.-J. Lv et al. Journal of Structural Biology xxx (xxxx) xxx–xxx

Fig. 7. SEM and TEM images of carbonate minerals in-

duced by strain GW-2a) Scanning electron microscopy
(SEM) image after 32 h of incubation showing spherical
minerals with bacterial extracellular secretions attached
to their surfaces. b) SEM image after 72 h of incubation
showing a single spherical mineral with a rough surface
and a diameter of approximately 2 μm. c) Transmission
electron microscopy (TEM) image after 16 h of incuba-
tion of a poorly crystallized spherical mineral. d)
Selected area electron diffraction (SAED) image of the
edge of the spherical mineral indicated by the arrow in
c). The diffuse rings correspond to amorphous calcium
carbonate (ACC). e) TEM image after 16 h of incubation
showing a spherical mineral. f) SAED image of vaterite
reflections with d = 0.294 nm, indicated by the arrow in
e). This demonstrates that the spherical mineral is poorly
crystallized vaterite. g) TEM image after 72 h of in-
cubation showing a well-crystallized spherical mineral.
h) SAED image of spots corresponding to vaterite re-
flections of crystal plane with d = 0.619 nm, indicated
by the arrow in e). i) Energy dispersive spectroscopy
(EDS) image corresponding to the encircled area in e)
showing that the spherical mineral is composed of
CaCO3.

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J.-J. Lv et al. Journal of Structural Biology xxx (xxxx) xxx–xxx

a
V V C V
60d V V
1250

1000 30d
Intensity (Counts)

750
20d

500

10d

250

0d

0
10 20 30 40 50 60
2ș(°)

5000
C
b
4000
Intensity (Counts)

3000

2000

10d C C C C CC C

1000
V V
V V V
0d V

0
10 20 30 40 50 60
2ș(°)
Fig. 8. X-ray diffraction patterns of a) biogenic and b) abiotic vaterite from aging experiments.

solid impurities in the biomineralization system, bacteria can reduce
the interfacial energy (rSL) by serving as a kind of nucleation site. The
rapid growth of bacteria led to an increase in CO32− content and bac-
terial density in the system, which increased the supersaturation of the
solution (S) and reduced the interfacial energy (rSL). During the middle
and late stages of experiments under shaking condition, the extreme
supersaturation of CaCO3 and enough nucleation sites favor vaterite
formation because the increased supersaturation and reduce interfacial
energy may decrease the nucleation energy barrier of vaterite.
4.3. ACC is a precursor of vaterite
In X-ray diffraction experiments, several diffraction peaks belonging
to vaterite were observed after 16 h of incubation, whereas in FT-IR
analysis of samples collected at the same time point, absorption peaks
around 1083, 875, and 1490–1420 cm−1 corresponded to ACC vibra-
tions sample (Weng, 2010). TEM and selected area electron diffraction
(SAED) experiments confirmed that samples taken after 16 h of in-
cubation were composed mainly of poorly crystallized vaterite and
ACC, whereas samples taken after 72 h of incubation contained well-

6
J.-J. Lv et al.
120
Precipitate amount (mg)
80
40
y = 0.0661x - 0.482
r=0.87 (P<0.01)
0
7.0 7.5 8.0 8.5
pH value
Fig. 10. Correlation analysis between pH value and precipitate amount.
crystallized vaterite. These results suggest that ACC is a precursor of
vaterite.
The Ostwald–Lussac step rule suggests that the phase that is syn-
thesized first has a maximum solubility under conditions of continuous
precipitation. Afterwards, other phases crystallize in order decreasing
solubility. Of the three anhydrous polymorphs of CaCO3, calcite has the
lowest solubility, whereas vaterite has the highest solubility. Therefore,
for the CaCO3 series, the crystallization order is ACC → vaterite →
aragonite → calcite (Cui et al., 2007).
4.4. Organic matters prevented vaterite from aging
Usually, vaterite might transform into stable phases in certain
conditions after the formation. There are two instances in which va-
terite transformed into calcite within 20–25 h at room temperature
(Kralj et al., 1997) and into aragonite within 1 h at 60 °C (Ogino et al.,
1987). However, the results of this paper show that vaterite retains
steady in the experimental system until the end of the biominerlization
experiment (72 h). Moreover, XRD patterns indicate that vaterite
transformation had not occurred in air even for 300 days and in
Journal of Structural Biology xxx (xxxx) xxx–xxx
Fig. 9. Scanning electron microscope images of vaterite
before and after aging in distilled water. a) Biogenic vaterite
spherical morphology before aging. b) Biogenic vaterite
spherical morphology after aging for 60 days. c) Abiotic
vaterite spherical morphology before aging. d) Abiotic va-
terite rhombohedral morphology after aging for10 days.
distilled water for 60 days (Fig. 9). While, the chemically synthesized
vaterite transformed into calcite, and their spherule-like morphology
became rhombohedron-like in the distilled water for 10 days.
Biofilms are multicellular communities that were so far thought
to be held together solely by a self-produced organic extracellular
matrix. And an intrinsic rise in carbon dioxide levels within the
biofilm is a strong trigger for the initiation of calcite-dependent
patterning (Yaara et al., 2016). In this paper, biofilms contained
organic matters (may be polysaccharides and proteins) tend to be a
trigger for the initiation of vaterite-dependent patterning. Although
the organic matters wrapped on the surface of vaterite has been
washed away by centrifugal separation of the liquid and solid
phases, the organic matters enwrapped by vaterite during their
nucleation and growth may restrain vaterite from transformation
into stable phase (Sawada, 1997; Vdović and Kralj, 2000). To
9.0 confirm our hypothesis, a group of extraction experiments with
organic solvent were carried out. After extraction, the solution with
biogenic vaterite was obviously turbid while those with abiotic
vaterite retained still clear. FT-IR patterns of the extracts from
biotic vaterite show that there are several absorption peaks corre-
sponding to diversiform organic functional groups (Fig. 11 and
Table 1), while there is no peak respect to chemical vaterite (Weng,
2010). It demonstrates that there might be something of organic
matters inside of biogenic vaterite, and this maybe the reason that
the vaterite induced by GW-2 strain did not undergo a phase
transformation but remained its stability.
5. Conclusions
In conclusion, our discussion about experimental results suggests
the following: the Lysinibacillus sp. strain GW-2 could induce the for-
mation of vaterite with spherule-like morphology under shaking con-
dition. SAED patterns indicate that ACC might be precursor of vaterite.
The aging experiments indicate that biogenic vaterite is more stable
than the abiotic vaterite in the distilled water. The possible reason for
stability of biogenic vaterite is that some bacterial secretion (for in-
stance, polysaccharides and proteins) were enwrapped by vaterite or
enwrapped vaterite, and they might restrain vaterite from transforming
into more stable phases.
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J.-J. Lv et al. Journal of Structural Biology xxx (xxxx) xxx–xxx

100 16h

98 P1 P2 P3 P4 P5 P6
96

2851.59
94
1464.96
1637.51 1061.42
92 2921.22

90

88

86
72h

95

90
2851.92
1466.50 1123.05
85 2923.39 1617.79
1653.95
80
3434.52

75

4000 3000 2000 1000

wavenumber (cm-1)

Fig. 11. FTIR patterns of extracts from biotic vaterite of the 16th and 72nd hour.

Table 1
Absorption peaks of extract from biotic vaterite of the 16th and 72nd hour by infrared spectrum detection(Weng, 2010; Qian et al., 2017).

Types of organic group Absorption peaks (cm−1) Vibration characteristics of groups Peak area in Fig. 11

Methyl groups 2921–2923 Stretching vibration of aliphatic methyl (eCH3) P2

2851 Stretching vibration of aliphatic methyl (eCH2e) P2
1464–1466 Asymmetric bending vibration of methyl and methylene P5

Nitrogen-containing groups 1637–1653 Stretching vibration of amide carbonyl P4

1617 Stretching vibration of aromatic eC]N P4

Oxygen – containing groups 3434 Stretching vibration of OeH P1

1123 Stretching vibration of alcoholic CeOH P6
1061 Stretching vibration of carbohydrate CeOH P6

Acknowledgments
This work was supported by the National Natural Science
Foundation of China (grant Nos: 41673083 and 41172308), the Key
Research Program of the Chinese Academy of Sciences (Grant No:
KZZD-EW-04-02) and by Open Project of the State Key Laboratory of
Loess and Quaternary Geology, CAS (grant No: SKLLQG1309). The
authors wish to thank Dr. Li Yan-ling and Ms. Luo Lian-chong at the
Nanjing Institute of Geography and Limnology, Chinese Academy of
Sciences for SEM observation.
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