Changes of chemical properties of humic acids from crude

and fungal transformed lignite

LianHua Dong, Quan Yuan, HongLi Yuan
*
College of Biological Science, Key Laboratory of Agro-Microbial Resource and Application, Ministry of Agrio,
China Agricultural University, Beijing 100094, PR China
Received 27 February 2006; received in revised form 19 May 2006; accepted 19 May 2006
Available online 27 June 2006
Abstract
The development of biological processes for fossil energy utilization has received increasing attention in recent years. There are abun-
dance of lignite resources in China and the lignite, a low-grade coal, can be transformed by a Penicillium sp. After fungal transformation,
the contents of humic acid and water-soluble humic material increased from 38.6% to 55.1%, and from less than 4.0% to 28.2%, respec-
tively. The dierences in chemical properties of crude lignite humic acid (aHA), fungal transformed lignite humic acid (bHA) and water-
soluble humic material from fungal transformed lignite (WS) were studied. Elemental analysis and size exclusion chromatography
showed that the N content of bHA increased by 47.36% compared with aHA, and the molecular mass of bHA was smaller than
aHA. And the WS with the smallest molecular mass contained most content of N. The
13
C NMR and FT-IR spectra of aHA and
bHA showed that aHA contained more aromatic structure than bHA.
2006 Elsevier Ltd. All rights reserved.
Keywords: Humic acids; Water-soluble humic material; Fungal transformation
1. Introduction
Humic acids are polyelectrolytic macromolecules which
play an important role in global carbon and nitrogen
cycling and in the regulation of the mobility and fate of
plant nutrients and environmental contaminants [13].
During the past few decades, there have been many
researches on humic substances, but their chemical struc-
ture is still not fully understood. To study the chemical nat-
ure of humic substances, a variety of chemical/physical
methods and spectroscopic techniques have been used,
such as elemental analysis, Fourier transformed infrared
spectrophotometry (FT-IR),
13
C nuclear magnetic reso-
nance with cross-polarization/ magic angle spinning (CP/
MAS
13
C NMR), and UV/VIS and uorescence spectro-
scopy.
Lignite, a low-grade coal, has a lignin-like structure and
contains humic acid and water-soluble humic material
including fulvic acids. It was reported that some microbes
could grow on coal and modify it with both non-enzymatic
and enzymatic processes taking place [4,5]. The non-enzy-
matic action may be due to the presence of various metab-
olites of an alkaline, surfactant and chelating nature [68].
Oxidative and non-oxidative enzymes are considered to be
the coal-degrading agents [914]. In recent years, more and
more research for producing several kinds of fuel and
industrial materials by biodegradation have been taken
into account [15,16]. It has been established that treatment
of low-rank coals with aerobic coal-solubilizing microor-
ganisms results in the production of highly polar, heteroge-
neous materials with a relatively high oxygen content [17].
There are abundant lignite resources in China and the
lignite with low caloric value and high ash content is piled
up as rubbish causing a serious waste of resources as well
as environmental pollution [18]. Research into biological
processes for the utilization of fossil energy has received
0016-2361/$ - see front matter 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.fuel.2006.05.027
*
Corresponding author. Tel.: +86 10 62733464; fax: +86 10 62731332.
E-mail address: hlyuan@cau.edu.cn (H. Yuan).
www.fuelrst.com
Fuel 85 (2006) 24022407
more and more attention in recent years. Microbial treat-
ment has been considered as an economically eective
and environmentally safe way of processing coal via degra-
dation of the macromolecular network into simpler, low
molecular mass products [1921]. In previous work on
microbial degradation of Chinese lignite, we isolated a
strain of Penicillium sp. P6 which is capable of degrading
lignite eectively. After fungal transformation, the contents
of humic acid and water-soluble humic material (WS) in
the lignite greatly increased and the products of lignite
degraded by microbes had a better bioactivity than the ori-
ginal extractant from crude lignite [22,23]. It is well known
that humic acids have widespread utilization in agriculture,
for example, they have been used as organic fertilizer and
as a feed additive. By fungal transformation, we can get
much more and better bioactivity humic acids.
The present work was intended to study the dierences
in chemical properties among aHA, bHA, and WS. The
humic acids and WS were quantied and characterized
using elemental analysis, UV/VIS spectrophotometry, size
exclusion chromatography (SEC), micro-FT-IR and solid-
state CP/MAS
13
C NMR spectroscopy.
2. Materials and methods
2.1. Fungal transformation
The Penicillium sp. P6 was isolated from a coal soil mix-
ture collected from the Qiantong coal-processing factory,
Liaoning Province, northeast China, and deposited in the
China General Microbiological Culture Collection Center,
Beijing, China (CGMCCNO. 0866). It is maintained on a
yeast extract-malt agar [24]. Slants inoculated with P6 were
incubated at 28 C for activation for three days. Both the
spore and mycelia of Penicillium P6 were suspended in dis-
tilled water and the homogenate was then inoculated into
500 mL asks containing 100 mL culture uid (sucrose,
20.00 g; peptone, 5.00 g; KH
2
PO
4
, 0.10 g; K
2
HPO
4
,
0.10 g; CaCO
3
, 0.10 g; NaCl, 0.10 g; MgSO
4
7H
2
O,
0.04 g; (NH
4
)
2
SO
4
, 0.25 g; distilled water, 1000 mL; pH
6.57.0) and shaking at 28 C for three days at 150 rpm.
Then inoculated with 10% of mycelia into the lignite cul-
ture (powder of bean cake, 5.00 g; bran, 2.50 g; (NH
4
)
2
SO
4,
0.50 g; lignite, 15.00 g) and incubated at 28 C for one
week.
2.2. Sample preparation of lignite
The crude lignite was collected from the Huolingele
Minerals Administration Coalmine, Inner Mongolian
Autonomous Region, northwest China. Air-dried crude
lignite or biodegraded lignite was pulverized and sieved
with a 70-mesh sieve. Two grams of lignite powder were
suspended in 100 mL 0.1 M NaOH and stirred at 20 C
for 24 h, then centrifuged at 6000g for 15 min. The super-
natant was ltered through Whatman no. 1 paper and
the pH was adjusted to 2.0 with 6.0 M HCl. The solution
was allowed to settle for at least 12 h and was centrifuged
at 8000g for 5 min to precipitate the humic acid (aHA)
which was washed with distilled water three times and
dried at 60 C.
Biodegraded lignite were suspended in 100 mL dis-
tilled water and then stirred and centrifuged at 8000g for
15 min. The precipitate was collected to extract humic acid
(bHA; the extract procedure was same as that for crude lig-
nite humic acid). The supernatant was ltered and the l-
trate was vacuum-ltered through 0.45 lm pore-size
cellulose nitrate and then the ltrate was transferred to a
dialysis tube (800012000 Da) to be dialysed for 24 h. The
dialyzed water-soluble humic material (WS) was dried at
60 C, weighed and stored in a vacuum-dried chamber.
The aHA, bHA and WS samples obtained from the
above processes were treated with dilute HF to remove
Fe and other paramagnetic materials. Briey, each sample,
weighting 5 g, was placed in a 50 mL polyethylene beaker.
The sample was shaken with 40 mL 10% (v/v) HF for 30 s
and the suspension was allowed to settle for at least 12 h.
The rise of temperature that was by exothermic reaction
of HF with minerals did not exceed 40 C. The supernatant
was removed with a tube attached to a plastic syringe to
prevent the loss of ne materials by decanting. The HF
treatment as described above was repeated twice, after
which the remaining sediment was washed with distilled
water and vacuum-ltered on a 0.45 lm pore-size cellulose
nitrate to remove HF. Finally, the isolated pellet was
freeze-dried. All samples were stored at 4 C until used.
2.3. Size exclusion chromatography (SEC)
SEC was performed on solutions of the humic sub-
stances with a concentration of 500 mg L
1
in 0.414 M
TrisHCl buer solution at pH 9.0 using a Sephadex G-
100 medium gel (Code No. 17-0060-02 Pharmacia Biotech
AB). The diameter of the column and the height of the gel
were 16 mm and 90 cm, respectively, and the ow rate was
approximately 18 mL h
1
. The elution proles were moni-
tored using UV absorbance at 400 nm.
2.4. Chemical analysis
Elemental analysis (C, H, N and S) of the three samples
was carried out using a Vario-EL Elemental Analyzer
(Germany). Each treatment was performed in triplicate
and then mixed well to analysis three times. The light
absorbance of samples in 0.05 M NaHCO
3
was measured
in the range from 200 to 800 nm with a Perkin Elmer
Lambda 35 UV/VIS Spectrometer (USA). The E
4
/E
6
ratio
(the absorbance at 465 nm divided by that at 665 nm) was
calculated [25].
2.5. Solid-state CP/MAS
13
C NMR
The solid-state CP/MAS
13
C NMR spectra of the three
demineralized samples were obtained with a Bruker av-300
L. Dong et al. / Fuel 85 (2006) 24022407 2403
spectrometer (Switzerland) at a frequency of 75.47 MHz
with magic angle spinning at 4 kHz with a contact time
of 3 ms and a pulse delay of 5 s. Approximately 2290 scans
were performed for each spectrum. The C chemical shifts
were related to tetramethylsilane (0 ppm) as external stan-
dard. For quantication, the spectra were divided into dif-
ferent chemical shift regions assigned to specic carbon
groups: the assignments are shown in Table 2.
2.6. Micro-FT-IR spectroscopy
As the samples are too hard to pulverize, we use local
micro-FT-IR of sliced specimen instead of KBr wafer with
the homogeneous and representative samples. Micro-FT-
IR spectra of local areas of sliced specimen were measured
using Nicolet Magna-IR 750 spectrophotometer (USA),
connected to a Nicolet NicPlan IR microscope and a
MCT detector. The resolution was 4 cm
1
and the spectral
range was 4000650 cm
1
.
3. Results
3.1. Extraction and purication of humic acids and
water-soluble humic material
The humic acid content of the crude lignite was 38.6%
and that of the water-soluble humic material was much lit-
tle (less than 4.0%). However, after fungal transformation,
the content of humic acid and water-soluble humic mate-
rial reached 55.1% and 28.2%, respectively.
3.2. Analysis by SEC
SEC was used to separate the single sample of humic
material by size. The Sephadex G-100 has a fractionation
range of 40001500000 Da for globular proteins and
1000150000 Da for dextran. Since humic substances are
more spherical than dextran, the fractionation range for
humic materials is possibly between dextran and globular
proteins, with a greater similarity to dextran. As no exter-
nal calibration standard was used, the SEC results in
Fig. 1 were only qualitative. However, relatively high
and low average molecular mass (MM) fractions from
each sample were recognized by normalizing to the total
peak area [26].
For the humic acids two well-separated peaks were
observed. For aHA, there was a high MM elution peak
and a low MM elution peak, and the eluent volume of
low average MM peak was 142 mL. In contrast, the eluent
volume of low average MM peak for bHA was 162 mL.
The SEC of the WS was dierent: only WS displayed
the low MM elution peak. Qualitative evaluation of the
average MM derived from the SECs are as expected:
WS < bHA < aHA. As the curve of WS had no high aver-
age MM elution peak, it was considered that WS had the
lowest average MW compared to the other humic
samples.
3.3. Elemental analysis and UVVIS
The elemental analysis results for aHA, bHA and WS
are shown in Table 1. Since the ash contents of the puried
humic acids and water-soluble humic material were negligi-
ble, the dierence to 100% was assigned to the oxygen con-
tent. The results for the three samples showed a clear trend:
N and O contents increased from aHA to WS indicating
that the contents of N or O functional groups increased
during the process of fungal transformation. The H/C
ratios were 0.78 for aHA, 0.83 for bHA and 0.89 for WS,
which was consistent with previous reports: the lower
H/C ratio of humic acids, the higher the MM and the
amount of aromatic structures [18,25].
Generally, the E
4
/E
6
ratio is expected to decrease with
increasing MM and content of condensed aromatic rings
[27]. In addition, the ratio increases with the increase in
oxygen content [25]. In Table 1, the E
4
/E
6
ratios for
aHA, bHA and WS increased in this order. The E
4
/E
6
mea-
surements also revealed that WS should have the lowest
MM range and bHA had smaller MM than aHA, in agree-
ment with the SEC analysis.
3.4. The analysis of
13
C NMR spectra
More detailed information about the structure of the
humic substances was obtained using solid-state CP/MAS
13
C NMR spectroscopy. The spectra of aHA, bHA and
WS are presented in Fig. 2 and the relative intensities of
the chemical shift regions are summarized in Table 2.
In general, every sample showed peaks in the resonance
areas of aliphatic carbon (0110 ppm), aromatic C
(110160 ppm), and carbonyl C (160220 ppm). This
0 50 100 150 200 250 300 350 400
Elution volume (ml)
aHA
bHA
WS
200 kDa 80 kDa 50 kDa
Fig. 1. Size exclusion chromatograms of aHA (humic acids of crude
lignite), bHA (humic acids of degraded lignite) and WS (water-soluble
humic substances of degraded lignite), expressed as the relative absor-
bances at 400 nm as function of elution volume given in mL.
2404 L. Dong et al. / Fuel 85 (2006) 24022407
suggested that the bulk properties of the carbon function-
alities of each sample were similar to one another. The
spectra had peaks at 30 ppm in the alkyl C (050 ppm)
range. This peak was likely due to aliphatic carbons in
alkyl chains [28,29]. In the O-alkyl C (50110 ppm) region,
signals for aliphatic carbon substituted by oxygen and
nitrogen are usually observed [30,31]. In this region, we
observed a weak signal at 73 ppm only in the spectrum of
WS, which was consistent with the elemental data, where
WS contained the most oxygen and nitrogen. In the aro-
matic region (110160 ppm), there were strong peaks at
128 ppm in the spectra of all three samples, which was
assigned to aromatic units in lignin [32,33]. The peak at
173 ppm in the carbonyl C region was attributed to car-
boxyl C (160190 ppm).
However, the relative intensities of the dierent carbon
shifts diered for the three samples. The spectra data for
the humic acids and WS were analyzed quantitatively,
according to the literature [31,34,35], which divided the
spectra into three regions as in Table 2. A comparison indi-
cated that WS contained more carboxyl carbon and less
aromatic carbon in agreement with the elemental data
which showed that WS contained more oxygen and less
carbon. The C
arom
/C
alip
values for aHA and bHA revealed
the change occurring during in the process of fungal trans-
formation: the spectrum of aHA (with the large MM) was
dominated by aromatic carbon, while in the spectra of
bHA (lower MM), the percentage of aromatic carbon
decreased and the percentage of aliphatic and carboxyl car-
bon increased.
Table 1
Elemental composition (%), atomic ratios, and some spectroscopic characteristic of aHA, bHA and WS
Samples Elemental analysis UV/VIS
Element (%) Atomic ratio
C H N O
b
S Ash H/C
c
O/C
c
E
4
/E
6
aHA 56.1
a
3.7 1.5 38.0 0.6 0.1 0.79 0.51 6.41
bHA 53.9 3.7 2.2 39.7 0.5 ND 0.82 0.55 6.98
WS 49.7 3.7 2.5 43.6 0.5 ND 0.89 0.66 8.81
a
Results are presented as mean of three analyses.
b
Calculated as dierence to 100%.
c
The atomic ratio.
250 200 150 100 50 0 - 50
ppm
WS
bHA
aHA
Fig. 2. The solid-state cross-polarization magic-angle spinning
13
C
nuclear magnetic resonance spectra of the humic substances showing
intensity as function of chemical shift, given in parts per million (ppm).
Table 2
Distribution of carbon in aHA, bHA and WS determined by solid-state
CP/MAS
13
C NMR spectroscopy
Samples Percentage distribution of carbon
090 ppm 110160 ppm 160190 ppm (110160)/(090)
Aliphatic C Aromatic C Carboxyl C C
arom
/C
alip
aHA 26.8
a
56.9 16.3 2.13
bHA 31.2 50.2 18.6 1.61
WS 31.3 43.2 24.9 1.38
a
Results are presented as mean of three analyses.
4000 3500 3000 2500 2000 1500 1000 500
Wavenumbers (cm
-1
)
WS
bHA
aHA
Fig. 3. Fourier-transform infrared spectra of the humic substances.
L. Dong et al. / Fuel 85 (2006) 24022407 2405
3.5. Micro-FT-IR spectroscopy analysis
Fig. 3 shows the micro-FT-IR spectra of the three sam-
ples. All samples exhibited the typical absorption bands of
humic material [18,36]. Although absorption magnitudes of
FT-IR spectra cannot be compared directly, the dierences
between the micro-FT-IR spectra of the humic acids and
WS can easily be seen. The relative peak intensities, which
were dierent in the samples, reected the relative amount
of each functional group. The aliphatic, carboxyl and
polysaccharide-like character represented by absorption
bands at 2932 cm
1
(aliphatic CH stretching), 1720 cm
1
(C@O stretching of COOH and ketones) and 1037 cm
1
(CO stretch of polysaccharide-like components) became
stronger with decreasing MM which was also consistent
with the NMR results: more aliphatic and carboxyl carbon
in bHA and WS.
4. Discussion
The SEC results showed that the MM of the humic acid
from degraded lignite (bHA) was smaller than that of the
humic acid from crude lignite (aHA). The abscissa of the
SEC spectra in Fig. 1, reected the relative absorbance with
the elution volume, and as no external standard was
applied in the study, it was impossible to quantify absolute
size ranges of the two peak eluents. Furthermore, a calcu-
lation of the absolute concentration would require known
absorptivities of the two size fractions, as the capacity for
light absorbance generally diers for humic materials
according to the nature of chromophores present within
the dierent size fractions. In spite of the diculties in
the application of size exclusion chromatography for
humic substances, so far it is the eective and general
method to estimate the MM of them and the measured
peak elution volumes relate to the MM for humic acids
[3739].
It was reported that the smaller humic acid size fractions
contained more functional groups than the larger ones
[3,40]. This might be caused by the less amount of aromatic
carbon and a lower degree of condensation in the smaller
ones, as indicated by the E
4
/E
6
ratio, which increased with
decreasing MM. The E
4
/E
6
ratio has also been shown to
increase with increase in the oxygen content [25,41]. It is
interesting to note that the smaller humic acid size fraction
(bHA) contained more N and O than aHA, based on the
elemental data in Table 1. And more aliphatic and carboxyl
carbon structure for bHA, as determined with
13
C NMR
spectroscopy, corresponded more closely to these results.
The comparison of micro-FT-IR spectroscopy for aHA
and bHA showed similar trends as those revealed by the
NMR results. All the results indicate that the lignite struc-
ture might have changed in the process of transformation
by Penicillium sp. P6. The structure of lignite could be
destroyed and transformed by the lignin peroxidase [42],
and maybe a part of the alkyl moiety of the aliphatic chain
structure was transformed into carboxyl or COH groups
by enzymatic-oxidation. This may be why the content of
WS, with the smallest MM and most abundant carboxyl
groups, increased in the degraded lignite. Our previous
experiment showed that the products of lignite by micro-
bial degradation had a better bioactivity than the extract-
ant from crude lignite [22].
There are abundant lignite resources in China, with
reserves reaching 130 million tons [43], but their utiliza-
tion is very limited. Since the products of degraded lignite
have better bioactivity, the use of them is widespread. Also
fungal transformation is proved to be an eective way in
enhancing the content of lignite humic acid and water-sol-
uble humic material.
The elemental data also showed that the nitrogen con-
tent of bHA and WS was much higher than for aHA. It
is interesting to note that this was also found in earlier
report [12]. However, the mechanism of increasing nitrogen
content is still unknown. As we added some nitrogen nutri-
ments to the culture medium, either the microbes or the
humic acids themselves play an important role in nitrogen
xation. We deduce that humic acids have the ability to
carry out nitrogen xation, a similar presumption being
reported by Young et al. [44]. Detailed analysis is under-
way in our laboratory to make clarify the mechanism of
nitrogen xation.
5. Conclusions
All the above data show that after transformed by Pen-
icillium sp. P6, the content of humic acids and WS in lignite
increased. In addition, the MM of the humic acid
decreased, while the oxygen and nitrogen content
increased. The chemical structure of the crude lignite humic
acid changed: by way of a decrease in the content of the
dominant aromatic carbon and an increase in the carboxyl
and aliphatic carbon content.
Acknowledgement
This research was part of project No. 30370040 sup-
ported by the National Science Foundation of China and
No. 2003AA241170 supported by the Ministry of Science
and Technology of China (the 863 program).
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