M INING

SCIENCE AND
TECHNOLOGY
Mining Science and Technology 19 (2009) 0358–0362
www.elsevier.com/locate/jcumt

Bio-solubilization of Chinese lignite I:

extra-cellular protein analysis
TAO Xiu-xiang1, PAN Lan-ying2, SHI Kai-yi1, CHEN-hui1, YIN Su-dong3, LUO Zhen-fu1
1
School of Chemical Engineering and Technology, China University of Mining & Technology, Xuzhou, Jiangsu 221116, China
2
School of Material Science and Engineering, Henan Polytechnic University, Jiaozuo, Henan 454003, China
3
Department of Mechanical and Manufacturing Engineering, Centre for Environmental Engineering Research and Education,
University of Calgary, Calgary, Alberta T2N 1N4, Canada

Abstract: A white rot fungus strain, Trichoderma sp. AH, was isolated from rotten wood in Fushun and used to study the
mechanism of lignite bio-solubilization. The results showed that nitric acid pretreated Fushun lignite was solubilized by T. sp. AH
and that extracellular proteins from T. sp. AH were correlated with the lignite bio-solubilization results. In the presence of Fushun
lignite the extracellular protein concentration from T. sp. AH was 4.5 g/L while the concentration was 3 g/L in the absence of
Fushun lignite. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of the extracellular proteins detected at
least four new protein bands after the T. sp. AH had solubilized the lignite. Enzyme color reactions showed that extracellular
proteins from T. sp. AH mainly consisted of phenol-oxidases, but that lignin decomposition enzymes such as laccase, peroxidase
and manganese peroxidases were not present.
Keywords: lignite; bio-solubilization; extracellular proteins; color reaction; white rot fungi

1 Introduction cular, enzyme-protein is the key factor. Therefore, in

Low rank coal (LRC: lignite and brown coal) plays
an important role in the energy supply of the world
and accounts for one third of all coal reserves.
However, because of its low heating value, high
moisture content and high ash content LRC is treated
as a cheap and dirty coal[1]. Several conversion
technologies have been studied to try and increase the
economic value, and to clean, LRC such as gasifica-
tion or liquefaction[2–3]. So far these processes still
require high temperature and pressure conditions that
result in high energy consumption. A more promising
technology is microbial solubilization of lignite,
which proceeds at ambient temperature and pressure.
Since the pioneer in the area, Fakoussa, found that
some microorganisms could use LRC as an energy
source for their own growth while solubilizing the
LRC, microbial lignite solubilization has been widely
investigated around the world[4]. A large number of
different microorganisms such as bacteria, comycetes
and ascomycetes have now been isolated and used to
solubilize LRC[4–5]. Some researchers proposed that
enzymes, chelates or alkali are involved in the
mechanism of lignite bio-solubilization[6–8]. In parti-
Received 20 October 2008; accepted 05 December 2008
Projects 50874107 and 50374068 supported by the National Natural Science Foundation of China, and CPEUKF06-12 by the Foundation of Key Laboratory
of Coal Processing & Efficient Utilization, Ministry of Education of China
Corresponding author. Tel: +86-516-83883194; E-mail address: taoxx163@163.com
this study we applied a new isolate named Trichoder-
ma sp. AH to the solubilization of Fushun LRC, with
an emphasis on the extracellular proteins or enzymes
that might affect the process.
2 Materials and methods
2.1 Fungus isolation and purification
Trichoderma sp. AH was isolated from rotten wood
in forest in Xuzhou, China.
Each sample was placed in 100 mL of sterile water
and shaken at 110 rpm for 10 min at room temper-
ature. The suspension was then diluted (10 or 100
times) and 0.2 mL aliquots were inoculated onto
minimal-salts medium agar plates containing 1.5 w%
Fushun LRC particles (<0.1 mm) as the sole carbon
source. The plates were kept in a biological incubator.
Isolated strains were then purified and cultured using
PDA plates consisting of 200 mL potato extract, 20 g
glucose, 20 g agar and 250 mg chloramphenicol
mixed in 1 liter of water. Trichoderma sp. AH was
one of the highly active strains and was used for the
following studies.
TAO Xiu-xiang et al Bio-solubilization of Chinese lignite I: extra-cellular protein analysis 359

2.2 Preparation of lignite tion where lignite had been added to an unheated
protein solution.
LRC from a Fushun opencast coal mine (early
2.4.3 Effect of LRC on extracellular protein excre-
Cenozoic, Tertiary, below 100 m) was used. The basic
tion from T. sp. AH
elemental composition is (w%): C 74.43, H 5.26, N
T. sp. AH was cultured in the presence and absence
1.31, S 0.49, O 9.07. The LRC samples were crushed
of Fushun LRC. Then the extracellular proteins
to pieces and sieved into 1.5–2.5 mm sized fractions.
produced from these two different cultures were used
Subsequently, the LRC particles were pretreated in
to solubilize Fushun LRC: bio-solubilization results
three different ways: 1) oxidization for 24 h by 8 M
were determined by UV-Vis analysis. In addition,
HNO3 in the ratio 15:40 g/mL. After oxidizing, the
these extracellular proteins were analyzed using SDS-
LRC was washed with sterile water until the pH value
PAGE electrophoresis.
of the filtrate was 5, it was then heated for 48 h at 60
°C; 2) oxidization for 2 h by 3% H2O2 (v/v) at room 2.5 Extracellular enzyme assays
temperature in the ratio of 15:40 (g/mL); 3)
2.5.1 Phenol-oxidase
microwave treatment using a 500 W microwave oven
The Bavendamm color-reaction is widely used to
for 5 min.
identify rot fungi[9]. Rot fungi cultured on agar plates
2.3 Bio-solubilization conditions containing tannin show a brown diffusion band on the
surface of the culture media. Absence of the brown
The isolated T. sp. AH was first inoculated onto a
diffusion band indicates the absence of phenol-
standard methods agar (SMA) culture medium that
oxidase. Tannin (4 mM/L) was added to the PDA
contained (per liter): 40 g maltose, 10 g peptone and
culture and the diameter of the mycelium growth and
18 g agar. The inoculated plates then were placed into
the brown circles were recorded every day.
an incubator at 27.5 °C until T. sp. AH mycelium had
2.5.2 Laccase
grown to cover the plates. Subsequently 1 g LRC
Excretion of laccase by T. sp. AH was established
sterilized with 75% alcohol (v/v) was put onto the
by adding guaiacol (0.5 g) to 3 mL of 95% alcohol
surface of the T. sp. AH. Once the lignite was
and then pouring the mixture onto the edge of the
converted into a black liquid by the T. sp. AH, the
mycelium. The medium turns a light red to indicate
black liquid was collected using a sterile injector. It
that laccase excrets from the fungi.
was then stored at 4 °C until further use.
2.5.3 Peroxidase
At the same time, T. sp. AH was inoculated into a
The fading method was used to identify the
liquid mixture of 1.5% (W/v) LRC and SMB culture
presence of peroxidase. Aniline blue (0.1 g/L) was
medium, the composition of which was the same as
added to the PDA culture medium overgrown with T.
that of the SMA but without the 18 g of agar, in
sp. AH. Fading of the plates shows the presence of
Erlenmeyer flasks. These samples were kept at 27.5
peroxidase.
°C under agitation at 110 r/min.
2.5.4 Manganese peroxidase (MnP)
2.4 Detection of extracellular proteins MnCl2·4H2O (0.1g/L) was added to a PDA culture
medium and T. sp. AH was then inoculated on the
2.4.1 Measurement of extracellular proteins
medium and cultured for two weeks. During the
To study the extracellular enzymes we first mea-
culture time the development of black-brown spots
sured the extracellular protein content. On different
was monitored. MnP catalyzes the oxidation of
days, the hypha body was separated from the SMB
MnCl2 to MnO2 thereby producing a black-brown
suspension using filter paper. The small mycelia were
spot.
then completely removed by filtering through a
micro-porous membrane (0.45 ȝm). 0.1 mL of sterile
filtrate was transferred to a test tube and 5 mL 3 Results and discussion
Coomassie brilliant blue G250 was added to the test
tube. The protein content was measured using 3.1 Preliminary morphology analysis of T. sp. AH
colorimetric analysis at a wavelength of 595 nm. Mycelia of T. sp. AH were white during their
Distilled water was used as a control. growth period and turned green in the late growth
2.4.2 Effect of extracellular proteins on lignite bio- stage. Observation using a microscope at 400
solubilization magnifications (Fig. 1) of mycelia showed that T. sp.
The influence of extracellular proteins on bio- AH mycelia are branched and separated. The spores
solubilization was examined by first heating 150 mL were transparent but not separated and had a
of protein solution from the T. sp. AH in a water bath characteristic conidial head. According to morpho-
at 100 °C for 30 min to denature the proteins. Then 1 logic analysis, T. sp. AH is preliminarily identified as
g of Fushun LRC was added to this solution. After 7 Trichoderma sp.
days, the solution was analyzed by UV-Vis spect-
rometry at 240 nm and compared to another prepara-
360 Mining Science and Technology Vol.19 No.3

3.2 Solubilization of different oxidized LRC sam- solubilization in our tests: the first stage was from the
ples by T. sp. AH 1st to the 5th days and the second was from the 6th to
the 12th days. During the first stage the solution was
LRC oxidized by Nitric acid (NA-LRC), hydrogen
neutral but it became acidic in the second stage.
peroxide (HP-LRC) or microwave radiation (MW-
Therefore, an alkaline mechanism cannot explain the
LRC) was placed onto plates covered by T. sp. AH.
bio-solubilization of Fushun LRC by T. sp. AH.
After ten days of solubilization (Fig. 2) the plate
containing NA-LRC had darkened. A black liquid
appeared on the surface of the mycelia on this plate
during the second day. The role of nitric acid

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oxidization pretreatment in T. sp. AH solubilizing of
Fushun LRC has been explained in another paper
written by our research team (Part II).

Fig. 3 UV-Vis spectra of the black liquid produced by T. sp.

AH in the SMB medium

 
S+

 
Fig. 1 Micrograph of Mycelia of T. sp. AH
(400 magnification) 



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I T. sp. AH+MW-LRC 

II T. sp. AH+NA-LRC       
III T. sp. AH
IV T. sp. AH+HP-LRC
Fig. 2 Solubilization results using differently oxidized
LRC samples: appearance at the 10th day
3.3 UV-Vis analysis of the black liquid products
Samples of the black liquid product were removed
from culture plates on different days using a sterile
micro-injector. The samples were then scanned with a
UV-Vis spectrophotometer. The spectra (Fig. 3) exhi-
bited maximum absorption at about 240 nm, which
indicates that alkyl substituted unsaturated aldehyde
and ketone groups are in the black liquid products.
Based on the relationship of the UV-Vis absorbance
of these black liquid products taken on different days
(Fig. 4) the maximum absorbance was confirmed to
be at 240 nm. The absorbance was used to charac-
terize the conversion ratio during Fushun LRC
bio-solubilization.
3.4 Conversion of Fushun LRC by T. sp. AH in
SMB
Fig. 4 shows that T. sp. AH took approximately 12
days to convert Fushun LRC. On the 7th day the
conversion ratio increased to the largest number. The
conversion process showed two stages of lignite
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Fig. 4 pH and absorbance at 240 nm as a function of
bio-solubilization of Fushun LRC versus time
3.5 Concentration variation of extracellular pro-
teins produced by T. sp. AH
The reactions of a microorganism to its enviro-
nment are mainly based on extracellular enzyme
catalysis. Thus, the composition of the extracellular
proteins indirectly reflects the excretion of extra-
cellular enzymes. In this study, we first compared the
extracellular protein content of SMB incubated T. sp.
AH (a non-converting condition) to that of SMB
incubation of T. sp. AH with added Fushun NA-LRC
(the converting condition). Fig. 5 shows that the
concentration of extracellular proteins under the
conversion condition was higher. For example, the
protein content under conversion conditions was 4.5
g/L while under non-conversion conditions it was 3
g/L. In both conditions the variation in protein
concentration over time had the same trend. This
result is due to the greater quantity of enzymes
excreted in the presence of the LRC. In addition,
variations in the conversion ratio (absorbance at 240
nm) and the content of extracellular proteins were the
same (Fig. 6). This indicates that the effect of T. sp.
AH on Fushun NA-LRC is related to the extracellular
proteins.
TAO Xiu-xiang et al Bio-solubilization of Chinese lignite I: extra-cellular protein analysis 361

LRC. The absorbance at 240 nm increased to 1.5.

3URWHLQFRQFHQWUDWLRQ However, when the proteins were denatured by
heating neither raw Fushun LRC nor NA-LRC was
solubilized. In summary, the extracellular proteins
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produced by T. sp. AH have the ability to solubilize

Fushun LRC, but once the proteins are denatured they
can no longer solubilize the LRC.

3.7 Analysis of T. sp. AH extracellular proteins

by SDS-PAGE
Fig. 5 Variation of extracellular protein concentration during
bio-conversion of lignite by T. sp. AH
SDS-PAGE technology was applied to the
investigation of protein excretion under lignite bio-
solubilization conversion, and non-conversion, condi-
tions. After T. sp. AH was cultured in SMB media
(one with added LRC and the other without) for 10
3URWHLQFRQFHQWUDWLRQ

days the proteins were separated and purified from

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each culture medium and then analyzed by SDS-

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Fig. 6 Variation of protein concentration and percent
conversion during bio-conversion of lignite by T. sp.
AH on different days
3.6 Bio-solubilization of Fushun NA-LRC by ext-
racellular proteins produced by T. sp. AH
The results above show that Fushun NA-LRC is
solubilized by T. sp. AH. To understand if LRC
solubilization was caused by an enzyme, two experi-
ments were designed: in one case bio-solubilization
was performed using extracellular proteins produced
by T. sp. AH for a period of seven days, in the other
case denatured extracellular proteins were used. The
LRC bio-solubilization results were measured by a
UV-Vis spectrometer.
The results are shown in Fig. 7 and indicate that in
the absence of Fushun NA-LRC, which acts as a
revulsant, the extracellular proteins produced by T. sp.
AH solubilized neither raw Fushun LRC nor NA-
LRC: the absorbency at 240 nm was less than 0.6. On
the other hand, in the presence of Fushun NA-LRC
the extracellular proteins produced by the T. sp. AH
did solubilize both raw Fushun LRC and NA-
PAGE. The results (Fig. 8) show thatprotein bands
from the sample produced under conversion condition
are darker than those from non-conversion conditions.
This implies that more extracellular proteins were
excreted under the conversion conditions. On the
other hand, the protein bands were located in almost
the same position regardless of the sample, which
indicates that most extracellular proteins had the
same molecular weight. However, four new proteins
bands were present under conversion conditions so
some new proteins were formed. These bands are not
very dark. In addition, as indicated in Fig. 5, more
proteins were induced in the presence of the LRC.
These new extracellular proteins could be the
efficient enzymes that solubilize the Fushun LRC.
1 Non-lignite
bio-solubilization
condition
2 Lignite bio-solubilization
condition
Fig. 8 Analysis of T. sp. AH extracellular proteins by
SDS-PAGE
3.8 Color reactions of T. sp. AH and P. versicolor
Color reactions were used to investigate the
enzymes excreted from T. sp. AH in comparison to
those from P. versicolor. A brown diffusion band
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Fig. 7 Bio-solubilization of Fushun NA-LRC by
extracellular proteins produced by T. sp. AH
caused by T. sp. AH appeared 24 h after inoculation.
Mycelia had covered the plate after 60 h. The ratio of
mycelia diameter to brown band diameter was 1.8
(>1). Subsequently, the brown band spread over the
entire plate. It took 12 h for P. versicolor to color the
plate and 120 h for the brown band to spread over the
plate. The ratio of diameters of the mycelium to the
brown band was 0.84 (<1). This shows that both
362 Mining Science and Technology
fungi belonged to the white-rot fungi.
P. versicolor showed light red on PDA plates with
added guaiacol as did T. sp. AH. This implies that
besides few laccases, T. sp. AH also produces other
phenol-oxidases. A PDA culture medium with added
aniline blue was completely de-colored by P.
versicolor. However, T. sp. AH had no effect on
aniline blue.
A medium containing MnCl2·4H2O showed the
appearance of black brown spots after inoculation
with P. versicolor. But no change was apparent on a
medium inoculated with T. sp. AH. These results
indicate that P. versicolor produces whole lignin
peroxidases while T. sp. AH excretes few peroxidases.
The lignin peroxidases had limited impact on the T.
sp. AH solubilization of Fushun LRC.
4 Conclusions
1) Results from morphologic analysis allow the
isolate to be preliminarily identified as Trichoderma
sp, Moniliaceae, moniliales, called T. sp. AH.
2) T. sp. AH solubilized Fushun nitric-acid-
oxidized lignite. A black liquid appeared on the
surface of the mycelia on the second day. The liquid
had an absorbance maximum at about 240 nm.
3) The conversion ratio of Fushun NA-LRC by T.
sp. AH was correlated to the concentration of
excreted proteins. The variation in absorbance at 240
nm and in extracellular protein concentration had the
same trend.
4) The Fushun NA-LRC induced an excretion of
extracellular proteins: under conversion conditions
the protein content was 4.5 g/L. Under non-conver-
sion conditions it was only 3 g/L. SDS-PAGE results
showed that at least four new protein bands appear
under conversion conditions.
5) Color reactions indicate that extracellular
enzymes from T. sp. AH consist mainly of phenol-
oxidases while lignin decomposition enzymes such as
laccase, peroxidase or manganese peroxidases were
Vol.19 No.3
not detected.
Acknowledgements
The study was supported by the National Natural
Science Foundation of China (50874107 and
50374068) and also by the Foundation of Key
Laboratory of Coal Processing & Efficient Utilization,
Ministry of Education of China (CPEUKF06-12),
both of which are gratefully acknowledged.
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