Received 25 October 2008; accepted 30 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
Bio-solubilization of Chinese lignite II:
protein adsorption onto the lignite surface
YIN Su-dong
1
, TAO Xiu-xiang
2
, SHI Kai-yi
2
1
Department of Mechanical Engineering, University of Calgary, Calgary, Alberta T2N 1N4, Canada
2
School of Chemical Engineering and Technology, China University of Mining & Technology, Xuzhou, Jiangsu 221116, China
Abstract: Lignite bio-solubilization is a promising technology for converting solid lignite into oil. This study concerns the adsorp-
tion of lignite-solubilizing enzymes onto the lignite surface. Adsorption capacity, infrared spectral analysis and driving forces
analysis are studied as a way to help understand the bio-solubilization mechanism. The results show that the amount of lignite
bio-solubilization is proportional to the amount of adsorbed lignite-solubilizing enzymes. An increase in lignite-solubilizing enzyme
adsorption of 10% leads to a 7% increase in lignite bio-solubilization. However, limited amounts of enzymes can be adsorbed by
the lignite, thus resulting in low percentages of bio-solubilization. Infrared spectral analysis shows that side chains, such as hy-
droxyl and carbonyl, of the lignite structure are the main, and necessary, structures where lignite-solubilizing enzymes attachto the
lignite. Furthermore, driving force analysis indicates that the electrostatic force between lignite and enzymes is the main adsorption
mechanism. The forces are influenced by solution pH levels, the zeta potential of the lignite and the isoelectric points of the en-
zymes.
Keywords: lignite; bio-solubilization; adsorption; enzyme
1 Introduction
Coal is becoming a more important energy re-
source in the world due to limited petroleum and
natural gas reserves. According to International En-
ergy Outlook 2007 of U.S. Department of Energy, the
reserves of coal, petroleum and natural gas at the end
of 2007 were 462.6, 164.5 and 163.3 trillion tones oil
equivalent, respectively. Assuming 2007 consumption
rates, the coal reserves would last for 146 years while
petroleum will be depleted in 50 years and natural gas
in 63 years. After petroleum and natural gas reserves
are depleted coal will dominate the fossil energy
market. Naturally, efficient utilization of coal is be-
coming a more important research topic, especially
for lignite, which accounts for 47.3% of the world
coal reserves.
Of world lignite production 96.4% is used for elec-
tricity and heat generation by combustion, which has
resulted in serious air pollution. The harmful emis-
sions from lignite combustion are mainly sulfur ox-
ides (SO
x
), nitrogen oxides (NO
x
), carbon dioxide
(CO
2
) and trace elements. Lignite combustion ac-
counted for 25% of the sulfur oxides emissions, 10%
of the nitrogen oxides emissions, 17.2% of the carbon
dioxide emissions and 33% of the atmospheric mer-
cury emissions in Canada in 2005. In the United
States, the average emission rates from lignite-fired
generation are 2249 lbs/MWh of carbon dioxide, 13
lbs/MWh of sulfur dioxide and 6 lbs/MWh of nitro-
gen oxides. In China, about 87% of the sulfur oxides
emissions, 67% of the nitrogen oxides emissions and
71% of the carbon dioxide emitted come from lignite
combustion
[1]
. Therefore, combustion is not a good
technology for lignite utilization with respect to en-
vironmental protection. New technologies are ur-
gently needed.
Bio-solubilization of lignite is a promising tech-
nology that applies microorganisms to solubilize solid
lignite to provide a clean, cost effective liquid energy
source. In 1982, Cohen first reported that American
lignite could be solubilized by the fungi Polyporus
versicolor and Poria monticol
[2]
. Later, in 1991,
Catcheside reported that Australian lignite was solu-
bilized by Coriolus versicolor, Phanerochaete chry-
sosporium, and five other species
[3]
. In 1992, bio-
solubilization of German lignite using seven basidio-
mycetes was investigated and confirmed by Resis. In
Mining Science and Technology 19 (2009) 03630368
MINI NG
SCIENCE AND
TECHNOLOGY
www.elsevier.com/locate/jcumt
Mining Science and Technology Vol.19 No.3 364
2002, Machnikowska found that Polish lignite was
solubilized by a strain of P. Putida. In 2003, Basaran
and his co-workers successfully solubilized Turkish
lignite to form a black liquid using Corilous versi-
color fungus. Recently, Shi and his colleagues solubi-
lized Chinese lignite by a fungus
[47]
.
The bio-solubilization product (a black liquid) re-
tains 97.5% of the raw lignite heating value, suggest-
ing almost no energy loss during the bio-solubiliza-
tion process and that this process efficiently transfers
the energy stored in the solid lignite into the liquid
oil
[8]
. Compared with thermal liquefaction of lignite,
bio-solubilization has the distinct advantages of: 1)
operation at atmospheric temperature and pressure; 2)
conversion of lignite into a single-phase product
without a large quantity of byproducts; and 3) the fact
that microorganisms can obtain hydrogen from water
and do not require another external source of hydro-
gen to perform lignite solubilization
[9]
. In addition,
sincethe lignite bio-solubilization product is free of
sulfur or nitrogen; it will not produce SO
x
and NO
x
during combustion and is thus a clean energy source.
For these reasons, bio-solubilization of lignite has
attracted considerable interest around the world.
However, low solubilization yields and long con-
version times hinder the development of lignite
bio-solubilization. To the authors knowledge, the
maximum bio-solubilization percentage yield has
only been 34%. The shortest conversion time still
requires 10 days. The maximum bio-solubilization
time reported required two months. Because lignite
bio-solubilization is mainly induced by enzymes se-
creted by the microorganism most researchers are
trying to find and isolate more efficient lig-
nite-solubilizing enzymes. To date, no apparent im-
provement has been achieved.
The adsorption of enzymes onto the lignite is, in
fact, another important factor that influences bio-
solubilization efficiency. During the typical process
of enzyme degradation of a substrate, the enzyme
must arrive at and adsorb to the substrate for degrada-
tion reactions to occur. If some enzymes can effi-
ciently solubilize lignite but are not easily adsorbed
by the lignite, their bio-solubilization efficiency must
be low. Duinhoven et al, investigated enzyme adsorp-
tion at a solid-liquid interface
[1011]
. Their results
show that enzyme adsorption depends on the nature
of the solid surface. Things like the surface zeta po-
tential and the electrostatic properties of the enzyme
affect enzyme performance. They showed that under
proper conditions the adsorption capacity of an en-
zyme on the same solid surface could be increased by
150 times. It may be that low enzyme adsorption af-
finity to the lignite surface is the factor that results in
low lignite bio-solubilization efficiencies.
In this paper, we focus on the adsorption of lig-
nite-solubilizing enzymes onto lignite surfaces. The
first objective is to investigate the relationship be-
tween enzyme adsorption and bio-solubilization re-
sults. Then we investigate the maximum adsorption
capacity of lignite-solubilizing enzymes onto lignite
under different conditions. Finally, the mechanism of
enzyme adsorption onto the lignite will be investi-
gated through infrared spectral and driving force
analysis.
2 Experimental
2.1 Lignite samples
Two types of lignite were used in this study. The
first was raw lignite and the other was acid pretreated
lignite. The raw lignite samples were mined from
Fushun, China, one of four main Chinese lignite
sources. The lignite samples were ground and sepa-
rated into sections. Lignite particles with a diameter
of 0.20.5 mm were used for the experiments. Acid
pretreated lignite samples were obtained by putting
lignite particles into a 0.8 mol/L nitric acid solution at
a concentration of 2 grams of lignite per milliliter of
nitric acid. The pretreatment lasted for 12 hours at
room temperature and was followed by distilled water
washing until the filtrate became neutral. The nitric
acid pretreated lignite particles were then dried in an
oven at 60C for 48 hours and stored at 4 C in a
refrigerator before subsequently being used in
bio-solubilization experiments. The basic elemental
composition of both samples is shown in Table 1.
Table 1 Elemental composition of lignite samples (%)
Weight fraction Raw lignite Nitric acid treated lignite
C 74.43 68.03
H 5.26 4.35
N 1.31 3.58
S 0.49 0.31
O 9.08 12.85
Ash 2.30 2.10
Note: Samples were dried to remove water before analysis.
2.2 Measurement of the zeta potential of lignite
samples
Since the zeta potential of a solid surface is rele-
vant to enzyme adsorption
[1013]
the zeta potential of
lignite suspended in solutions of pH ranging from 1.5
to 7.0 was measured using a JS94 micro-electropho-
resis apparatus. One gram of lignite particles 0.2 to
0.5 mm in diameter was added to 400 mL of distilled
water. This heterogeneous solution was well agitated
and then adjusted to the required pH using either HCl
or NaOH. The solution was agitated during adjust-
ment to get a uniform pH. The heterogeneous solu-
tion was then allowed to stand for 5 minutes. Subse-
quently, one mL of supernatant was transferred to the
micro-electrophoresis apparatus for the zeta-potential
measurement. The zeta-potential of the lignite was
measured 3 times at each pH and the average value is
used in the study.
YIN Su-dong et al Bio-solubilization of Chinese lignite II: protein adsorption onto the lignite surface 365
2.3 Preparation of enzyme solutions
Enzyme solutions were prepared using extracellu-
lar enzymes secreted by a fungus that was isolated
from decaying wood. Previous study had confirmed
that these extracellular enzymes can solubilize Fu-
shun lignite. Preparation of the enzyme solution re-
quired first culturing the fungus in a liquid culture
medium (10 grams of ammonium nitrate, 40 grams of
maltose and 1000 mL of distilled water) for 7 days in
a flask. The culture medium was then passed through
a membrane filter (0.45 m pore size) to remove the
fungal hypha and obtain a raw fungal extracellular
enzyme solution. The raw enzyme solution was then
added to a (NH
4
)
2
SO
4
solution (80% of the saturation
concentration) to precipitate the enzymes. After cen-
trifugation, the enzyme precipitate was collected and
dissolved in distilled water to obtain a pure enzyme
solution. The concentration of enzyme was deter-
mined using the Coomassie brilliant blue R-350
method.
2.4 Enzyme adsorption onto the lignite
Enzyme adsorption onto the lignite was determined
from the protein mass loss in the supernatant. It was
calculated by
WL
V C C
AD

=
) 1 0 (
(1)
where AD is the enzyme adsorption (mg/g lignite);
C0 is the initial enzyme concentration (mg/mL); C1 is
the final enzyme concentration after enzyme adsorp-
tion onto the lignite (mg/mL); V is the volume of en-
zyme solution (mL); and, WL is the mass of the lig-
nite added to the enzyme solution (g).
The enzyme solution was first adjusted to the re-
quired pH value using dilute NaOH or HCl. Then raw
lignite particles, or nitric acid treated lignite particles,
with a diameter from 0.2 to 0.5 mm were added to the
enzyme solution. The ratio of lignite to enzyme solu-
tion was 1 g/200 mL. The temperature was kept at
27.5C. The time when lignite was added to the so-
lution was recorded as the zero time and the enzyme
concentration in the supernatant was measured at 10
minute intervals, using the Coomassie brilliant blue
R-350 method, until no further change of protein
concentration occurred (Fig. 1). The enzyme adsorp-
tion onto the lignite was then calculated based on the
initial and final enzyme concentrations using Eq.(1).
Fig. 1 Illustration of apparatus for measurement of
enzyme adsorption onto lignite
2.5 Measurement of lignite bio-solubilization
The results of lignite bio-solubilization after en-
zyme adsorption were measured by the ultraviolet
adsorption at 240 nm wavelength (A240 nm), which
is the distinct adsorption wavelength of lignite
bio-solubilization products. To eliminate the effect of
lignite solubilization by water a blank test was per-
formed containing only lignite particles and distilled
water but no enzyme.
2.6 Infrared spectral analysis
To investigate the interaction between enzyme and
lignite during the adsorption process, raw lignite, ni-
tric acid treated lignite and lignite after enzyme ad-
sorption were analyzed by infrared spectroscopy
(AVATAR 360, Nicolet) over a wavelength range
from 4000 to 500 cm
1
. The samples were prepared as
pellets of lignite in KBr in a 1:100 ratio.
3 Results and discussion
3.1 Enzyme adsorption effects on lignite bio-
solubilization
The relationship between enzyme adsorption and
lignite bio-solubilization (A240 nm) is illustrated in
Fig. 2. A linear fit to that data gives the correlation
Y=0.76764*X, with an R
2
value of 0.99221. Thus lig-
nite bio-solubilization was proportional to the amount
of enzyme adsorption. In particular, no bio-solubili-
zation occurred when there was no enzyme adsorp-
tion onto the lignite. Once enzyme had been adsorbed
by the lignite, bio-solubilization increased with in-
creasing enzyme adsorption. In this study, an increase
of 10% adsorbed enzyme lead to an increase in lignite
bio-solubilization of 7%. This is consistent with other
enzyme conversion processes. Ooshima and co-
workers also found that the adsorption of cellulase
onto cellulose was positively correlated to cellulose
degradation
[12]
. The Medve research team investi-
gated adsorption of two different types of cellobioy-
hydrolase onto cellulose and also showed a linear
correlation between the cellulose conversion and
A
2
4
0

(
n
m
)
Fig. 2 Lignite bio-solubilization results (A240 nm) as a
function of enzyme adsorption on the lignite: 27.5C,
pH of 5.4, one gram of nitric acid treated lignite
Mining Science and Technology Vol.19 No.3 366
amount of adsorbed enzyme
[13]
. Enzyme catalysis rate
is expected to depend on both the substrate and en-
zyme concentrations. When the amount of enzyme
attached to a substrate increases the local concentra-
tion increases and catalysis is promoted, which leads
to a better catalysis result. Therefore, the amount of
enzyme adsorbed on the lignite can influence lignite
bio-solubilization results and the maximum adsorp-
tion capacity of the enzyme will largely determine
lignite bio-solubilization results.
3.2 Enzyme adsorption capacity on lignite
The maximum enzyme adsorption on lignite at
different pH conditions is presented in Fig. 3. Notice
that the lignite enzyme adsorption goes through a
maximum. Hence, it is difficult to improve bio-solu-
bilization by adding more lignite-solubilizing en-
zymes without considering the maximum in enzyme
adsorption. This also partly explains why other kinds
of lignite-solubilizing enzymes that have been found
are still inefficient and do not improve lignite bio-
solubilization.
0
2
4
6
8
10
12
14
16
18
A
d
s
o
r
p
t
i
o
n

o
I

e
n
z
y
m
e

o
n

t
h
e

l
i
g
n
i
t
e

(
m
g
/
g

l
i
g
n
i
t
e
)
pH
2.0 2.4 3.4 4.8 5.4 8.0
Raw ligniteBSA
Nitric acid treated ligniteBSA
Raw lignitelignite-solubilising enzymes
Nitric acid treated lignitelignite-
solubilising enzymes
Fig. 3 Enzyme adsorption levels on lignite at
27.5 C versus pH level
Fig. 3 also shows that nitric acid treated lignite ad-
sorbed more lignite-solubilizing enzymes than did the
raw lignite over the pH range of 2.08.0. This indi-
cates that proper chemical modification of the lignite
can improve enzyme adsorption on to it. Furthermore,
based on the analysis in Section 3.1 where lignite
bio-solubilization is shown to be proportional to en-
zyme adsorption levels, it appears nitric acid treated
lignite would more easily be solubilized than raw
lignite.
Fig. 3 shows that lignite-solubilizing enzyme ad-
sorption is influenced by pH level. The maximum
enzyme adsorption of 2 mg/g (nitric acid treated lig-
nite) seen in this study occurred at a pH of 5.4. At
other pH levels enzyme adsorption ranged from 0.7 to
1.0 mg/g (nitric acid treated lignite).
At the same time, we investigated the adsorption
capacity of bovine serum albumin (BSA), a non-lig-
nite-solubilizing enzyme, onto lignite, Fig. 3. The
nitric acid treated lignite also adsorbed more BSA
than did raw lignite, which indicates that lignite
structure does impact enzyme adsorption. But in con-
trast to solubilizing enzyme adsorption, the pH level
for greatest BSA adsorption was 3.4 rather than 5.4.
This indicates that enzyme properties also influence
adsorption capacity. Therefore, enzyme adsorption
capacity depends not only on lignite structure but also
on enzyme properties.
The relationship between lignite-solubilizing en-
zyme adsorption onto lignite and the equilibrium en-
zyme concentration in solution was investigated to
help understand adsorption onto nitric acid treated
lignite. The results shown in Fig. 4 indicate that en-
zyme adsorption initially increased with the increase
in equilibrium enzyme concentration. Enzyme ad-
sorption became constant at the maximum adsorption
capacity of 2.0 mg/g lignite as the solution concentra-
tion continues to increase. The Langmuir equation
was applied to this data. The Langmuir equation, of-
ten used to study adsorption onto solids, is given as
e 0 e 0
1 1 1
Q Q KC Q
= + (2)
where Q
e
is the enzyme adsorption capacity on the
lignite; Q
0
is the maximum enzyme adsorption capac-
ity on the lignite; C
e
is the equilibrium concentration
of enzyme in solution; and, K is a constant. Fig. 5
shows that the enzyme adsorption fitted the Langmuir
equation well with an R
2
of 0.9789.
E
n
z
y
m
e

a
d
s
o
r
p
t
i
o
n

c
a
p
a
c
i
t
y

o
I

n
i
t
r
i
c

a
c
i
d

t
r
e
a
t
e
d

l
i
g
n
i
t
e

(
m
g
/
g

l
i
g
n
i
t
e
)
Fig. 4 Enzyme adsorption on lignite versus free enzyme
concentration:
(27.5C, 1 gram of nitric acid treated lignite, 20 mL of
1.5 g/L lignite-solubilizing enzyme solution)
1

/

4
e
Fig. 5 Langmuir adsorption plot of lignite-solubilizing
enzyme onto nitric acid treated lignite
YIN Su-dong et al Bio-solubilization of Chinese lignite II: protein adsorption onto the lignite surface 367
The Langmuir equation is based on two assump-
tions: 1) the adsorption forms a monolayer on the
solid surface; 2) the solid supplies specific enzyme
adsorption sites. So, in this study, the nitric acid
treated lignite is assumed to only adsorb a monolayer
of lignite-solubilizing enzymes and it has special
chemical structures on the surface where the enzymes
adsorb. In other words, nitric acid treatment creates
additional enzyme adsorption points on the raw lig-
nite surface. These then adsorb more lignite-solubi-
lizing enzymes, which leads to the better bio-solubi-
lization results.
3.3 Infrared spectral analysis of enzyme adsorp-
tion onto lignite
As mentioned in Section 3.2, nitric acid treated
lignite adsorbed more enzymes than did the raw lig-
nite. To investigate the influence of the lignite surface
on enzyme adsorption, infrared spectroscopy was
used to characterize the raw lignite, the nitric acid
treated lignite and the nitric acid treated lignite after
enzyme adsorption. The results shown in Fig. 6 indi-
cate that raw lignite mainly contains hydroxyl (3450
cm
1
), cyclane (2925 cm
1
), carbonyl (1600 cm
1
) and
ether (10001300 cm
1
) moieties. After treatment
with nitric acid the content of hydroxyl, carbonyl, and
ether moieties increased. When the nitric acid treated
lignite was further treated with lignite-solubilizing
enzyme the hydroxyl and carbonyl content decreased
Wavenumber (cm
1
)
4000 500 1000 1500 2000 2500 3000 3500
20
40
60
80
100
(b) Nitric acid treated lignite
(c) Nitric acid treated lignite aIter lignite-
solubilising enzyme adsorption
(a) Raw lignite
Wavenumber (cm
1
)
4000 500 1000 1500 2000 2500 3000 3500
20
40
60
80
100
Wavenumber (cm
1
)
4000 500 1000 1500 2000 2500 3000 3500
20
40
60
80
100
Fig. 6 Infrared spectra of raw lignite, nitric acid treated
lignite and nitric acid treated lignite after exposure to
lignite-solubilizing enzymes
sharply thus indicating that these two functional
groups are the adsorption sites for the enzymes.
Therefore, enzyme adsorption onto lignite requires
specific chemical structures like hydroxyl and car-
bonyl. This result also supports and explains the
Langmuir adsorption analysis. On the other hand, the
result also indicates that the low bio-solubilization
percentage of raw lignite is largely due to the exis-
tence of few adsorption sites in the raw lignite. Lim-
ited adsorption sites on the lignite result in low lignite
bio-solubilization levels.
3.4 Driving force analysis of enzyme adsorption
onto lignite surfaces
According to Duinhoven et al. the main driving
force for enzyme-solid interactions is an electrostatic
force
[1011]
. So the electrostatic properties of lignite
and enzymes were investigated.
The electrostatic properties of lignite samples are
presented in Fig. 7. The isoelectric points of the raw
lignite and the nitric acid treated lignite are at a pH of
2.5 and a pH of 1.5, respectively. In other words, the
raw lignite contains negative charge when the pH is
larger than 2 while the nitric acid treated lignite is
negatively charged when the pH is larger than 1.5.
Furthermore, within the pH range from 1.5 to 7 the
nitric acid treated lignite is more negatively charged
than is the raw lignite. The infrared spectra in Section
3.3 suggest that the only structural difference between
nitric acid treated lignite and raw lignite is the num-
ber of carboxyl, carbonyl and ether groups. Hence,
the extra negative charge in the nitric acid treated
lignite is attributed to more carboxyls, carbonyls and
ethers.
Z
e
t
a
-
p
o
t
e
n
t
i
a
l

(
m
V
)
Fig. 7 Zeta potential of lignite
We used BSA as a model enzyme due to the ab-
sence of information concerning the isoelectric point
of the lignite solubilizing enzyme. BSA has a similar
adsorption trend on lignite compared to lignite-solu-
bilizing enzyme (see Fig. 3). The isoelectric point of
BSA is well known to be pH of 4.6. So when the pH
is greater than 4.6 BSA is negatively charged. When
the pH is less than 4.6 it is positively charged.
Considering the lignite electric charge, the BSA
electric charge and the BSA adsorption onto lignite
(see Fig. 8), note that the maximum adsorption is
over the pH range from the lignite isoelectric point to
Mining Science and Technology Vol.19 No.3 368
the BSA isoelectric point. Within this pH range the
lignite was negatively charged but the BSA was posi-
tively charged. Hence electrostatic forces promoted
BSA adsorption onto the lignite. At other pH values
enzyme adsorption is inhibited because BSA and lig-
nite have the same electric charge. For instance when
the pH is lower than the lignite isoelectric point the
lignite and the BSA are both positively charged.
When the pH is higher than the BSA isoelectric point
both the lignite and the BSA are negatively charged.
Furthermore, the nitric acid treated lignite is more
negatively charged than the raw lignite (see Fig. 7) so
the nitric acid treated lignite adsorbs more BSA than
does the raw lignite when the BSA was positive.
Fig. 8 Electrostatic force analysis of enzyme adsorption
onto nitric acid treated lignite
(Isoelectric points of BSA and nitric acid treated lignite were pH of 4.6 and
pH of 1.5, respectively; Zone A: lignite and BSA were positive, then en-
zyme adsorption was inhibited; Zone B: lignite was negative but BSA was
positive, then enzyme adsorption was promoted; Zone C: lignite and BSA
were negative, then enzyme adsorption was inhibited.)
Similarly, nitric acid treated lignite adsorbs more
positive lignite-solubilizing enzymes than does the
raw lignite. This leads to higher percentages of
bio-solubilization. Therefore, electrostatic forces play
an important role in the mechanism of enzyme ad-
sorption onto lignite and the solution pH is the key
factor.
4 Conclusions
This study investigated enzyme adsorption onto
lignite. Experimental data show that lignite bio-solu-
bilization is proportional to the degree to which lig-
nite-solubilizing enzymes adsorb onto the lignite sur-
face. A 10% increase in enzyme adsorption leads to a
7% increase in lignite bio-solubilization. However,
there are limits to the amount of adsorbed enzymes
on the lignite. This results in a low percentage of
solubilization. Infrared spectral analysis showed that
side chains, such as hydroxyl and carbonyl, of the
lignite structure were the main and required structures
where lignite-solubilizing enzyme adsorption occurs
on the lignite. Lignite contains few side chains, re-
sulting in limited enzyme adsorption. Driving force
analysis found that the electrostatic forces between
lignite and enzyme were the main adsorption mecha-
nism. When the pH level of the enzyme solution was
higher than the lignite isoelectric point, but lower
than the enzyme isoelectric point, electrostatic forces
did promote enzyme adsorption.
Therefore, in the future it is possible to improve
lignite bio-solubilization results by increasing the
number of enzyme adsorption sites on the lignite and
by enhancing the attractive electrostatic forces be-
tween lignite and lignite-solubilizing enzymes.
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