393
14
Microbial Degradation and
Modification of Coal
Dr. Martin Hofrichter1
Dr. RenØ M. Fakoussa2
1
Department of Applied Chemistry and Microbiology, University of Helsinki, Viikki
Biocenter, P.O. Box 56, 00014 University of Helsinki, Finland, Tel: ‡ 358-919159321,
Fax: ‡ 358-919159322, E-mail: [email protected]
2
Institute of Microbiology and Biotechnology, Rheinische Friedrich WilhelmsUniversity of Bonn, Meckenheimer Allee 168, 53115 Bonn, Germany,
Tel: ‡ 49-228737219, Fax: ‡ 49-228737576
1
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
394
2
Historical Outline . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
397
3
3.1
3.2
Modification of Hard Coal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Fungi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
398
398
399
4
4.1
4.2
4.3
4.3.1
4.3.2
4.3.3
4.4
4.4.1
4.4.2
4.4.3
4.4.4
4.4.5
Bioconversion of Brown Coal . . . . . . . . . . . . . . . . . . .
Mechanisms: Solubilization, Depolymerization, Utilization
Organisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Solubilization of Brown Coal . . . . . . . . . . . . . . . . . .
Alkaline Solubilization . . . . . . . . . . . . . . . . . . . . . .
Solubilizing Effects of Chelators . . . . . . . . . . . . . . . .
Involvement of Hydrolases in Brown Coal Solubilization . .
Depolymerization of Brown Coal by Oxidative Enzymes . . .
Lignin Peroxidase (LiP ) . . . . . . . . . . . . . . . . . . . . .
Manganese Peroxidase (MnP ) . . . . . . . . . . . . . . . . . .
Other Peroxidases . . . . . . . . . . . . . . . . . . . . . . . . .
Laccases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Use of Decarboxylases for the Hydrophobation of Lignite . .
.
.
.
.
.
.
.
.
.
.
.
.
.
403
403
406
406
406
408
409
410
410
412
414
415
417
5
Anaerobic and other Approaches to Convert Coal . . . . . . . . . . . . . . . . .
417
6
Outlook and Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
418
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
394
14 Microbial Degradation and Modification of Coal
7
Patents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
419
8
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
421
ABCDE-system
ABTS
DMF
ESR
GC-MS
GPC
3-HAA
HRP
LiP
MnP
MW
NADPH
NMR
PAH
pI
SBP
SDS
THF
microbial effector system consisting of alkaline substances, oxidative)
biocatalysts, chelators, detergents and esterases
2,2'-azinobis(3-ethylbenzthiazoline-6-sulphonate)
N,N-dimethylformamide
electron spin resonance
gas chromatography-mass spectrometry
gel permeation chromatography
3-hydroxyanthranilic acid
horseradish peroxidase
lignin peroxidase
manganese peroxidase
molecular weight
reduced nicotinamide adenine dinucleotide
nuclear magnetic resonance
polycyclic aromatic hydrocarbon
isoelectric point
soy bean peroxidase
sodium dodecyl sulfate
tetrahydrofuran
1
Introduction
Relatively few microbiologists, and perhaps
even fewer geochemists and fuel scientists,
have seriously considered that microorganisms might be able to modify the physicochemical structure of coal. There are two
main reasons for this. Microbiologists usually prefer simple sugars, organic acids and
the like as substrates for microbial activity,
and they try to avoid the use of too complex
substrates such as coal. On the other hand,
the resistance of geochemists and fuel scientists stems from an equally specialized
knowledge of the physico-chemical processes
and extreme conditions (temperature, pressure) which were involved in the genesis of
coal (coalification) and are also required for
the industrial coal conversion.
Nevertheless, there are several reasons to
investigate microbial activities towards coal.
Besides crude oil, coal is the most important
fossil fuel and thus, a basic energy as well as a
raw material source. The worldwide coal
deposits are considerably larger than those of
oil and therefore, coal could become again the
main resource of raw materials (feedstock)
for the chemical industry (see Chapter 19). In
this context, new conversion technologies for
coal are urgently needed to reduce environmental damages caused by the classic carbochemistry processes. One approach, which is
still at the fundamental research stage, might
be the utilization of biotechnological processes to convert coal into value-added products which can be used for further biotechnological or chemical synthesis. Another reason
to study the microbial conversion of coal is
attributed to environmental problems of
1 Introduction
former coal mining areas (e.g. the huge opencast mines in East Germany). When coal
mining finishes in a region, the landscape
that remains is usually devastated, with
infertile soil that has to be recultivated. The
degradative activities of microbes towards the
residual coal may be of significance if soil
fertility is to be improved and intact soils recreated by mobilizing the humic substances
in coal (see Chapter 9). In addition to these
economic and ecological considerations,
there is also a general interest on the part of
coal chemists with regard to the degradative
activities of microorganisms, which might be
helpful in elucidation of the coal structure by
gradually decomposing the coal components.
Although coals have been of major economic importance for more than 100 years,
the structure of coals ± low rank coals and
even hard coal ± is still under discussion ( Van
Krevelen, 1993; see also Chapters 14 and 15 in
this volume). Research into this field has
been inadequate, especially in the case of
brown coal ( ˆ lignite, low-rank coal; Stefanova et al., 1993), and only a few structural
models of coals are to be found in the
literature. The so-called `coal structure'
changes with the rank of the coal. Figure 1
provides a survey of different coal structure
models (modified from Schumacher, 1997).
Schulten and Schnitzer (1993) have published the first detailed structural model of a
humic acid from brown coal; in addition,
several different structures of the so-called
fulvic acids, which are also present in soils,
are discussed by Stevenson (1994). Since
research into coal microbiology focuses on
brown coal (lignite), we must face the fact that
lignite has an even more complex structure
than hard coal because it consists of several
distinct classes of constituents. These include
the mainly hydrophobic bitumen, the alkalisoluble humic and fulvic acids, and the
insoluble residue designated as matrix or
humine. In spite of the great economic
importance of brown coal, there have been
few investigations of the humic substances in
lignite, compared for example with research
into humic acids from water or soil (Stefanova
et al., 1993; Stevenson, 1994). Even though
some Australian, American and East European lignites have been characterized (Wildenhain, 1969; Hatcher et al., 1981, 1988;
Verheyen and Johns, 1981; Verheyen et al.,
1982, 1985; Chaffee et al., 1983; Künstner
et al., 1986; Hatcher, 1990), these results
cannot be transferred to lignites of other
origins, because the coal diagenesis differs
strongly with each coal deposit depending on
plant input, coal generating conditions, etc.
(Stach et al., 1982; van Krevelen, 1993). Until
recently, there was neither a reliable measurement of the molecular weight (MW) distribution of humic acids from lignite (Henning
et al., 1997) nor any publication about this
important basic characteristic. Only for microbiologists has the MW of lignite compounds become a research topic. Linehan
et al. (1991) described how the apparent MWs
were influenced by the method used. For the
same humic acid solution, they determined
average MWs between 340,340 and
800,000 Daltons (Da), depending on the
method. Only Hofrichter and Fritsche
(1996) have established a rapid method to
compare the MWs of humic acids by gel
permeation chromatography (GPC ) using
simple HPLC equipment.
In the first instance, coal appears to be
resistant to microbial degradation; under
normal circumstances, no extensive microbial growth can be seen on coal pieces
collected in open-cast or underground mines.
However, when the conditions for microbes
are improved (humidity, minerals, additional
carbon source), growth of both fungi and
bacteria on coal can be achieved. This chapter
aims to demonstrate that, even though coal is
indeed comparatively resistant to microbial
attack, there are microorganisms which are
395
Fig. 1
Typical structure models for coals of different rank [modified after Schumacher (1997), using Wender (1976), Mallya and Zingaro (1984) and Pätz et al. (1989)]
396
14 Microbial Degradation and Modification of Coal
2 Historical Outline
capable of modifying the coal structure by
different mechanisms. With respect to the
origin of coal from fossil lignocelluloses, the
activities of ligninolytic fungi and their
extracellular enzymes are considered in particular. The chapter focuses on the microbial
conversion of brown coal and the two main
transformation principles: solubilization
and depolymerization, since most studies
were carried out with this type of coal. In
addition, a summary of the microbial modification of hard coal is also provided.
2
Historical Outline
The idea that coals might be acted on by
microbes or utilized as growth substrate is not
new. For example, as early as 1908 Potter
reported that bacteria acted as biocatalytic
agents in the oxidation of amorphous brown
coal (Potter, 1908). Two years later, Galle
(1910) first isolated pure cultures of bacteria
grown on brown coal samples, whilst some
years later Fischer and Fuchs (1927a,b)
published two articles about the growth of
fungi on various types of coal. They had
observed by chance that white and greenish
mycelia had arisen readily on untreated,
moist coal samples stored in the laboratory.
This observation prompted them to study this
phenomenon in greater detail, and resulted
in the discovery of various filamentous microfungi (molds) that were able to colonize
brown coal; later, even coal-briquettes, coke
and hard coal were found to be growth
substrates for these fungi. Microscopic studies indicated that the microfungi responsible
belonged to the deuteromycetes (Penicillium
spp., Aspergillus spp.) as well as to the yeastlike fungi (Torula spp.). In addition, Fischer
and Fuchs referred to their correspondence
with a Dutch colleague (D.W. Kreulen,
Amsterdam) who observed the development
of the zygomycete Mucor mucedo on bitumenrich brown coal (`Fettschlammkohle') and
speculated that the fungus somehow oxidized the coal to assimilate and solubilize part
of the humic substances during this process
(Fischer and Fuchs, 1927b). First detailed
investigations into the microflora of natural
coal deposits were carried out by Lieske and
Hofmann (1928), and resulted in the description of a wide range of microorganisms in
mining areas. The same author first considered the idea of (bio)technological applications of coal and coal-colonizing microbes, for
example, the use of coal as fertilizer in
agriculture (Lieske, 1929, 1931). All these
investigations came to a preliminary end in
1932, marked by the publication of the
summarizing article `Biologie und Kohleforschung' (Biology and coal research; Fischer,
1932).
Unfortunately, no further interest was
shown in this field until 1981, when RenØ
Fakoussa (working at the institute in Mülheim where Fischer and Fuchs had worked
some 50 years earlier) demonstrated that
certain bacteria were able to utilize organic
extracts of hard coal as sole carbon source and
to solubilize part of the native coal, resulting
in the formation of colored liquids (Fakoussa,
1981). The same author also recognized the
biotechnological potential of coal-modifying
microorganisms, and published the first
detailed considerations into this topic in
1983 (Fakoussa and Trüper, 1983). At almost
the same time, in the USA Cohen and
Gabriele (1982) found that wood-decaying
basidiomycetes (white-rot and brown-rot
fungi) could form black droplets from leonardite particles (a special kind of highly
oxidized brown coal). Both findings were the
starting point for a number of intensive
research programs that were conducted in the
US, and later also in Germany, Spain and
Australia, to find suitable microorganisms
for the biological conversion of coal (prefer-
397
398
14 Microbial Degradation and Modification of Coal
entially brown coal) into useful products such
as chemicals and fuels. The progress in this
field during the past 20 years is summarized
in Table 1 (Fakoussa and Hofrichter, 1999).
Another aspect of coal microbiology that is
also of biotechnological significance concerns the bacterial desulfurization of coal,
and this topic is detailed in Chapter 15.
Tab. 1
Year
3
Modification of Hard Coal
3.1
Bacteria
Certain bacteria can grow by utilizing hard
coal as sole carbon source. In a screening of
about 3100 cultivation experiments, micro-
Advances in coal microbiology and biotechnology during the past two decades
Achievement
1981 Effects on hard coals by bacteria (Pseudomonas
spp.), simultaneous biotenside secretion
1982 Solubilization of lignite to droplets on agar plates
by action of wood-decaying basidiomycetous
fungi
1986 Acceleration of solubilization by pretreatment of
coal (fungi ‡ bacteria)
1987 First solubilization mechanism elucidated: production of alkaline substances (fungi ‡ bacteria)
1988 Second solubilization mechanism elucidated:
production of chelating agents (fungi)
1989 First product on market: solubilized lignite as
fertilizer
1991 Evidence that chelators alone are not responsible
for all effects
1994 Decolorization and reduction of MW of soluble
lignite derived humic acids proves catalytic, i.e.,
enzymatic attack (basidiomycetous fungi)
1991 Improved analysis by 13C-solid state NMR, MW
determination, e.g., ultrafiltration, gel permeation chromatography
1996 In-vitro systems shown preferentially to polymerize humic acids without regulation of the
fungus (laccase)
1997 In-vitro systems based on fungal Mn peroxidase
was shown to depolymerize humic acids and to
attack coal particles, including the matrix
1997 Involvement of unspecific esterases in the solubilization of coal (deuteromycetous fungi)
1997 First fine chemical produced successfully from
heterogeneous humic acid mixtures by bacterial
pure cultures: polyhydroxyalkanoates (PHA,
`Bioplastic')
Reference(s)
Fakoussa (1981)
Cohen and Gabriele (1982)
Scott et al. (1986), Grethlein (1990) and others
Quigley et al. (1987, 1988a, 1989a)
Cohen et al. (1990), Quigley et al. (1988b, 1989b)
Arctech, Virginia (USA )
Fakoussa and Willmann (1991), Fakoussa (1994)
Willmann (1994), Ralph and Catcheside (1994),
Hofrichter and Fritsche (1996, 1997a)
Willmann and Fakoussa (1991), Polman and
Quigley (1991), Ralph and Catcheside (1996),
Hofrichter and Fritsche (1996, 1997a), Henning
et al. (1997) and others
Willmann (1994), Frost (1996) and others
Hofrichter and Fritsche (1997b), Hofrichter et al.
(1999)
Hölker et al (1997, 1999)
Steinbüchel and Füchtenbusch (1997), Füchtenbusch and Steinbüchel (1999)
3 Modification of Hard Coal
organisms from suitable locations were enriched using five types of hard coal, each with
different volatile matter. The coals were not
chemically pretreated, but ground into pieces
of an average size of 2.5 mm in order to
increase the surface area. Forest fire regions
(0.5 to 20 years old) were used as screening
locations, since the charcoal structure is
somehow related to that of hard coal. Growth
was observed in 0.2% of all cases, which is an
exceptionally low rate.
Initially, pleomorphic bacteria were isolated, probably belonging to the group of
mycobacteria or nocardia. These pleomorphic bacteria had extremely hydrophobic cell
walls. Thus, the cell aggregations could only
float on the surface of the medium liquid and
could only be dispersed by using detergents
or oily substances. The poor growth rates, the
distance to the (sedimented) coal particles
and investigation of the fate of substances in
the culture supernatant indicated that these
bacteria only grow on water-soluble coal
substances, which are released through surface pores (Fakoussa, 1981, 1990). Such
strains can also be observed on old solutions
of humic acids obtained from lignite.
During this screening, a more interesting
bacterium was enriched and subsequently
identified as Pseudomonas fluorescens. This
strain showed some remarkable properties:
. The bacterium released a very effective
surfactant into the medium, which lowered the surface tension to about
25.5 mN m±2. This value is even lower than
that of a saturated solution of sodium
dodecyl sulfate (SDS ) (for comparison, the
surface tension of water is 72.6 mN m±2,
while that of acetone is 23.5 mN m±2).
. The coal particles were altered during the
cultivation in several characteristics, such
as color, wettability, and extractability.
. After some time the culture supernatant
turned brown in color, indicating that coal
substances were being released. When
isolated, these substances had molecular
weights of 50,000 ± 100,000 Da. Infrared
spectra and esterification experiments
showed a high content of carboxylic and
hydroxyl groups, which is consistent with
an oxidative attack on the coal particles
(Fakoussa, 1988).
It can be assumed that any secreted enzyme
converted the coal substances to a more
hydrophilic status, i.e., they become more
water-soluble, and were then taken up by the
bacterium. Control experiments with different surfactants indicated that these molecules adhere to the hydrophobic surface of the
hard coal particles, thus clogging the pores.
Consequently, the use of pure water will
result in a more extensive extraction of hard
coal than would a solution of surfactant.
To summarize, the results of these studies
suggested that hard coals with a high content
of volatile organic matter are more easily
attacked by bacteria, though the bacterial
attack on the coal particles is not efficient
enough for application to commercial coal
conversion processes.
3.2
Fungi
A number of comprehensive screening programs have been carried out to identify fungal
strains capable of modifying the physicochemical structure of hard coal or derived
products (e.g., asphaltenes, organic hard coal
extracts). In addition to testing a large
number of bacteria (Section 3.1), Fakoussa
(1981, 1988, 1990) also investigated whether
yeasts and filamentous fungi from suitable
locations (e.g., former forest fire sites, hard
coal samples) could utilize hard coal as sole
source of carbon and energy and release
brown-colored substances from powdered
hard coal. As a result of these studies, three
filamentous fungi and two yeasts were en-
399
400
14 Microbial Degradation and Modification of Coal
riched that would grow (albeit at an exceptionally slow rate) with powdered hard coal as
sole carbon source. Attachment of coal
particles (average size 2.5 mm) to the fungal
cell wall was observed both for filamentous
fungi and yeasts, and attributed to a hydrophobic interaction (Figure 2A,B ). Interestingly, when the fungal hyphae had just
germinated, there was no affinity to the coal;
however, hyphae in a more mature stage
showed many coal particles adhering to the
cell wall, and older hyphae even became
coated with a layer of coal. There was no
embedding of hard coal particles into mucilaginous substances.
Similar results have been reported by
Stewart et al. (1990), who investigated the
colonization of differently pretreated bituminous coals placed on agar plates by a number
of filamentous fungi. A Penicillium sp. strain
and a Cunninghamella sp. strain were found to
be the most active fungi. By using scanning
electron microscopy, an extensive surface
colonization (including conidia formation
and a tight attachment of fungal hyphae to the
coal particles) was observed. Interestingly,
only air-oxidized coal samples, which had
previously been exposed to 150 8C for 7 days,
were overgrown by the molds, whereas untreated particles showed only little evidence of
colonization. The gravimetric quantitative
analysis indicated that up to 10% of the preoxidized coal was converted to water-soluble
products.
An extensive screening involving more
than 750 strains of filamentous fungi was
carried out to select strains which modify an
untreated German hard coal (Bublitz et al.,
1994; Hofrichter et al., 1997a). Among the
strains tested were representatives of different taxonomic groups of filamentous fungi:
zygomycetes, ascomycetes and deuteromycetes, as well as basidiomycetes. The hard coal
particles were exposed for 6 weeks to agar
(A ) Germinating conidia-spore of a
filamentous microfungus (mold). Hard coal
particles start to attach at the tip of the young
hypha. (B ) Hypha of a filamentous microfungus
that grows in a liquid medium in the presence of
hard coal particles. Attachment of the coal
particles to the fungal hypha is clearly visible
(Fakoussa 1981, 1988)
Fig. 2
3 Modification of Hard Coal
plates which had been overgrown by the
fungalmycelia.Only sixof the 750strains tested
acted noticeably on the hard coal. Tight connections were developed between the fungal
hyphae or rhizomorphs and the coal particles
(Figures 3 and 4), which in turn were split
into smaller pieces. Moreover, the wettability
of the particles increased, this becoming
visible by the attachment of fungal guttation
droplets on the hydrophobic hard coal surface, suggesting either the secretion of fungal
surfactants or oxidation of the surface.
Interestingly, all hard coal `eroding fungi'
belonged to the litter-decomposing and wooddecaying basidiomycetes. The most active
fungus, Coprinus sclerotigenis, was studied in
more detail with respect to the formation of
low-molecular mass products from powdered
hard coal. 2-Hydroxybiphenyl, alkylated benzenes, polycyclic aromatic hydrocarbons
(PAHs) and branched alkanes were extracted
with tetrahydrofuran (THF ) from coal samples treated with this fungus (Figure 5). It
was assumed that the nonoxidized compounds (alkylated benzenes and alkanes,
PAHs) might be part of the mobile phase of
hard coal, and were liberated from the micropores through mechanic effects of the fungal
hyphae (biodeterioration of coal). In contrast,
the formation of 2-hydroxybiphenyl was
probably brought about by an enzymatic,
bond-cleaving process, because the same
compound was also detected after the fungal
treatment of hard coal-derived asphaltene
powder lacking micropores and a mobile
phase. However, it remained unclear which
enzyme was responsible for this, since the
most promising candidates ± ligninolytic
peroxidases and phenol oxidases ± seemed
to be lacking in Coprinus sclerotigenis.
Formation of rhizomorphs by Coprinus
sclerotigenis C142-1 which are tightly attached to
the surface of a hard coal piece (1 3 mm)
(Hofrichter et al., 1997a)
Fig. 3
Mycelium of Coprinus sclerotigenis C142-1
on the surface of a hard coal particle (scanning
electron micrograph; original magnification,
”1000) (Hofrichter et al., 1997a)
Fig. 4
401
402
14 Microbial Degradation and Modification of Coal
Gas chromatogram of a THF extract of powdered German hard coal ( < 200 mm) treated with the
basidiomycetous fungus Coprinus sclerotigenis (modified after Hofrichter et al., 1997a). The liberated
compounds were putatively identified by mass spectroscopy (GC-MS analysis). Except for 2-hydroxybiphenyl
(12), the substances most likely originate from the coal micropores (according to Hofrichter et al., 1997a). (1),
1-methyl-2-ethylbenzene; (2), 1,3-dimethylbenzene; (3), 1,2-dimethylbenzene; (4), 1,2-diethylbenzene; (5), 1,4diethylbenzene; (6), 1,3,4,5-tetramethylbenzene; (7), 1,3,4, 5-tetramethylbenzene; (8), naphthalene; (9), 3,4,5trimethylbenzyl alcohol; (10), biphenyl; (11), 1,2-dimethylnaphthalene; (12), 2-hydroxybiphenyl; (13), 2,6,10trimethylundecane; (14), 1,7-di(tert-butyl)-4-methylheptane; (15), tetramethyl-biphenyl; (16), fluorene; (17),
pyrene
Fig. 5
Powdered Polish hard coal and its chloroform extracts were exposed to the basidiomycetous fungus Piptoporus betulinus belonging
to the wood-decaying white-rot fungi (Osipowicz et al., 1994). The agitated cultures used
contained maltose and peptone as actual
growth substrates, while the coal served as cosubstrate. Under these conditions, the fungus acidified the medium drastically during
its growth (from pH 6.0 to 1.0!), which was
associated with the production of organic
acids. Piptoporus betulinus acted on both the
native coal and its organic extract, this being
demonstrated on the basis of spectroscopic
data. According to the latter, the biotransformation caused dearomatization and depolymerization of the coal substrates. Thus, a
decrease in aromatic absorption in the UV
spectra and a reduction in intensity of the
broad signal of aromatic protons, together
with an increase in alkene protons in the 1H
NMR spectra were observed. On the basis of
the IR spectra, it was concluded that the
biotransformation process was also accompanied by decarboxylation reactions. Furthermore, high-performance size-exclusion chromatography (HPSEC ) using chloroform as
the solvent revealed a substantial shift in
molecular mass distribution towards lower
values. Attempts to explain the alterations of
hard coal caused by the fungus were not
made. Another white-rot fungus, Panus tigrinus, was found to convert an asphaltene ±
obtained through the hydrogenation of German hard coal ± in a similar manner. The
asphaltene, representing a complex mixture
of aromatic compounds with molecular
masses between 0.25 and 0.6 kDa was exposed to solid-state cultures of the fungus
growing on wood shavings (Hofrichter et al.,
1997a). As the result of a 6-week incubation,
the predominant molecular masses of the
4 Bioconversion of Brown Coal
asphaltene decreased from 0.4 to 0.3 kDa, and
a new low-molecular mass fraction (0.1 kDa)
consisting probably of monoaromatic compounds was formed. It was concluded that the
ligninolytic enzyme system of Panus tigrinus
attacked the asphaltene unspecifically while
it degraded the lignin in the wood shavings.
Because manganese (Mn) peroxidase is the
predominant ligninolytic enzyme of this
fungus (Maltseva et al., 1991) and one of
the most powerful peroxidases (see Section
4.4.2), it was proposed that this enzyme is
involved in the asphaltene depolymerization
by this fungus.
The most effective modification of hard
coal observed so far has been reported for
Spanish bituminous and sub-bituminous
coals which were converted under co-metabolic conditions (Sabouraud maltose broth
was used as growth medium) to a tar-like
mass by the action of a deuteromycetous
fungus isolated from native hard coal samples (Monistrol and Laborda, 1994). Unfortunately, the strain lost its hard coal-solubilizing
ability over the following years, probably due
to spontaneous degeneration processes. The
authors, however, succeeded in isolating new
molds (Trichoderma sp. M2, Penicillium sp.
M4) with hard coal-modifying and -solubilizing activities, although the formation of tarlike products was not observed again (Laborda et al., 1997, 1999). The authors detected
oxidative and hydrolytic enzyme activities in
fungal supernatants which had been grown
in the presence of hard coal, but the actual role
of these enzymes (phenoloxidases, peroxidases, esterases) in hard coal transformation
is still not clear.
In summary, hard coal is much more
resistant towards fungal attack than lower
rank coals, this being a result of its higher
hydrophobicity, the higher proportion of
condensed aromatic rings, and the lower
oxygen content (Fakoussa and Trüper, 1983).
Nevertheless, there are some fungal organ-
isms ± both deuteromycetes and basidiomycetes ± which are capable of modifying the
physico-chemical structure of hard coal and
even liberating low-molecular mass compounds. The biochemical processes, however, underlying these phenomena are only
poorly understood. It is most likely that a
string of factors, e.g., mechanical effects of
fungal hyphae, surface-active substances
(surfactants), and extracellular enzymes (hydrolytic and/or oxidative) are responsible for
the structural changes.
4
Bioconversion of Brown Coal
4.1
Mechanisms: Solubilization,
Depolymerization, Utilization
Distinction must be made between two
different principles of the structural modification of brown coal ( ˆ low-rank coal and
lignite), namely solubilization and depolymerization (Catcheside and Ralph, 1997;
Hofrichter et al., 1997b; Fritsche et al.,
1999; Klein et al., 1999) (Figure 6). The
solubilization of brown coal, which leads to
the formation of black liquids (Figure 7), is a
mainly nonenzymatic desolving process that
occurs preferentially at higher pH values (pH
7 ± 10) and is due to the microbial formation
of alkaline substances and/or chelating
agents and surfactants. In addition, recent
studies have provided a novel indication that
certain hydrolytic enzymes may enforce the
solubilization process (for details, see below).
Coal solubilization does not result in a
substantial decrease in the molecular mass
of coal humic substances; on the contrary, it
may even be accompanied with polymerizing
reactions and an increase in the predominant
molecular mass (Hofrichter et al., 1997b).
403
404
14 Microbial Degradation and Modification of Coal
Fig. 6
1998)
The two main structural modifications of brown coal by microorganisms (modified after Fritsche et al.,
Solubilization of brown coal by a filamentous microfungus (Alternaria sp.). The black
droplet was formed from a brown coal piece (1
3 mm) placed on a pre-grown agar plate
Fig. 7
The term `liquefaction', although often used
in earlier publications, should be avoided in
connection with microbial activities towards
coal, because it is already occupied by the
`pure' chemical processes of coal conversion
(see Chapter 17).
The depolymerization of brown coal or
derived macromolecules (coal humic acids)
is an enzymatic process that occurs at lower
pH values (pH 3 ± 6) and results in the
cleavage of bonds inside the coal molecule,
leading to the formation of yellowish, fulvic
acid-like substances (`bleaching') with lower
molecular masses. As an example, Figure 8
shows the decolorization of an agar plate
containing a high-molecular mass humic
acid from coal as a test substrate.
In general, coal solubilization ± and probably also depolymerization of coal ± are
facilitated when oxidatively pretreated or
naturally highly oxidized coals (e.g., weathered lignite, leonardite) are exposed to microbes (Scott et al., 1986; Ward et al., 1988;
Hofrichter et al., 1997b). Artificial oxidative
pretreatment can be performed in the laboratory using nitric acid (HNO3), hydrogen
peroxide (H2O2), ozone (O3), or radiation
(Strandberg and Lewis, 1987; Kitamura et al.,
1993; Achi, 1993; Gazsó, 1996; Henning
et al., 1997; Hofrichter et al., 1997b).
In addition to the structural modifications
mentioned above, a number of microorganisms (bacteria, molds, yeasts) is able to grow
on brown coal by utilizing parts of the mobile
4 Bioconversion of Brown Coal
Depolymerization of high-molecular
weight coal substances by a ligninolytic fungus
(Nematoloma frowardii b19). The agar plates
contained coal humic acids extracted with NaOH
from Rhenish brown coal (modified after Hofrichter and Fritsche, 1996). Right: control without fungus; center: 10-day-old culture; left: 20day-old culture
Fig. 8
phase (Hodek, 1994), which comprises a
complex mixture of low-molecular mass
aromatics and wax-like aliphatics, as sole
carbon source (Kucher and Turovskii, 1977;
Ward, 1985; Ralph and Catcheside, 1993;
Willmann, 1994; see also Section 2). At
present, no information is available about
the exact nature of these organic compounds,
but studies using low-molecular mass models have indicated that they include substances such as phenols, benzoic acids, biphenyls
and biphenylethers, as well as various cycloalkanes, n-alkanes and n-alkanols (Engesser
et al., 1994; Ralph and Catcheside, 1994a;
Schumacher and Fakoussa, 1999; Fritsche
and Hofrichter, 2000). Residual cellulose and
hemicelluloses that can be found in certain
brown coals (e.g., xylite ˆ coal that preserved
the wood structure) might be an additional
carbon source for microorganisms (Cohen
and Gabriele, 1982). Usually, the microorganisms grow slowly on coal particles, but the
growth is noticeably stimulated when natu-
rally weathered or chemically pre-oxidized
coals are used ( Ward, 1985) (Figure 9); the
oxidation most likely enhances the bioavailability of available compounds. Furthermore,
the addition of mineral solutions (N, P, S,
metal ions or mineral salts) stimulates the
microbial growth, indicating limitations of
essential elements in native coal. In some
cases, the utilization of brown coal might be
accompanied by its solubilization. Thus,
Hölker et al. (1999b) have reported that
14
CO2 was evolved from radioactively labeled
coal (14C-methoxylated German lignite) by a
coal-solubilizing fungus.
Fakoussa (1991) has proposed the so-called
ABC-system, that was later extended to the
ABCDE-system (Fakoussa and Hofrichter,
1999), to describe all possible mechanisms
which are involved in brown coal bioconversion:
. Alkaline substances
. Biocatalysts (oxidative enzymes)
Growth of a filamentous microfungus
(Penicillium sp.) on a piece of brown coal.
Almost the entire surface of the coal particle is
covered with conidia-forming mycelium. The
fungus utilizes part of the mobile phase of coal
as carbon source
Fig. 9
405
406
14 Microbial Degradation and Modification of Coal
The so-called ABCDE-mechanism of biological conversion of brown coal (modified after Fakoussa,
1991). The arrows indicate structures that can be attacked by different microbial agents. A: alkaline substances;
B: biocatalysts (oxidative enzymes); C: chelators; D: detergents; E: esterases
Fig. 10
. Chelators
. Detergents (surfactants, biotensides)
. Esterases
These mechanisms are illustrated in Figure 10, using the example of a brown coal
model structure.
4.2
Organisms
While the process of brown coal solubilization is typical for microfungi (molds, yeasts)
and bacteria (actinomycetes, pseudomonads), the depolymerization of coal is evidently limited to the ligninolytic basidiomycetes (wood-decaying and litter-decomposing
fungi, ˆ white-rot fungi; see Figure 4). Nevertheless, there is some overlap of these
abilities. Thus, some white-rot fungi (e.g.,
Trametes (Coriolus) versicolor, Phanerochaete
chrysosporium), which are active decolorizers
of coal humic substances (Ralph and Catcheside, 1994b; Frost, 1996), can also solubilize
coal, but under other conditions and/or by
using other mechanisms (Cohen and Gabriele, 1982; Torzilli and Isbister, 1994).
Microorganisms utilizing part of coal as
growth substrate can be found among many
groups of aerobic microorganisms, and it
should also be mentioned that brown coalderived products are used as fertilizers and
soil conditioners in agriculture (Yang et al.,
1985; Fortun et al., 1986; Gyori, 1986; Aleksandrov et al., 1988). Table 2 provides a
concise overview of microorganisms which
either solubilize or depolymerize brown coal,
or utilize it as a growth substrate.
4.3
Solubilization of Brown Coal
4.3.1
Alkaline Solubilization
The alkaline solubilization of brown coal was
the first discovered mechanism of microbial
coal conversion (Quigley et al., 1987, 1988a),
and was later confirmed by several authors
(Maka et al., 1989; Runnion and Combie,
1990; Torzilli and Isbister, 1994). This phenomenon is attributed to the high content of
carboxylic groups in the coal humic acids,
which can be deprotonated at higher pH (>8),
4 Bioconversion of Brown Coal
Selected microorganisms that have solubilizing or depolymerizing activities towards brown coal, or
utilize it as a growth substrate
Tab. 2
Organisms
Bacteria
Actinomycetes
Streptomyces spp.
Streptomyces setonii
Eubacteria
Bacillus sp.
Bacillus lichiniformis
Pseudomonas cepacia
Basidiomycetous fungi
Wood-decaying white-rot fungi
Clitocybula dusenni
Nematoloma frowardii
Phanerochaete chrysosporium
Trametes (Coriolus) versicolor
Litter-decomposing fungi
Agrocybe praecox
Stropharia rugosoannulata
Isolates RBS 1k1, RBS 1b1
Wood-decaying brown-rot fungi
Poria monticola
Effects on brown coal and derived
products
References
Solubilization
Solubilization
Gupta et al. (1988)
Strandberg and Lewis (1987)
Solubilization
Solubilization
Solubilization (depolymerization)
Quigley et al. (1989a)
Polman et al. (1994a)
Crawford and Gupta (1991)
Depolymerization
Depolymerization
Solubilization, depolymerization
Solubilization, utilization, depolymerization
Ziegenhagen and Hofrichter (1998)
Hofrichter and Fritsche (1996)
Torzilli and Isbister (1994), Ralph
and Catcheside (1994)
Cohen and Gabriele (1982), Frost
(1996), Fakoussa and Frost (1999)
Steffen et al. (1999)
Hofrichter and Fritsche (1996)
Willmann (1994), Willmann and
Fakoussa (1997a,b)
Depolymerization
Depolymerization
Depolymerization
Solubilization, utilization
Cohen and Gabriele (1982)
Deuteromycetous and ascomycetous fungi
Solubilization
Alternaria sp.
Utilization
Aspergillus terreus
Solubilization
Fusarium oxysporum
Solubilization
Neurospora crassa
Solubilization, utilization
Paecilomyces spp.
Solubilization, utilization
Penicillium citrinum
Solubilization
Trichoderma atroviride
Hofrichter et al. (1997b)
Ward (1985)
Hölker et al. (1995)
Patel et al. (1996)
Ward (1985), Scott et al. (1986)
Polman et al. (1994b)
Hölker et al. (1997b, 1999a)
Yeast-like fungi
Candida bombicola
Candida sp.
Candida tropicalis
Solubilization
Utilization
Utilization
Breckenridge and Polman (1994)
Ward (1985)
Kucher and Turovskii (1977)
Zygomycetous fungi
Cunninghamella sp.
Solubilization
Mucor lausannesis
Utilization
Ward and Sanders (1989), Ward
(1993)
Ward (1985)
1
Enriched and isolated from brown coal from an open-cast mining region.
407