J. Microbiol. Biotechnol. (2011), 21(11), 1101–1108
http://dx.doi.org/10.4014/jmb.1106.06005
First published online 10 September 2011
Evaluation of the Coal-Degrading Ability of Rhizobium and Chelatococcus
Strains Isolated from the Formation Water of an Indian Coal Bed
Singh, Durgesh Narain and Anil Kumar Tripathi*
School of Biotechnology, Banaras Hindu University, Varanasi-221005, Uttar Pradesh, India
Received: June 3, 2011 / Revised: July 5, 2011 / Accepted: July 19, 2011
The rise in global energy demand has prompted researches
on developing strategies for transforming coal into a
cleaner fuel. This requires isolation of microbes with the
capability to degrade complex coal into simpler substrates
to support methanogenesis in the coal beds. In this study,
aerobic bacteria were isolated from an Indian coal bed
that can solubilize and utilize coal as the sole source of
carbon. The six bacterial isolates capable of growing on
coal agar medium were identified on the basis of their 16S
rRNA gene sequences, which clustered into two groups;
Group I isolates belonged to the genus Rhizobium, whereas
Group II isolates were identified as Chelatococcus species.
Out of the 4 methods of whole genome fingerprinting
(ERIC-PCR, REP-PCR, BOX-PCR, and RAPD), REPPCR showed maximum differentiation among strains
within each group. Only Chelatococcus strains showed the
ability to solubilize and utilize coal as the sole source of
carbon. On the basis of 16S rRNA gene sequence and the
ability to utilize different carbon sources, the Chelatococcus
strains showed maximum similarity to C. daeguensis. This
is the first report showing occurrence of Rhizobium and
Chelatococcus strains in an Indian coal bed, and the
ability of Chelatococcus isolates to solubilize and utilize
coal as a sole source of carbon for their growth.
Keywords: Coal bed, coal solubilization, rep-PCR, 16S rRNA
gene sequence, Chelatococcus
Coal is one of Earth’s most abundant fossil fuels, which
contributes nearly 30% of global energy consumption [19].
The burning of coal creates a variety of problems to the
environment including the release of atmosphere-polluting
sulfur and nitrogen oxides, and leftover ash containing
toxic metals, but it cannot be replaced or minimized by any
other more efficient fuel in the near future. Although direct
*Corresponding author
Phone: +91 9451525811; Fax: +91-542-2368693;
E-mail: [email protected]
thermochemical liquefaction can convert coal into a cleaner
liquid fuel, it is an energy intensive process requiring high
temperatures and pressures, and, hence, is not cost effective
[26]. In order to address the environmental concerns
associated with the use of coal, alternative methods of
coal-processing are being explored that would extend
utilization of coal-derived biofuels as an alternative source
of energy. It has been predicted that a microbial, enzymatic,
or enzyme-mimetic technology would have greater advantage
compared with the physical and chemical technologies for
coal conversion.
Coal is a solid rock, which is often dominated by
recalcitrant, partially aromatic, and largely lignin-derived
macromolecules that tend to be relatively resistant to
microbial degradation. It has a highly heterogeneous
structure; lignite being more complex than the hard coal.
Since lignin was the main parent material in the formation
of lignite, typical structures of the original lignin are preserved
in lignite, which is also designated as demethylated and
dehydrated lignin [9, 17, 18]. The lignite consists of several
distinct compound classes: mainly hydrophobic bitumen,
the alkali-soluble humic and fulvic acids, and insoluble
residue known as the matrix or humine. The aromatic
structures of coal derived from cellulose and lignin are
often interlinked by oxygen bridges and contain numerous
oxygen-containing moieties (e.g., carboxyl, hydroxyl, or
ketone functional groups). These oxygen-containing functional
groups can be targeted by fermentative microbes, providing
important intermediates of degradation, such as succinate,
propionate, acetate, CO2, and H2, which can be utilized by
methanogens to produce methane. The rate-limiting step
of coal biodegradation is the initial fragmentation of a
macromolecular, polycyclic, lignin-derived aromatic network
of coal.
Lignin can be degraded by some fungi and bacteria
that produce unspecific extracellular enzymes [8, 14]. Up
to 40% of the weight of some coals can be dissolved using
extracted microbial enzymes [32]. There are several
reports on the ability of filamentous fungi to solubilize
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Singh and Tripathi
solid particles of low-rank hard coal into black liquid
droplets [5, 11, 14, 21], but there are very few reports on
the ability of bacteria to degrade or utilize coal as the sole
source of carbon and energy [7, 11-13, 20]. In recent
years, bacterial communities present in the coalbeds, which
can convert coal into the substrates that can be utilized by
methanogens, have been described with the help of
cultivation-independent methods [23, 28].
Coal beds contain water (known as formation water),
which facilitates coal degradation by bacteria at the coalwater interface. Thus, the water extracted from the coal
beds might harbour unique microbial communities possessing
the ability to solubilize and degrade coal and other
polyaromatic hydrocarbons. In the present study, bacteria
were isolated from the formation water collected from a
coal bed located in eastern India. The isolates were identified
using molecular tools of taxonomy and characterized for
their ability to solubilize and utilize coal as a sole source of
carbon and energy. This study is the first attempt from
India towards the isolation and characterization of bacteria
from a coal bed.
5 min at 72oC. The amplified product was analyzed in 0.8% agarose
gel at 5 V/cm for 4 h and was visualized under UV light with an
Alpha imager (Alpha Innotech Corporation, UK).
MATERIALS AND METHODS
Genomic Fingerprinting
rep–PCR. Primers corresponding to the conserved motifs in bacterial
repetitive elements (REP, ERIC, and BOX) were used for PCR
amplification to generate genomic fingerprints to differentiate the
isolates further at the species, subspecies, or strain levels. All the six
isolates were subjected to genomic fingerprinting using primer sets
corresponding to REP, ERIC, and BOX elements. The PCR protocols
with REP, ERIC, and BOX primers are collectively referred to as
rep-PCR. The primer pairs REP1R-I (5'-IIIICGICGICATCIGGC-3')
and REP2-I (5'-ICGICTTATCIGGCCTAC-3') (where I is inosine);
ERICIR (5'-ATGTAAGCTCCTGGGGATTCAC-3') and ERIC2 (5'AAGTAAGTGACTGGGGTGAGCG-3'); and BOXAlR (5'-CTACG
GCAAGGCGACGCTGACG-3') [25] primer sequence corresponding
to BOXA, a subunit of BOX element, were used to amplify REP-,
ERIC-, and BOX-like elements from the isolates DNA. Approximately
50 ng of DNA was used as template, and PCR amplification reactions
were performed with a Veriti 96-well Thermal cycler (Applied Biosystem,
USA) using the following cycles: an initial denaturation at 95oC for
7 min; 35 cycles of denaturation at 94oC for 1 min, annealing at 44,
57, or 60oC for 1 min with REP, ERIC, or BOX primer, respectively;
and extension at 72oC for 1 min with single extension cycle at 72oC
for 10 min before cooling at 4oC. A reaction containing all the
components of PCR except genomic DNA was included as negative
control in all the thermal cycles run. The 10 µl of PCR products
was loaded on agarose [1.5% (w/v)] gel and amplification products
were visualized under a UV light with Alpha imager.
RAPD. Amplification was performed in 25 µl with 20-50 ng of
genomic DNA as template, 4 µM of 10 mer primer (5'-GTGTGCCCCA3') [36], 2.5 U of Taq DNA polymerase (Genei, Banglore) with its
1× buffer, and 200 µM of each dNTPs. The reaction condition
included an initial denaturation of 7 min at 95oC, followed by 40
cycles of 1 min at 94oC, 1 min at 35oC, and 2 min at 72oC, with a final
extension of 5 min at 72oC. An 8-10 µl aliquot of each amplification
mixture was analyzed by agarose gel (2.5%) electrophoresis.
Collection of Sample
The sample was collected directly from the pipe extruding formation
water from a depth of 700 m from a coal bed methane (CBM)producing well located at Parbatpur in the Jharia coalfield of eastern
India. Samples were collected in autoclaved bottles, which were
brought to the laboratory under cooled condition. At the time of
collection, the temperature of the formation water was 42oC and pH
was 7.2; chloride and sulfate contents were 180 ppm and 22 ppm,
respectively.
Isolation of Coal-Solubilizing Bacteria
For isolation of coal-solubilizing bacteria, formation water was
serially diluted with sterile saline (0.89% NaCl) and spread onto coal
agar medium [22] containing (per liter) KH2PO4, 1.2 g; Na2HPO4·12H2O,
10.8 g; MgSO4·7H2O, 0.04 g; FeSO4·7H2O, 0.02 g; MnCl2·4H2O,
0.02 g; NH4C1, 3 g; lignite coal, 50 g; agar, 15 g; and cycloheximide,
0.5 g; pH 7.0. The lignite coal was oxidized with 10% hydrogen
peroxide before using it in the coal agar medium [22]. The plates
were incubated at 37oC for 3 days.
Nucleic Acid Extraction and 16S rRNA Gene Amplification
Total genomic DNA was extracted from the isolates using a Wizard
genomic DNA purification kit (Promega, Madison, WI, USA). The 16S
rRNA gene was amplified using universal bacterial primers 8f (5'AGA GTT TGA TYM TGG CTC AG- 3' and 1495r (5'-CTA CGG
CTA CCT TGT TAC G -3') [16]. A 50 µl reaction mixture included
20-50 ng of bacterial DNA as template, 200 µM of each primer,
and 1.0 unit of Taq DNA polymerase (Genei, Bangalore). The
reactions were performed on a Veriti 96-well Thermal cycler (Applied
Biosystem, USA), and the reaction condition included an initial
denaturation of 5 min at 95oC followed by 35 cycles of 1 min at
94oC, 1 min at 51oC, and 1 min at 72oC, with a final extension of
Sequencing of 16S rRNA Genes
The amplified 16S rRNA gene was purified with a Wizard SV gel
PCR purification kit (Promega, USA) and quantified by using a ND1000 Spectrophotometer (NanoDrop, Wilmington, DE, USA). Direct
sequencing was performed with three primers, 8f (5'-AGA GTT
TGA TYM TGG CTC AG- 3'), 1495r (5'-CTA CGG CTA CCT
TGT TAC G -3') [16], and 561f (5'-AATTACTGGGCGTAAAG -3')
[2] using the BigDye Terminator v3.1 Cycle sequencing kit (Applied
Biosystems) in an ABI Prism 310 automated DNA Sequencer
(Applied Biosystems, Rotkreuz, Switzerland).
Analysis of 16S rRNA Gene Sequence
The 16S rRNA gene sequence of the six isolates was edited using
Bioedit software version 3.1 to make a complete sequence. The almost
complete sequence (1,400 nt) was compared with the nucleotide
sequences present in the NCBI database using the standard nucleotide
BLAST search [1]. The programme ClustalW [35] was used to
align the 16S rRNA gene of the isolates with the similar sequences
retrieved from the NCBI database to construct a phylogenetic tree,
as described by Chowdhary et al. [4].
STUDY OF COAL-DEGRADING BACTERIA FROM COAL BED
1103
Carbon Source Utilization Ability of the Isolates
Bacterial inocula were prepared by growing the isolates on Biolog
universal growth agar (BUG; Biolog) medium for 24 h. The cultures
from the Biolog universal growth agar medium were collected using
sterile swabs and resuspended in 0.85% saline. The inoculum
density was adjusted with a Biolog turbidimeter within the limit of
GN MicroPlate turbidity standards supplied by the manufacturer. The
cell suspension was poured into a sterile disposable plastic reservoir,
and Biolog-GN MicroPlates (Biolog Inc., Hayward, CA, USA)
containing 95 different carbon sources were inoculated with 150 µl
of bacterial suspension, and the microplates were incubated for 24 h
at 37oC. Substrate utilization patterns were compared with Microlog
database release 4.01 software.
pellets were incubated at 37oC for 4 h with 1 ml of 1 N NaOH. [33].
After centrifugation, 200 µl of supernatant was used for estimation
of protein according to the Lowry method of protein estimation.
Evaluation of the Ability of Isolates to Utilize and Solubilize Coal
In order to check coal utilization and solubilization capabilities, isolates
were grown in mineral salt broth (MSB) supplemented with and
without 5% coal. The constituent of MSB were g/l KH2PO4, 1.2 g;
Na2HPO4·12H2O, 10.8 g; MgSO4·7H2O, 0.04 g; FeSO4·7H2O, 0.02 g;
MnCI·4H2O, 0.02 g; and NH4C1, 3.0 g. Flasks were inoculated with
equal number of overnight-grown log phase culture (same optical
density) and incubated at 37oC under shaking condition of 200 rpm.
The culture broth from each flask was taken at a regular interval and
centrifuged to pellet down cells and the supernatant was used for
spectrophotometric monitoring of coal solubilization at 450 nm [15,
37]. To determine the growth of isolates utilizing coal as sole carbon
source the protein concentration of cultures grown in MSB supplemented
with and without 5% coal was measured. Cell pellets harvested at
regular intervals of time were treated with 10% trichloroacetic acid
for 30 min at 50oC. After incubation, tubes were centrifuged and
Isolation of Bacteria Capable of Growing on Coal Agar
In order to isolate bacteria capable of degrading coal and using
coal components as the sole carbon source, we inoculated
formation water collected from a 700 m depth of a coal bed
onto coal agar plates containing minimal medium with oxidized
coal [5% (w/v)] as the sole carbon source and cycloheximide
(0.5 mg/ml) to suppress the growth of fungi. Since the pH
of the formation water was 7.2, the pH of the agarized
medium was also maintained at 7.0. Aerobic incubation of
the plates at 37oC for 3 days produced bacterial colonies
with different morphologies. Fourteen bacterial colonies
were selected, which were thought to represent the
morphological diversity on coal agar. Clonal cultures of 14
isolates (Isolate #1 - Isolate #14) were generated by repeated
dilution streaking on coal agar. When the 14 isolates were
Nucleotide Sequence Accession Numbers
The 16S rRNA gene sequences of the six formation water isolates
have been deposited in GenBank with accession numbers shown in
brackets: Isolate #1 (JN182697), #2 (JN182699 ), #3 (JN182698),
#4 (JN182700), #5 (JN182701), and #6 (JN182702).
RESULTS AND DISCUSSION
Fig. 1. Phylogenetic (neighbor-joining) tree based on 16S rRNA gene sequence (>1,400 nt) showing relationship of the 6 formation
water isolates with related strains.
Significant bootstrap probability values are indicated at each branch point. Rhodococcus sp. YL-1 and E. coli were included as out-group members.
1104
Singh and Tripathi
subcultured on coal agar, 6 of them showed relatively
better and faster growth and were selected for further
study. It might be possible that the remaining 8 colonies
utilized the metabolic products produced as a result of coal
solubilization by other microorganisms. Thus, when they
were present in pure form in a medium with coal as the
sole carbon source, they were not able to proliferate.
Identification of the Isolates on the Basis of 16S rRNA
Gene Sequence
The 6 selected isolates were identified using 16S rRNA
gene sequencing. The identification based on comparison
of the 16S rRNA gene sequence of the isolates with that of
other bacterial sequences existing in the GenBank database
showed that isolates #1, 3, and 5 showed 99% sequence
identity, whereas isolate #4 showed 98% identity to
Rhizobium sp. 525W (Accession No. AB262326). However,
isolates #2 and 6 showed 98% sequence identity to
Chelatococcus daeguensis (Accession No. EF584507). A
phylogenetic tree based on the 16S rRNA gene sequences
of our isolates and other related sequences clustered
isolates into two groups; Group I isolates showing close
similarity to the genus Rhizobium (isolates #1, 3, 4, and 5),
and Group II isolates showing maximum similarity to the
genus Chelatococcus (isolates #2 and 6) (Fig. 1).
Genomic Fingerprinting of the Bacterial Isolates
Since the 16S rRNA gene is a very small fraction of the whole
genome, the techniques of whole genome fingerprinting
are considered more sensitive for differentiating the isolates
to the species, subspecies, and strain levels [34]. Hence,
we used whole genome fingerprinting methods, namely,
rep-PCR (REP-, ERIC-, and BOX-PCR) and RAPD, to
differentiate the isolates of the two groups from each other
at the strain level. The genomic fingerprints of the isolates
revealed distinct patterns with 5-15 fragments in the range
of 100-3,000 bp size and revealed differences among the
isolates. Out of the 4 genomic fingerprinting methods used
in this study, REP-PCR showed maximum differentiation
among the isolates, followed by ERIC-PCR and RAPD,
which were also able to show some differentiation within
each of the two groups, whereas BOX-PCR failed to show
any notable differentiation (Fig. 2).
Keeping in view the fact that rhizobia are normally
known to form nodules in legumes, occurrence of the
members of the genus Rhizobium in Indian coal bed was
surprising. However, the presence of Rhizobium and
Bradyrhizobium strains in the coal beds has already been
shown with the help of cultivation-independent methods
[23, 28]. Agrobacterium sp. was also reported from the
formation water of an oil field in China [24]. These
organisms are known to be associated with plant roots, and
it seems that during the process of coalification, the plantassociated bacteria were also buried along with the
remnants of plants under the Earth’s surface and were
trapped in the coal seams. They might have survived the
slow process of wood coalification to lignite, adapting
themselves to the extreme conditions of coal seams [29].
Comparison of the Chelatococcus Strains with Other
Species
The two strains of Chelatococcus isolated in this study
from the formation water of the coal bed showed almost
identical carbon source utilization abilities (Table 1). Like
Chelatococcus asaccharovorans, both of them were ureasepositive, but C. daeguensis was urease-negative [38]. Similar
to C. asaccharovorans and C. sambhunathii, but unlike C.
daeguensis, both the isolates also utilized nitrilotriacetate
Fig. 2. Genomic fingerprinting of the 6 isolates of formation water showing differentiation among the isolates within each group.
Lane M contains a 1 kb ladder (NEB), and the number above each lane indicates the isolate number.
STUDY OF COAL-DEGRADING BACTERIA FROM COAL BED
1105
Table 1. List of carbon sources utilized by the formation water isolates # 2, 6 and other described members of the genus Chelatococcus.
Carbon source
Isolate
#2
#6
Chelatococcus
Chelatococcus
Chelatococcus
sambhunathii
asaccharovorans
daeguensis
DSM 1867
Nitrilotriacetate
D-Galactose
Rhamnose
Sorbitol
Inositol
Sucrose
Adonitol
Glycerol
Arabinose
Xylitol
Mannitol
Cellobiose
Mannose
+
+
+
+
+
+
+
+
+
+
+
-
+
+
+
+
+
+
+
+
+
+
+
-
+
-
T
T
DSM 6462
CCUG 54519
+
-
+
+
+
+
+
+
+
+
+
+
+
+
T
(+), Positive; (-), Negative.
(NTA) as a carbon source. Unlike C. asaccharovorans and
C. sambhunathii, both our isolates were similar to C.
daeguensis in utilizing galactose, rhamnose, sorbitol, inositol,
sucrose, adonitol, glycerol, arabinose, xylitol, and mannitol.
However, unlike C. daeguensis, they could not utilize
cellobiose and mannose [27, 38]. In view of the above
characteristics and the phylogenetic tree based on the 16S
rRNA gene sequences, C. daeguensis appears to be the
most closely related, validly described species.
Chelatococcus asaccharovorans is known to utilize the
metal-chelating NTA as a sole source of carbon, energy, and
nitrogen. It was described as a chelating coccus, a bacterium
that forms claw-like complexes with divalent cations [10].
It is likely that inside the coal bed, the bacterial consortia
involved in coal degradation produce metal ion chelators
[14, 20], which are utilized by Chelatococcus as a source
of carbon, energy, and nitrogen, or else Chelatococcus might
be directly involved in the utilization and solubilization of
coal by producing metal ion chelators, which lead to the
weakening of coal matrix [3] and release of substance in
the supernatant, rendering it the yellowish brown colour.
C. daeguensis has been shown to grow under anaerobic
condition on trypticase soy agar plate supplemented with
0.01% potassium nitrate [38].
Growth of the Coal Bed Isolates on Coal as Sole
Carbon Source
For characterizing the coal utilization ability of the
isolates, their growth was monitored in minimal medium
supplemented with or without coal as the sole carbon source.
Since coal components create problems in measuring
growth on the basis of turbidity or absorbance at 560 or
600 nm, we estimated the protein content of the culture at
different stages of growth. A comparison of the growth of
the isolates revealed that the Rhizobium isolates showed
very little growth in minimal medium supplemented with
or without coal, whereas Chelatococcus isolates showed
much higher growth in the medium supplemented with 5%
coal compared with the medium without coal (Fig. 3).
From the growth pattern of the isolates, it became clear
that Rhizobium isolates were inefficient users of coal as a
carbon source. The fact that Chelatococcus isolates grew
much better in coal-supplemented medium than that
without coal indicated that Chelatococcus isolates were
able to utilize some components of coal as a carbon source
for their growth.
Coal Solubilization Ability of the Coal Bed Isolates
In some cases, the utilization of brown coal is accompanied
by its solubilization. Evolution of 14CO2 from radioactively
labeled coal (14C-methoxylated german lignite) by a coalsolubilizing fungi was also used as an indicator of their
coal utilization ability [20]. The browning of the supernatant
of the cultures grown on hard coal was used for the first
time to show coal solubilization [11, 12]. We found that
Chelatococcus isolates demonstrated coal solubilization,
as shown by an increase in absorbance at 450 nm and
browning of the culture supernatant (Fig. 4). However,
Rhizobium isolates did not show such coal solubilization
ability. Thus, it can be concluded that Chelatococcus strains
isolated in this study are capable of the solubilization and
utilization of coal. Biosolubilization of coal involves
various mechanisms such as production of alkaline substances,
biocatalysts, metal ions chelators, detergents, and esterases
[14, 20]. The microorganisms growing on rich media were
shown to produce alkaline metabolic products, which lead
1106
Singh and Tripathi
Fig. 3. Utilization of coal as sole source of carbon by the 6 isolates of formation water grown in mineral salt broth (- ○ -) and in mineral
salt broth containing 5% oxidized coal (- ● -) (error bars indicate SD).
to the ionization of acidic groups in low-rank coal to
render coal humates water-soluble [30]. Removal of metal
ions from the coal leads to the cleavage of ionic linkages
between structural elements and an increase in the number
of free acidic groups, enhancing the hydrophilicity of the
coal [3].
Fig. 4. Spectrophotometric comparison of the coal solubilization
ability of the 6 formation water isolates (error bars indicate SD).
In a preliminary characterization, we found that Rhizobium
isolates produced some kind of biosurfactants, which are
known to increase the surface area of hydrophobic waterinsoluble substrates to increase their bioavailability. It is
likely that Rhizobium isolates participate in the process of
degradation of hydrocarbons by producing bioemulsifiers
and thereby making the components of coal more watersoluble. In a consortium, the biosurfactant-producing
bacteria provide emulsifier (surfactant) to other bacteria
that carry out degradation of hydrocarbons. This is why the
cultures of Acinetobacter radioresistens, which produce
bioemulsifier but are unable to use hydrocarbon as a carbon
source, were included in the mixture of oil-degrading
bacteria to enhance oil bioremediation [31]. Therefore,
further study is required to elucidate the role of Rhizobium
isolates in coal degradation.
Advances in the understanding of coal biosolubilization
processes and the discovery of enzymatic degradation of
coal macromolecules offer new possibilities for utilizing
the carbon from low-rank coals. Several Gram-positive
and Gram-negative bacteria have been shown to produce
non-oxidative hydrolytic enzymes, which might be involved
in the depolymerization of humic acids obtained from
STUDY OF COAL-DEGRADING BACTERIA FROM COAL BED
12.
13.
14.
Acknowledgments
We thank the Institute of Reservoir Studies, Oil and
Natural Gas Commission (ONGC) for their assistance in
the collection of formation water from the Jharia coal bed.
D.N.S. was supported by a Senior Research Fellowship
from the University Grants Commission, New Delhi.
15.
16.
17.
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