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J. Microbiol. Biotechnol.

(2011), 21(11), 11011108
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 thermochemical liquefaction can convert coal into a cleaner
on developing strategies for transforming coal into a liquid fuel, it is an energy intensive process requiring high
cleaner fuel. This requires isolation of microbes with the temperatures and pressures, and, hence, is not cost effective
capability to degrade complex coal into simpler substrates [26]. In order to address the environmental concerns
to support methanogenesis in the coal beds. In this study, associated with the use of coal, alternative methods of
aerobic bacteria were isolated from an Indian coal bed coal-processing are being explored that would extend
that can solubilize and utilize coal as the sole source of utilization of coal-derived biofuels as an alternative source
carbon. The six bacterial isolates capable of growing on of energy. It has been predicted that a microbial, enzymatic,
coal agar medium were identified on the basis of their 16S or enzyme-mimetic technology would have greater advantage
rRNA gene sequences, which clustered into two groups; compared with the physical and chemical technologies for
Group I isolates belonged to the genus Rhizobium, whereas coal conversion.
Group II isolates were identified as Chelatococcus species. Coal is a solid rock, which is often dominated by
Out of the 4 methods of whole genome fingerprinting recalcitrant, partially aromatic, and largely lignin-derived
(ERIC-PCR, REP-PCR, BOX-PCR, and RAPD), REP- macromolecules that tend to be relatively resistant to
PCR showed maximum differentiation among strains microbial degradation. It has a highly heterogeneous
within each group. Only Chelatococcus strains showed the structure; lignite being more complex than the hard coal.
ability to solubilize and utilize coal as the sole source of Since lignin was the main parent material in the formation
carbon. On the basis of 16S rRNA gene sequence and the of lignite, typical structures of the original lignin are preserved
ability to utilize different carbon sources, the Chelatococcus in lignite, which is also designated as demethylated and
strains showed maximum similarity to C. daeguensis. This dehydrated lignin [9, 17, 18]. The lignite consists of several
is the first report showing occurrence of Rhizobium and distinct compound classes: mainly hydrophobic bitumen,
Chelatococcus strains in an Indian coal bed, and the the alkali-soluble humic and fulvic acids, and insoluble
ability of Chelatococcus isolates to solubilize and utilize residue known as the matrix or humine. The aromatic
coal as a sole source of carbon for their growth. structures of coal derived from cellulose and lignin are
Keywords: Coal bed, coal solubilization, rep-PCR, 16S rRNA often interlinked by oxygen bridges and contain numerous
gene sequence, Chelatococcus 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,
Coal is one of Earths most abundant fossil fuels, which propionate, acetate, CO2, and H2, which can be utilized by
contributes nearly 30% of global energy consumption [19]. methanogens to produce methane. The rate-limiting step
The burning of coal creates a variety of problems to the of coal biodegradation is the initial fragmentation of a
environment including the release of atmosphere-polluting macromolecular, polycyclic, lignin-derived aromatic network
sulfur and nitrogen oxides, and leftover ash containing of coal.
toxic metals, but it cannot be replaced or minimized by any Lignin can be degraded by some fungi and bacteria
other more efficient fuel in the near future. Although direct that produce unspecific extracellular enzymes [8, 14]. Up
*Corresponding author to 40% of the weight of some coals can be dissolved using
Phone: +91 9451525811; Fax: +91-542-2368693; extracted microbial enzymes [32]. There are several
E-mail: reports on the ability of filamentous fungi to solubilize
1102 Singh and Tripathi

solid particles of low-rank hard coal into black liquid 5 min at 72oC. The amplified product was analyzed in 0.8% agarose
droplets [5, 11, 14, 21], but there are very few reports on gel at 5 V/cm for 4 h and was visualized under UV light with an
the ability of bacteria to degrade or utilize coal as the sole Alpha imager (Alpha Innotech Corporation, UK).
source of carbon and energy [7, 11-13, 20]. In recent
Sequencing of 16S rRNA Genes
years, bacterial communities present in the coalbeds, which
The amplified 16S rRNA gene was purified with a Wizard SV gel
can convert coal into the substrates that can be utilized by PCR purification kit (Promega, USA) and quantified by using a ND-
methanogens, have been described with the help of 1000 Spectrophotometer (NanoDrop, Wilmington, DE, USA). Direct
cultivation-independent methods [23, 28]. sequencing was performed with three primers, 8f (5'-AGA GTT
Coal beds contain water (known as formation water), TGA TYM TGG CTC AG- 3'), 1495r (5'-CTA CGG CTA CCT
which facilitates coal degradation by bacteria at the coal- TGT TAC G -3') [16], and 561f (5'-AATTACTGGGCGTAAAG -3')
water interface. Thus, the water extracted from the coal [2] using the BigDye Terminator v3.1 Cycle sequencing kit (Applied
beds might harbour unique microbial communities possessing Biosystems) in an ABI Prism 310 automated DNA Sequencer
the ability to solubilize and degrade coal and other (Applied Biosystems, Rotkreuz, Switzerland).
polyaromatic hydrocarbons. In the present study, bacteria
were isolated from the formation water collected from a Analysis of 16S rRNA Gene Sequence
The 16S rRNA gene sequence of the six isolates was edited using
coal bed located in eastern India. The isolates were identified
Bioedit software version 3.1 to make a complete sequence. The almost
using molecular tools of taxonomy and characterized for complete sequence (1,400 nt) was compared with the nucleotide
their ability to solubilize and utilize coal as a sole source of sequences present in the NCBI database using the standard nucleotide
carbon and energy. This study is the first attempt from BLAST search [1]. The programme ClustalW [35] was used to
India towards the isolation and characterization of bacteria align the 16S rRNA gene of the isolates with the similar sequences
from a coal bed. retrieved from the NCBI database to construct a phylogenetic tree,
as described by Chowdhary et al. [4].

MATERIALS AND METHODS Genomic Fingerprinting

repPCR. Primers corresponding to the conserved motifs in bacterial
Collection of Sample repetitive elements (REP, ERIC, and BOX) were used for PCR
The sample was collected directly from the pipe extruding formation amplification to generate genomic fingerprints to differentiate the
water from a depth of 700 m from a coal bed methane (CBM)- isolates further at the species, subspecies, or strain levels. All the six
producing well located at Parbatpur in the Jharia coalfield of eastern isolates were subjected to genomic fingerprinting using primer sets
India. Samples were collected in autoclaved bottles, which were corresponding to REP, ERIC, and BOX elements. The PCR protocols
brought to the laboratory under cooled condition. At the time of with REP, ERIC, and BOX primers are collectively referred to as
collection, the temperature of the formation water was 42oC and pH rep-PCR. The primer pairs REP1R-I (5'-IIIICGICGICATCIGGC-3')
was 7.2; chloride and sulfate contents were 180 ppm and 22 ppm, and REP2-I (5'-ICGICTTATCIGGCCTAC-3') (where I is inosine);
respectively. ERICIR (5'-ATGTAAGCTCCTGGGGATTCAC-3') and ERIC2 (5'-
Isolation of Coal-Solubilizing Bacteria GCAAGGCGACGCTGACG-3') [25] primer sequence corresponding
For isolation of coal-solubilizing bacteria, formation water was to BOXA, a subunit of BOX element, were used to amplify REP-,
serially diluted with sterile saline (0.89% NaCl) and spread onto coal ERIC-, and BOX-like elements from the isolates DNA. Approximately
agar medium [22] containing (per liter) KH2PO4, 1.2 g; Na2HPO412H2O, 50 ng of DNA was used as template, and PCR amplification reactions
10.8 g; MgSO47H2O, 0.04 g; FeSO47H2O, 0.02 g; MnCl24H2O, were performed with a Veriti 96-well Thermal cycler (Applied Biosystem,
0.02 g; NH4C1, 3 g; lignite coal, 50 g; agar, 15 g; and cycloheximide, USA) using the following cycles: an initial denaturation at 95oC for
0.5 g; pH 7.0. The lignite coal was oxidized with 10% hydrogen 7 min; 35 cycles of denaturation at 94oC for 1 min, annealing at 44,
peroxide before using it in the coal agar medium [22]. The plates 57, or 60oC for 1 min with REP, ERIC, or BOX primer, respectively;
were incubated at 37oC for 3 days. 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
Nucleic Acid Extraction and 16S rRNA Gene Amplification components of PCR except genomic DNA was included as negative
Total genomic DNA was extracted from the isolates using a Wizard control in all the thermal cycles run. The 10 l of PCR products
genomic DNA purification kit (Promega, Madison, WI, USA). The 16S was loaded on agarose [1.5% (w/v)] gel and amplification products
rRNA gene was amplified using universal bacterial primers 8f (5'- were visualized under a UV light with Alpha imager.
AGA GTT TGA TYM TGG CTC AG- 3' and 1495r (5'-CTA CGG RAPD. Amplification was performed in 25 l with 20-50 ng of
CTA CCT TGT TAC G -3') [16]. A 50 l reaction mixture included genomic DNA as template, 4 M of 10 mer primer (5'-GTGTGCCCCA-
20-50 ng of bacterial DNA as template, 200 M of each primer, 3') [36], 2.5 U of Taq DNA polymerase (Genei, Banglore) with its
and 1.0 unit of Taq DNA polymerase (Genei, Bangalore). The 1 buffer, and 200 M of each dNTPs. The reaction condition
reactions were performed on a Veriti 96-well Thermal cycler (Applied included an initial denaturation of 7 min at 95oC, followed by 40
Biosystem, USA), and the reaction condition included an initial cycles of 1 min at 94oC, 1 min at 35oC, and 2 min at 72oC, with a final
denaturation of 5 min at 95oC followed by 35 cycles of 1 min at extension of 5 min at 72oC. An 8-10 l aliquot of each amplification
94oC, 1 min at 51oC, and 1 min at 72oC, with a final extension of mixture was analyzed by agarose gel (2.5%) electrophoresis.

Carbon Source Utilization Ability of the Isolates pellets were incubated at 37oC for 4 h with 1 ml of 1 N NaOH. [33].
Bacterial inocula were prepared by growing the isolates on Biolog After centrifugation, 200 l of supernatant was used for estimation
universal growth agar (BUG; Biolog) medium for 24 h. The cultures of protein according to the Lowry method of protein estimation.
from the Biolog universal growth agar medium were collected using
sterile swabs and resuspended in 0.85% saline. The inoculum Nucleotide Sequence Accession Numbers
density was adjusted with a Biolog turbidimeter within the limit of The 16S rRNA gene sequences of the six formation water isolates
GN MicroPlate turbidity standards supplied by the manufacturer. The have been deposited in GenBank with accession numbers shown in
cell suspension was poured into a sterile disposable plastic reservoir, brackets: Isolate #1 (JN182697), #2 (JN182699 ), #3 (JN182698),
and Biolog-GN MicroPlates (Biolog Inc., Hayward, CA, USA) #4 (JN182700), #5 (JN182701), and #6 (JN182702).
containing 95 different carbon sources were inoculated with 150 l
of bacterial suspension, and the microplates were incubated for 24 h
at 37oC. Substrate utilization patterns were compared with Microlog RESULTS AND DISCUSSION
database release 4.01 software.
Isolation of Bacteria Capable of Growing on Coal Agar
Evaluation of the Ability of Isolates to Utilize and Solubilize Coal
In order to check coal utilization and solubilization capabilities, isolates
In order to isolate bacteria capable of degrading coal and using
were grown in mineral salt broth (MSB) supplemented with and coal components as the sole carbon source, we inoculated
without 5% coal. The constituent of MSB were g/l KH2PO4, 1.2 g; formation water collected from a 700 m depth of a coal bed
Na2HPO412H2O, 10.8 g; MgSO47H2O, 0.04 g; FeSO47H2O, 0.02 g; onto coal agar plates containing minimal medium with oxidized
MnCI4H2O, 0.02 g; and NH4C1, 3.0 g. Flasks were inoculated with coal [5% (w/v)] as the sole carbon source and cycloheximide
equal number of overnight-grown log phase culture (same optical (0.5 mg/ml) to suppress the growth of fungi. Since the pH
density) and incubated at 37oC under shaking condition of 200 rpm. of the formation water was 7.2, the pH of the agarized
The culture broth from each flask was taken at a regular interval and medium was also maintained at 7.0. Aerobic incubation of
centrifuged to pellet down cells and the supernatant was used for the plates at 37oC for 3 days produced bacterial colonies
spectrophotometric monitoring of coal solubilization at 450 nm [15, with different morphologies. Fourteen bacterial colonies
37]. To determine the growth of isolates utilizing coal as sole carbon
were selected, which were thought to represent the
source the protein concentration of cultures grown in MSB supplemented
with and without 5% coal was measured. Cell pellets harvested at
morphological diversity on coal agar. Clonal cultures of 14
regular intervals of time were treated with 10% trichloroacetic acid isolates (Isolate #1 - Isolate #14) were generated by repeated
for 30 min at 50oC. After incubation, tubes were centrifuged and dilution streaking on coal agar. When the 14 isolates were

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 at the strain level. The genomic fingerprints of the isolates
better and faster growth and were selected for further revealed distinct patterns with 5-15 fragments in the range
study. It might be possible that the remaining 8 colonies of 100-3,000 bp size and revealed differences among the
utilized the metabolic products produced as a result of coal isolates. Out of the 4 genomic fingerprinting methods used
solubilization by other microorganisms. Thus, when they in this study, REP-PCR showed maximum differentiation
were present in pure form in a medium with coal as the among the isolates, followed by ERIC-PCR and RAPD,
sole carbon source, they were not able to proliferate. which were also able to show some differentiation within
each of the two groups, whereas BOX-PCR failed to show
Identification of the Isolates on the Basis of 16S rRNA any notable differentiation (Fig. 2).
Gene Sequence Keeping in view the fact that rhizobia are normally
The 6 selected isolates were identified using 16S rRNA known to form nodules in legumes, occurrence of the
gene sequencing. The identification based on comparison members of the genus Rhizobium in Indian coal bed was
of the 16S rRNA gene sequence of the isolates with that of surprising. However, the presence of Rhizobium and
other bacterial sequences existing in the GenBank database Bradyrhizobium strains in the coal beds has already been
showed that isolates #1, 3, and 5 showed 99% sequence shown with the help of cultivation-independent methods
identity, whereas isolate #4 showed 98% identity to [23, 28]. Agrobacterium sp. was also reported from the
Rhizobium sp. 525W (Accession No. AB262326). However, formation water of an oil field in China [24]. These
isolates #2 and 6 showed 98% sequence identity to organisms are known to be associated with plant roots, and
Chelatococcus daeguensis (Accession No. EF584507). A it seems that during the process of coalification, the plant-
phylogenetic tree based on the 16S rRNA gene sequences associated bacteria were also buried along with the
of our isolates and other related sequences clustered remnants of plants under the Earths surface and were
isolates into two groups; Group I isolates showing close trapped in the coal seams. They might have survived the
similarity to the genus Rhizobium (isolates #1, 3, 4, and 5), slow process of wood coalification to lignite, adapting
and Group II isolates showing maximum similarity to the themselves to the extreme conditions of coal seams [29].
genus Chelatococcus (isolates #2 and 6) (Fig. 1).
Comparison of the Chelatococcus Strains with Other
Genomic Fingerprinting of the Bacterial Isolates Species
Since the 16S rRNA gene is a very small fraction of the whole The two strains of Chelatococcus isolated in this study
genome, the techniques of whole genome fingerprinting from the formation water of the coal bed showed almost
are considered more sensitive for differentiating the isolates identical carbon source utilization abilities (Table 1). Like
to the species, subspecies, and strain levels [34]. Hence, Chelatococcus asaccharovorans, both of them were urease-
we used whole genome fingerprinting methods, namely, positive, but C. daeguensis was urease-negative [38]. Similar
rep-PCR (REP-, ERIC-, and BOX-PCR) and RAPD, to to C. asaccharovorans and C. sambhunathii, but unlike C.
differentiate the isolates of the two groups from each other 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.

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 Chelatococcus Chelatococcus Chelatococcus
#2 #6 sambhunathii asaccharovorans daeguensis
DSM 1867 DSM 6462 CCUG 54519
Nitrilotriacetate + + + + -
D-Galactose + + - - +
Rhamnose + + - - +
Sorbitol + + - - +
Inositol + + - - +
Sucrose + + - - +
Adonitol + + - - +
Glycerol + + - - +
Arabinose + + - - +
Xylitol + + - - +
Mannitol + + - - +
Cellobiose - - - - +
Mannose - - - - +
(+), Positive; (-), Negative.

(NTA) as a carbon source. Unlike C. asaccharovorans and different stages of growth. A comparison of the growth of
C. sambhunathii, both our isolates were similar to C. the isolates revealed that the Rhizobium isolates showed
daeguensis in utilizing galactose, rhamnose, sorbitol, inositol, very little growth in minimal medium supplemented with
sucrose, adonitol, glycerol, arabinose, xylitol, and mannitol. or without coal, whereas Chelatococcus isolates showed
However, unlike C. daeguensis, they could not utilize much higher growth in the medium supplemented with 5%
cellobiose and mannose [27, 38]. In view of the above coal compared with the medium without coal (Fig. 3).
characteristics and the phylogenetic tree based on the 16S From the growth pattern of the isolates, it became clear
rRNA gene sequences, C. daeguensis appears to be the that Rhizobium isolates were inefficient users of coal as a
most closely related, validly described species. carbon source. The fact that Chelatococcus isolates grew
Chelatococcus asaccharovorans is known to utilize the much better in coal-supplemented medium than that
metal-chelating NTA as a sole source of carbon, energy, and without coal indicated that Chelatococcus isolates were
nitrogen. It was described as a chelating coccus, a bacterium able to utilize some components of coal as a carbon source
that forms claw-like complexes with divalent cations [10]. for their growth.
It is likely that inside the coal bed, the bacterial consortia
involved in coal degradation produce metal ion chelators Coal Solubilization Ability of the Coal Bed Isolates
[14, 20], which are utilized by Chelatococcus as a source In some cases, the utilization of brown coal is accompanied
of carbon, energy, and nitrogen, or else Chelatococcus might by its solubilization. Evolution of 14CO2 from radioactively
be directly involved in the utilization and solubilization of labeled coal (14C-methoxylated german lignite) by a coal-
coal by producing metal ion chelators, which lead to the solubilizing fungi was also used as an indicator of their
weakening of coal matrix [3] and release of substance in coal utilization ability [20]. The browning of the supernatant
the supernatant, rendering it the yellowish brown colour. of the cultures grown on hard coal was used for the first
C. daeguensis has been shown to grow under anaerobic time to show coal solubilization [11, 12]. We found that
condition on trypticase soy agar plate supplemented with Chelatococcus isolates demonstrated coal solubilization,
0.01% potassium nitrate [38]. as shown by an increase in absorbance at 450 nm and
browning of the culture supernatant (Fig. 4). However,
Growth of the Coal Bed Isolates on Coal as Sole Rhizobium isolates did not show such coal solubilization
Carbon Source ability. Thus, it can be concluded that Chelatococcus strains
For characterizing the coal utilization ability of the isolated in this study are capable of the solubilization and
isolates, their growth was monitored in minimal medium utilization of coal. Biosolubilization of coal involves
supplemented with or without coal as the sole carbon source. various mechanisms such as production of alkaline substances,
Since coal components create problems in measuring biocatalysts, metal ions chelators, detergents, and esterases
growth on the basis of turbidity or absorbance at 560 or [14, 20]. The microorganisms growing on rich media were
600 nm, we estimated the protein content of the culture at 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 In a preliminary characterization, we found that Rhizobium
render coal humates water-soluble [30]. Removal of metal isolates produced some kind of biosurfactants, which are
ions from the coal leads to the cleavage of ionic linkages known to increase the surface area of hydrophobic water-
between structural elements and an increase in the number insoluble substrates to increase their bioavailability. It is
of free acidic groups, enhancing the hydrophilicity of the likely that Rhizobium isolates participate in the process of
coal [3]. degradation of hydrocarbons by producing bioemulsifiers
and thereby making the components of coal more water-
soluble. 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
Fig. 4. Spectrophotometric comparison of the coal solubilization non-oxidative hydrolytic enzymes, which might be involved
ability of the 6 formation water isolates (error bars indicate SD). in the depolymerization of humic acids obtained from

weathered lignite [6]. The bacteria progressively convert microbial decomposition of untreated hard coal. Prepared for
the coal polymers into solubilized coal, which can serve as U.S. Department of Energy, Pittsburgh Energy Technology
substrate for anaerobic bacteria to produce clean methane Center, 1987.
as fuel. This study is the first of its kind to describe the 12. Fakoussa, R. M. 1988. Production of water-soluble coal-
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