Geomicrobiology Journal

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Isolation and Characterization of Bacteria from

Coal Mines of Dara Adam Khel, Pakistan

Qaiser Jamal, Iftikhar Ahmed, Shafiq Ur Rehman, Saira Abbas, Kil Yong Kim
& Muhammad Anees

To cite this article: Qaiser Jamal, Iftikhar Ahmed, Shafiq Ur Rehman, Saira Abbas, Kil Yong Kim
& Muhammad Anees (2016) Isolation and Characterization of Bacteria from Coal Mines of Dara
Adam Khel, Pakistan, Geomicrobiology Journal, 33:1, 1-9, DOI: 10.1080/01490451.2014.964886

To link to this article: http://dx.doi.org/10.1080/01490451.2014.964886

Accepted author version posted online: 25

Sep 2014.
Published online: 25 Sep 2014.

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 Geomicrobiology Journal (2016) 33, 1–9
Copyright © Taylor & Francis Group, LLC
ISSN: 0149-0451 print / 1521-0529 online
DOI: 10.1080/01490451.2014.964886

Isolation and Characterization of Bacteria from Coal Mines

of Dara Adam Khel, Pakistan
QAISER JAMAL1, IFTIKHAR AHMED2, SHAFIQ UR REHMAN3, SAIRA ABBAS4,
KIL YONG KIM1 and MUHAMMAD ANEES4,*
Downloaded by [NWFP University of Engineering & Technology - Peshawar] at 22:18 27 September 2017

1
Institute of Environmentally-Friendly Agriculture, College of Agriculture and Life Sciences, Chonnam National University, Gwangju,
Korea
2
National Institute for Genomics and Advanced Biotechnology (NIGAB), National Agricultural Research Center (NARC),
Park Road, Islamabad, Pakistan
3
Department of Botany, Kohat University of Science and Technology, Kohat Pakistan
4
Department of Microbiology, Kohat University of Science and Technology, Kohat Pakistan

Received July 2014; Accepted September 2014

The coal fields of Pakistan and their microbiology have not been fully explored. Therefore, a study was conducted on the coal mines
of Dara Adam Khel located in the Federally Administered Tribal Areas of Pakistan. For this purpose, sampling was done from nine
different mines with varying depths. A total of 32 bacterial strains were isolated and their colony size, form, texture, color, margin,
elevation and opacity were noted. The majority of the strains (75%) were found Gram negative. The bacterial strains were then
characterized in detail by different biochemical tests including catalase, citrate, oxidase, indole, triple sugar iron, motility, methyl
red-Vogues Proskeur, nitrate reduction and phenylalanine deaminase, and an enormous physiological diversity was observed. The
Gram positive strains were further characterized on molecular level using 16S rRNA gene amplification and sequence analysis.
Based on molecular analysis, seven strains were identified as Bacillus tequilensis, B. cereus, Janibacter melonis, Kocuria atrinae,
B. anthracis, K. rosea and B. simplex. The other two strains (strains 6 and 41) had molecular similarity of only 98% and 97% with
Brachybacterium spp. and Arthrobacter spp. respectively. The phylogenetic analysis further suggested that the strains 6 and 41 may
be potential candidates for novel species; however, further work is needed for confirmation.
Keywords: Bacillus species, biochemical characterization, coal mines in Dara Adam Khel, Pakistan, Kocuria species

Introduction conversion of coal to methane (Orem et al. 2010), bioremedi-

Pakistan is a coal rich country with 185 billion tons of coal
(NEPRA 2004). Huge reserves of coal have been discovered
in Dara Adam Khel. However, large part of the coal reserves
in the Federally Administered Tribal Areas (FATA) and
Khyber Pakhtunkhwa (KPK) Province of Pakistan has not
been explored (Malkani 2012). The coal in these areas is clas-
sified as Sub-bituminous with low sulfur and ash contents.
The coal beds in Hangu District are thicker than rest of these
areas (NEPRA 2004). Although very little data has been
reported from different coal mines of Pakistan, the present
study was, however, based on the sampling from the coal
mines of Dara Adam Khel.
A number of phylogenetic studies on subsurface coal beds
have described active microbial communities especially that
of bacteria (Green et al. 2008; Shimizu et al. 2007). There are
several reasons to investigate microbial activities in coal
mines. These bacteria may possess versatile abilities including
*Address correspondence to Muhammad Anees, Department of
Microbiology, Kohat University of Science & Technology,
Kohat 26000, Pakistan; Email: dr.anees@kust.edu.pk
ation of heavy metals (Luo et al. 2011), biodegradation of
the toxic material from soil (Safekordi and Yaghmaei 2001)
and antimicrobial activities (Khulana and Niranjan 2012).
Extremely acidic environments (pH < 3) occur naturally in
the coal mining areas due to anthropogenic activities, and
microorganisms are present in such areas which are acid-tol-
erant or acidophilic (Johnson and McGinness 1991).
However, the idea is old, as in 1908, Potter reported that
bacteria act as catalytic agents in the oxidation of amorphous
brown coal and 2 years later, Galle (1910) could isolate pure
cultures of bacteria grown on brown coal samples. Further-
more, numerous experiments on coal solubilization by bacte-
ria have since been performed (Fakoussa and Hofrichter
1999; Laborda et al. 1997). Fakoussa investigated the ability
of bacteria to degrade and solubilize the organic phase of
hard coal (Fakoussa 1988). Moreover, the wood–rot fungi
could quantitatively solubilize the low-rank Leonardite
(Catcheside and Ralph 1999).
Various microorganisms including bacteria (e.g., Bacillus
sp. Proteobacteria, Streptomyces badius, Streptomyces setoni,
A. ferrooxidans and L. ferrooxidans), and fungi (e.g., Coriolus
versicolor, Phanerochaete chrysporium, Poria placent,
 2 Jamal et al.

Piptoporus betulinus, Coprinus sclerotigenis, Candida sp., Biochemical tests

Fusarium oxysporum, Aspergillus sp. and Trichoderma atro-
viride) are known to be present in coal mines (Faison 1991;
Holker et al. 1999; Laborda et al. 1997).
From the context given here, it is clear that coal mines can
be an important reservoir of useful bacteria. They represent
an extreme environment for microorganisms. The chance of
finding novel species is greater in extreme environments such
as coal mines because of their being less explored to date
(Roohi et al. 2014). Moreover, Pakistan is rich with reser-
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Standard biochemical tests were used to characterize bacte-
rial isolates (Hussain et al. 2013). Briefly, for the indole test,
cultures were incubated for 24 h on peptone broth and
Kovak’s solution was used as reagent (MacFaddin 2000).
For the methyl red and Voges-Proskauer test (MRVP), the
bacteria were inoculated onto buffered peptone-glucose broth
using methyl red and potassium hydroxide as indicators.
Starch hydrolysis was detected by growing bacteria in starch
agar medium with the addition of iodine for 24 to 48 h. For

voirs. The present study was, therefore, based on the isolation
and identification of the indigenous bacterial strains of coal
from different coal mines of Dara Adam Khel. The charac-
terization of the isolates was also done by using biochemical
assays. Selected strains were identified by molecular methods,
especially including the sequencing of bacterial strains by the
16S rRNA gene.
Methodology
Coal Sample Collection for Isolation of Bacteria
Coal samples were collected from nine coal mines of various
depths from Dara Adam Khel (33
410 1.5300 N, 71
310
4.1200 E), Pakistan (Table 1). Sampling was performed during
the period of October 2010. Samples were collected asepti-
cally in 500 g sterile autoclavable plastic boxes. Before
isolation, coal samples were crushed with the help of a
well-cleaned grinder and were completely powdered for serial
dilution to isolate of bacterial flora of coal. Serial dilutions
were performed for the isolation. Dilutions were grown on
Nutrient agar plates using the pour-plate method.
Phenotypic Characterization
Staining, morphology, and cultural characteristics
Cultures were examined for cell morphology and Gram reac-
tion after 24 h of growth in nutrient agar medium. A total of
32 isolates were selected. Colony morphology of all the iso-
lates was examined by using cultures that were grown for
2 days on nutrient agar (McClelland 2001). Characteristics
including form, texture, color, margin, elevation and opacity
were studied.
citrate and ammonia utilization tests, the bacteria were
grown on Simmons citrate agar for 24–48 h.
To determine whether the microbe produces the enzyme
phenylalanine deaminase, the bacteria were grown onto phe-
nylalanine agar, a nutrient medium enriched supplemented
with 0.2% phenylalanine and 10% ferric chloride as reagent
for 24-h-old cultures. Bacteria were screened out for enzyme
cytochrome oxidase when 24-h-old cultures were transferred
to filter paper and then were moistened with a few drops of
1% tetramethyl-p-phenylenediamine dihydrochloride. The
motility of the bacterial isolates was observed in semisolid
nutrient agar. Nitrate reduction was also tested by using stan-
dard assays (Chapin and Lauderdale 2007). A small amount
of 1% hydrogen peroxide was added to bacterial cultures for
the detection of catalase activity. To detect the bacteria that
can utilize any or all of the different sugars, dextrose, lactose
and sucrose were supplemented in TSI Agar after 24–72 h.
Molecular Identification of Bacterial Strains
The Genomic DNA was extracted by using colony-PCR
method by using TE buffer, absolute ethanol and 70% etha-
nol. Colonies from fresh bacterial cultures were suspended in
20 uL TE buffer and heated for 10 min at 95
C in PCR
machine followed by centrifuging for 6 min at 8000 rpm. The
supernatant was then used as template DNA for the amplifi-
cation of 16S rRNA gene.
The 16S rRNA gene of all the isolated strains were ampli-
fied by polymerase chain reaction (PCR) using primers 9F(50 -
GAGTTTGATCCTGGCTCAG-30 ) and 1510R (50 - GGC
TACCTTGTTACGA-30 ) using PreMix ExTaq (Takara,
Japan) as described previously (Ahmed et al. 2007). The
PCR was carried out by an initial denaturation step at 95
C

Table 1. Sampling from coal mines of Dara Adam Khel, Pakistan. ‘Isolates id’ were denoted to different bacterial isolates picked
from the respective mines
Mine name Owner Location Depth of mines Isolates id

Mine No 1 Fakaruddin Feroz Mela 800–900ft 25,41

Mine No 2 Saranzeb Feroz Mela 700–850 ft 6,9,17,18
Mine No 3 Khanzada Feroz Mela 800ft 15,24
Mine No 4 Rizwan Tandi Kala 900–950ft 5,8,13,43,2
Mine No 5 Ahmad Feroz Mela 600–700ft 14,19,29,30
Mine No 6 Abdur Rehman Tandi Kala 800ft 12,10,16,33,34,36
Mine No 7 Saranzeb Feroz Mela 1000ft 7,11,39
Mine No 8 Fakaruddin Tandi Kala 800–900ft 28,31,35
Mine No 9 Shakeel Feroz Mela 1000ft 4,20,32
 Bacteria from Coal Mines of Dara Adam Khel 3

for 1 min, followed by 30 cycles of denaturation at 95
C for Neighbor-joining tree and Maximum Parsimony Algorithm
20 s, annealing at 52
C for 40 s, and elongation at 72
C for methods were used to construct phylogenetic tree of the
1 min 30 s. Cycling was completed by a final elongation step sequenced strains (Marcheggiani et al. 2008).
at 72
C for 10 min. The amplified PCR products of 16S
rRNA gene of bacterial strains were purified and sequenced
using commercial service of Macrogen, Inc. Korea (www.
dna.macrogen.com).
Results
The sequences obtained were compared with the sequences
Morphological and Cultural Characteristics
present in the GenBank Database by using the BLAST pro-
gram of the National Center of Biotechnology Information The surface characteristics of bacterial isolates were found to
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(NCBI). Matching hit with e-values closest to 0.0 were chosen
for alignment. The alignment and editing was performed
using BIOEDIT, EZTaxon Server 2. The nearest taxa of the
16s rRNA sequence of isolates were identified by EZTaxon
server 2.1 (Kim et al. 2012). Reported and reference sequen-
ces were aligned using BIOEDIT and MEGA5. MEGA5
software package was used to generate phylogenetic
tree for the strains, which showed lower similarities with
the already identified sequences present in the databases.
be smooth, rough, dry, glistening and shiny. Most of the colo-
nies, which were selected visually based on differences with
the naked eye, were whitish and cream color, while a yellow
color was also observed. A margin of colonies of bacterial
isolates were found to be entire, undulate, filliform, lobate
and convex. Out 32 bacterial isolates, 75% were Gram nega-
tive. Most of the isolates were rod-shaped (78%), 25% isolates
were Gram positive rods and 6% isolates were Gram positive
cocci.

Table 2. The biochemical test results of the different isolates picked from the coal mines of Dara Adam Khel
Vogues Nitrate Starch Phenylalanine Triple
Strain Indole Methyl Proskauer Citrate Reduction Catalase Oxidase Motility hydrolysis Deaminase Sugar Iron
ID Test Red Test Test Test Test Test Test Test Test Test

2 C C ¡ ¡ C C ¡ C C ¡ C
4 ¡ ¡ C C ¡ ¡ ¡ C C C C
5 ¡ C C C ¡ ¡ ¡ ¡ C C C
6 ¡ C C ¡ ¡ C ¡ C ¡ ¡ C
7 ¡ C C C C ¡ ¡ C C ¡ ¡
8 C C C ¡ ¡ C ¡ C ¡ C C
9 ¡ C C C ¡ ¡ ¡ C ¡ C ¡
10 ¡ C C C ¡ C ¡ C ¡ ¡ ¡
11 ¡ C C ¡ ¡ ¡ ¡ C C ¡ C
12 ¡ C C ¡ C C ¡ C ¡ ¡ ¡
13 ¡ ¡ C C ¡ ¡ ¡ C ¡ C C
14 C C C C ¡ C ¡ C C ¡ ¡
15 ¡ C C C ¡ C ¡ C C ¡ C
16 ¡ C C C ¡ ¡ ¡ ¡ C ¡ C
17 C C C C ¡ ¡ ¡ C C C ¡
18 ¡ C C ¡ ¡ ¡ ¡ C C ¡ C
19 ¡ C C C ¡ ¡ ¡ ¡ C C C
20 ¡ ¡ C C ¡ C ¡ C C C ¡
24 C C C C ¡ C ¡ C C C C
25 C C C C ¡ C ¡ C C C ¡
28 C C C ¡ ¡ ¡ ¡ ¡ C ¡ ¡
29 C C C C ¡ ¡ ¡ C C ¡ C
30 ¡ C C ¡ ¡ ¡ ¡ C C C ¡
31 ¡ C C C ¡ ¡ ¡ C C C ¡
32 ¡ ¡ C ¡ ¡ C ¡ ¡ C C C
33 ¡ C C C C ¡ ¡ C C C ¡
34 ¡ C C ¡ ¡ C ¡ C C C C
35 ¡ C C ¡ ¡ C ¡ C C ¡ C
36 ¡ C C C C ¡ ¡ C C C C
39 ¡ C C ¡ C C ¡ C C ¡ C
41 ¡ C C C ¡ C ¡ C ¡ C ¡
43 ¡ ¡ C C ¡ C ¡ C ¡ ¡ C
‘C’ means the activity takes place and ‘¡‘ means the no activity.
 4 Jamal et al.

Table 3. Molecular identification of isolated bacterial strains isolated from the coal mines in Dara Adam Khel, Pakistan
Accession Number of Sequence
Strain From number of nucleotides of Closely related validly similarity(%) of 16S
ID Mine 16S rRNA gene 16SrRNA gene published taxa rRNA gene

41 Mine 1 AB753823 1531 Arthrobacter globiformis AB279890 97.770

6 Mine 2 AB753794 1405 Brachybacterium sacelli AJ415384 98.513
24 Mine 3 AB753818 1129 Bacillus cereus AE016877 99.911
2 Mine 4 AB753793 1120 Bacillus tequilensis HQ223107 99.911
30 Mine 5 AB753819 1137 Bacillus anthracis AB190217 100.000
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12 Mine 6 AB753795 944 Janibacter melonis AY522568 99.353

39 Mine 7 AB753822 969 Bacillus simplex AB363738 100.000
35 Mine 8 AB753821 1029 Kocuria atrinae FJ607311 100.000
32 Mine 9 AB753820 1155 Kocuria rosea X87756 99.653

5 Arthrobacter oxydans (X83408)

9 Arthrobacter polychromogenes (X80741)
6 Arthrobacter scleromae (AF330692)

8 Arthrobacter phenanthrenivorans (CP002379)

0.5%
Arthrobacter chlorophenolicus(CP001341)
Arthrobacter equi (FN673551)
9 6
7 Arthrobacter defluvii (AM409361)
10 Arthrobacter niigatensis (AB248526)
2
Arthrobacter sulfonivorans (AF235091)
Arthrobacter methylotrophus (AF235090)
4
9 Arthrobacter alkaliphilus (AB248527)
Arthrobacter flavus (AB299278)
1 Arthrobacter tec (AJ639829)
9
9 Arthrobacter paries (AJ639830)
6 Arthrobacter tumbae (AJ315069)
3
NCCP 773 (AB753823)
Arthrobacter globiformis (M23411)
2 Arthrobacter pascens (X80740)
7
5 Arthrobacter oryzae (AB279889)
9 Arthrobacter humicola (AB279890)
Arthrobacter crystallopoietes (X80738)
Arthrobacter woluwensis (X93353)
Arthrobacter rhombi (Y15885)
Arthrobacter cryotolerans (GQ406812)
6
Arthrobacter bergerei (AJ609630)
5
Arthrobacter kerguelensis (AJ606062)
8
Arthrobacter sulfureus (X83409)
7
6 Arthrobacter antarccus (AM931709)
9 Arthrobacter gangotriensis (AJ606061)
Micrococcus luteus (CP001628)

Fig. 1. Phylogenetic tree showing relationship of strain 41(NCCP 773) with closely related taxa contingent from 16S rRNA sequen-
ces. Tree was generated using the Neighbor-Joining. Bootstrap values (more than 50%), expressed as percentage of 1000 replications,
and are indicated at the nodes. Accession number of each type strain is shown in parentheses.
 Bacteria from Coal Mines of Dara Adam Khel
A variety of biochemical assays were carried out to
have a comprehensive view of the phenotypic characteris-
tics of bacteria (Table 2). It was observed that 75% of
bacterial isolates were indole-positive and 84% were acid
producers, while the rest of the isolates were capable of
producing neutral compounds in media. Overall, the bac-
terial isolates showed various biochemical attributes.
Briefly, bacterial isolates were positive for citrate utiliza-
tion (62.5%), nitrate reduction (19%), catalase test (50%),
oxidase (100%), motility test (85%), starch hydrolysis
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(75%), phenylalanine deaminase test (53%) and triple
sugar iron test (60%).
Molecular Characterization
Out of 9 strains that were identified on a molecular level,
4 (strains 2, 24, 30 and 39) were identified as Bacillus spp.
(Table 3). Strain 2 was identified as B. tequilensis. Strain
24 was identified as B. cereus, strain 30 as B. anthracis
and strain 39 as B. simplex. Strain 6 was identified as Bra-
chybacterium sacelli. Strain 12 was identified as Janibacter
melonis. Moreover, strain 32 was identified as Kocaria
rosea, and strain 35 as K. atrinae. Strain 41 was identified
as Arthrobacter globiformis. Furthermore, the phylogenetic
analyses supported the identification (Figures 1–6). All the
sequences were submitted to GenBank for further
referencing (Table 3).
Fig. 2. Phylogenetic tree showing inter-relationship of strain 41 (NCCP 773) with closely related taxa, inferred from 16S rRNA
sequences. Tree was generated using the Maximum-Parsimony Algorithm. Bootstrap values (more than 50%), expressed as percent-
age of 1000 replications, and are indicated at the nodes. Accession number of each type strain is shown in parentheses.
5
Discussion
Coal is the biggest resource for power generation globally
and a major anthropogenic source of CO2 production (Man-
cuso and Seavoy 1981). Although Pakistan is rich and has an
abundance of coal reserves, the coal production has been low
due to lack of facilities, inadequate financing and inadequate
modern coal exploration technical expertise and has not been
developed for the last three decades. Moreover, the coal
reserves in FATA and KPK province of Pakistan are not
fully researched. Furthermore, ecological data for microbiol-
ogy of coal is unavailable although such areas might contain
the potentially new bacterial species (Roohi et al. 2014).
Therefore, the present study was based on isolation, charac-
terization and identification of bacteria from the coal mines
in Dara Adam Khel, FATA Pakistan.
Purification of enrichment cultures of bacteria in addition
to the isolation and enumeration of bacteria from natural
samples were satisfactorily accomplished through conven-
tional plating techniques. Colony morphology is an impor-
tant aspect for the isolation of bacteria as in pure cultural
work. It is sometimes necessary to make suppositions because
bacterial colonies of the same morphology may represent the
same bacterial species. Therefore, in the present study, the
bacterial isolates were initially selected on the basis of differ-
ences in colony morphology. According to this study most of
the coal bacteria were Gram negative (75%).
 6 Jamal et al.
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Fig. 3. Phylogenetic tree showing inter-relationship of strain 41 (NCCP773) with closely related taxa, the, inferred from 16S rRNA
sequences. Tree was generated using the Maximum Likelihood algorithm and Bootstrap values (more than 50%), expressed as per-
centage of 1000 replications, and is indicated at the nodes. Accession number of each type strain is shown in parentheses.

Previous workers have demonstrated the existence and
nonexistence of Gram-positive bacteria in coal mines. Dugan
et al. (1968) were able to isolate two Gram-positive and five
Gram- negative bacteria from acidic (pH 2.8) mine drainage.
Johnson and McGinness (1991) isolated several Gram-posi-
tive bacteria, including Staphylococci, Micrococci, Coryne-
bacteria, and spore-forming bacilli, from the drainage of an
abandoned coal mine. Tuttle et al. (1968) isolated eight
Gram-negative bacilli and no Gram-positive bacteria from
an acidic, coal pile drainage. The low number of Gram-posi-
tive bacteria in coal may be due to a greater leak in the struc-
ture of the Gram-positive cell walls to toxic hydrogen ions.
Moreover, the biochemical characterization was also per-
formed in the present study as an essential part in characteri-
zation of bacteria. An enormous diversity could be found
using the variable standard biochemical assay (Table 2). The
bigger diversity at the metabolic level among the different
bacterial isolates from the coal mines of Dara Adam Khel
may suggest the presence of variable genotypes and higher
chances of finding potential novel bacterial strains. To con-
firm the hypothesis, molecular identification was performed
using 16S rRNA gene sequencing.
All the Gram-positive bacteria (9 out of 32) were identified
in the present study on a molecular basis. Bacillus tequilensis,
Bacillus cereus, Janibacter melonis, Kocuria atrinae, Bacillus
anthracis, Kocuria rosea, Bacillus simplex were identified in
coal mines of Dara Adam Khel based on molecular analysis.
Two strains (6 and 41) showed lower similarity of 97% with
Arthrobacter globiformis and 98% with Brachybacterium
sacelli respectively based on the NCBI BLAST analysis.
Strains 6 and 41 were, therefore, subjected to phylogenetic
analysis using variable algorithms, which suggested that the
 Bacteria from Coal Mines of Dara Adam Khel 7

100 Brachybacterium paraconglomeratum (AJ415377)

95 Brachybacterium conglomeratum (X91030)
92 Brachybacterium saurashtrense (EU937750)
0.5%
52 Brachybacterium faecium (CP001643)
Brachybacterium alimentarium (X91031)
79
97 Brachybacterium tyrofermentans (X91657)

76 Brachybacterium sacelli (AJ415384)

53 Brachybacterium fresconis (AJ415378)
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31
NCCP-762 (AB753794)
14 Brachybacterium muris (AJ537574)

28 Brachybacterium squillarum (GQ339911)

43 Brachybacterium rhamnosum (AJ415376)
83
Brachybacterium zhongshanense (EF125186)
Brachybacterium nesterenkovii (X91033)
Brachybacterium phenoliresistens (DQ822566)
Arthrobacter globiformis (M23411)

Fig. 4. Phylogenetic tree showing inter-relationship of strain 6 (NCCP 762) with closely related taxa, inferred from 16S rRNA
sequences. Tree was generated using the Neighbor-Joining Algorithm and Bootstrap values (more than 50%), expressed as percent-
age of 1000 replications, and is indicated at the nodes. Accession number of each type strain is shown in parentheses.

strains may be considered as candidates for novel species
(Figures 1–6).
However, further work is needed to confirm the hypothe-
sis. B. tequilensis was first time isolated and identified as novel
species from a sample taken from an approximately 2000-
year-old shaft-tomb located in the Mexican state of Jalisco,
near the city of Tequila (Gaston et al. 2006). Additionally,
Bacillus spp., Arthrobacter, Janibacter spp. isolated from the
mining soils could oxidize the Antimony present in the
mining soils showing high resistance (Shi et al. 2013).
Another strain CM100B of B. cereus was isolated from the
coal mines and showed high effectiveness in generation of
selenium nanoparticles (SNs) by transformation of toxic sele-
nite (SeO32¡) anions into red elemental selenium (Se0) under
aerobic conditions (Dhanjal and Cameotra 2010). Similarly,
Kocuria spp. were isolated from the coal amended fly ash, the
combustion remains of the coal, and was found resistant to
fly ash (Jabeen and Sinha 2012).

Brachybacterium paraconglomeratum (AJ415377)

98
81 Brachybacterium conglomeratum (X91030)
10 87 Brachybacterium saurashtrense (EU937750)
4 Brachybacterium faecium (CP001643)

66 Brachybacterium sacelli (AJ415384)

Brachybacterium fresconis (AJ415378)
74 Brachybacterium alimentarium (X91031)
23
88 Brachybacterium tyrofermentans (X91657)
13
NCCP-762 (AB753794)
Brachybacterium muris (AJ537574)
18 Brachybacterium squillarum (GQ339911)
28
36 Brachybacterium rhamnosum (AJ415376)
Brachybacterium nesterenkovii (X91033)
Brachybacterium zhongshanense (EF125186)
Brachybacterium phenoliresistens (DQ822566)
Arthrobacter globiformis (M23411)

Fig. 5. Phylogenetic tree showing inter-relationship of strain 6 (NCCP 762) with closely related taxa inferred from 16S rRNA sequen-
ces. Tree was generated using the Maximum-Parsimony Algorithm and Bootstrap values (more than 50%), expressed as percentage
of 1000 replications, and is indicated at the nodes. Accession number of each type strain is shown in parentheses.
 8 Jamal et al.

99 Brachybacterium paraconglomeratum (AJ415377)

82 Brachybacterium conglomeratum (X91030)
93 Brachybacterium saurashtrense (EU937750)
0.5%
Brachybacterium faecium (CP001643)

72 94 Brachybacterium alimentarium (X91031)

37 Brachybacterium tyrofermentans (X91657)
78 Brachybacterium fresconis (AJ415378)
Brachybacterium sacelli (AJ415384)
31 NCCP-762 (AB753794)
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Brachybacterium zhongshanense (EF125186)

72 Brachybacterium muris (AJ537574)

22
Brachybacterium squillarum (GQ339911)
36
43 Brachybacterium rhamnosum (AJ415376)
Brachybacterium nesterenkovii (X91033)
Brachybacterium phenoliresistens phenol (DQ822566)
Arthrobacter globiformis (M23411)

Fig. 6. Phylogenetic tree showing inter-relationship of strain 6 (NCCP 762) with closely related taxa inferred from 16S rRNA sequen-
ces. Tree was generated using the Maximum-Likelihood Algorithm. Bootstrap values (more than 50%), expressed as percentage of
1000 replications, and are indicated at the nodes. Accession number of each type strain is shown in parentheses.

In conclusion, the bacterial strains isolated from coal
mines in Dara Adam Khel showed diversity at physiologi-
cal and molecular level. Further work is, however, needed
for confirmation of novelty of strains from the mines.
Moreover, the mines may represent a gene pool that may
be tested for variable activities including bioremediation,
biodegradation and antimicrobial plant growth-promoting
abilities.
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