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Molecular Identification of Acidophilic

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Molecular Identification of Acidophilic Manganese

(Mn)-Solubilizing Bacteria from Mining Effluents
and Their Application in Mineral Beneficiation

A. S. Sanket, S. Ghosh, R. Sahoo, S. Nayak & A. P. Das

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Molecular Identification of Acidophilic Manganese (Mn)-Solubilizing Bacteria from Mining
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 GEOMICROBIOLOGY JOURNAL
2016, VOL. 0, NO. 0, 110
http://dx.doi.org/10.1080/01490451.2016.1141340

Molecular Identication of Acidophilic Manganese (Mn)-Solubilizing Bacteria from

Mining Efuents and Their Application in Mineral Beneciation
A. S. Sanket, S. Ghosh, R. Sahoo, S. Nayak, and A. P. Das
Bioengineering and Biomineral Processing Laboratory, Centre of Biotechnology, Siksha O Anusandhan University, Bhubaneswar, India

ABSTRACT ARTICLE HISTORY

The study of the microbial ecology in extreme acidic environments has provided an important foundation for Received November 2015
the development of mineral biotechnology. The present investigation reports the isolation, identication and Accepted January 2016
molecular characterization of indigenous manganese (Mn) solubilizing acidophilic bacterial strains from mine KEYWORDS
water samples from Odisha, India. Four morphologically distinct bacterial strains showing visible growth on 16S rRNA; acidophiles;
Mn-supplemented plates of varying pH were isolated and identied. Mn solubilizing ability of the isolates isolation; manganese;
was tested by growing them on Mn-supplemented agar plates. The appearance of lightening around the solubilization
growing colonies of all the isolates demonstrated their Mn solubilizing ability in the medium. 16 S rRNA
Downloaded by [Tripura University] at 03:44 11 July 2016

sequencing was carried out and the bacterial isolates were taxonomically classied as Enterobacter sp.
AMSB1, Bacillus cereus AMSB3, Bacillus nealsonii AMSB4 and Staphylococcus hominis AMSB5. The evolutionary
timeline was studied by constructing neighbor-joining phylogenetic trees. The ability of acidophilic
microorganisms to solubilize heavy metals is supported by ve basic mechanisms which include: enzymatic
conversion, metal efuxing, reduction in sensitivity of cellular targets, intra- or extracellular sequestration, and
permeability barrier exclusion. Such ecological studies undoubtedly will provide insights into Mn
biogeochemical processes occurring in leaching environments. The application of acidophilic microbiology in
mineral biorecovery and beneciation has a large future potential.

Introduction
Microbial ecology is dened as the study of the relationship
of microorganisms to their environment and to each other
(Johnson 2001). Bacteria have long been studied for their
roles in mineral accumulation and metal leaching. Acido-
philes in general are recognized as important for mineral
biotechnology and bioremediation (Johnson 1998).
Although most acidophiles are obligatory aerobic, there is
now emerging evidence that many of them are also faculta-
tive anaerobes (Thamdrup 2000). A variety of interactions
including competition, predation, mutualism and synergy
have been reported in these microorganisms (Tebo et al.
2005). Acidophilic microorganisms will continue to be the
focus of research due to the expansion of the mineral proc-
essing industry, the increased interest in eco-friendly meth-
ods of metal extraction, and concerns about the massive
pollution caused by mining wastes. Thus the application of
acidophilic microorganisms to mineral processing is devel-
oping into one of the most successful areas of mineral
biotechnology.
Metals play an important role in industrial processes such as
the vital role Mn has in casting iron and steel (Das et al.
2015a). The 12th most abundant element on Earth manganese
(Mn) exists in various oxidation states ranging from 2 to 4
(Zaratos et al. 2003; Lee et al. 2001; Das et al. 2015b).
Metal mining and processing lead to the production of huge
amounts of wastes (Bosecker et al. 1993; Lee et al. 2001) that
also damage vegetation and crops by changing the chemical
properties of the soil, thereby adversely affecting the farming
industry (Ghosh et al. 2015). Continued exploration for useful
microbes in mineral deposits and their positive exploitation in
bioremediation has the potential to open new arenas in the eld
of wastewater treatment and management. Microbial ecological
research has a future in commercialized biomining processes,
and could also provide solutions to emerging constraints in the
expanding eld of mineral biotechnology.
Therefore newer and efcient techniques are being investi-
gated for remediation of Mn contaminated soils and water
(Das et al. 2011; Ghosh and Das 2015). Both direct and indirect
mechanisms for metal recovery from wastes have been reported
(Das et al. 2012; Xin et al. 2012). Application of biorecovery has
been applied to release of Mn from Mn silicate ores (Konishi
et al. 1995), Mn sulde ores (Keeling et al. 2005; Rodriguez-
Valera et al. 1979), Mn oxide nodules (Tebo et al. 2005), and
spent ZnMn batteries (Xin et al. 2011).
Diversity of acidophilic microorganisms is being researched
because of their ability to grow under high stress conditions
such as low pH, inorganic media, and high metal ion concen-
trations (Johnson 2001). Response and adaption to stress is
supported and conrmed by the observation of sporulation in

CONTACT A. P. Das alokdas@soauniversity.ac.in Bioengineering and Biomineral Processing Laboratory, Centre of Biotechnology, Siksha O Anusandhan Uni-
versity, Khandagiri Square, Bhubaneswar, Odisha 751003, India.
Color versions of one or more of the gures in the article can be found online at www.tandfonline.com/ugmb.
2016 Taylor & Francis Group, LLC
 2 A. S. SANKET ET AL.
bacteria and also by alternation in synthesis of specic outer
membranous proteins (Leyer and Johnson 1993). Because
many industrial metals are highly soluble at acidic pH values,
indigenous acidophiles are generally exposed to high concen-
trations of metals. They are highly metal resistant and have
more efcient active resistance systems than are present in neu-
trophils (Dopson et al. 2003). Some can tolerate high metal
concentrations due to their intrinsic tolerance not related to
specic genes for resistance.
The present investigation aims to: (1) isolate and identify
native acidophilic Mn solubilizing bacteria from mining efu-
ents in Odisha, India; (2) screen the isolates for Mn solubilizing
ability; and (3) suggest possible mechanisms for Mn solubiliza-
tion in extreme conditions. The results of this study provide
interesting insights into the potential use of native acidophilic
bacterial strains from mining efuents for bioremediation of
mining wastes.
Experimental procedures
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Sample collection
Mn mining efuents were collected randomly from different
Mn mining deposits in Odisha, India, for isolation of Mn-solu-
bilizing acidophilic bacteria. The samples were collected in ster-
ile Falcon tubes during the month of June (summer), the air
temperature ranged from 30 to 35 C, and the collected water
samples had temperatures ranging from 28 to 30 C. They were
stored at 4 C for further culture isolation studies. Representa-
tive sections of the collected samples were pooled to form one
single sample which was further studied for their physicochem-
ical parameters of pH, conductivity, total dissolved solids and
total suspended solids.
Medium of growth and bacterial isolation
Spread plate technique was used for the isolation of bacteria
from the pooled sample. Nutrient agar plates supplemented
with 50 mM MnO2 with varying pH between 2 and 4 were
used as bacterial isolation. About 10 M HCl and 10 M NaOH
were used to optimize the pH of the medium. A dilution series
of the sample was prepared in 10 ml sterile water (101 to
105) and 0.1 ml from the nal dilution was used for spreading.
The plates were incubated for 48 h at 30 C and the growth of
colonies with different morphologies was observed. Axenic cul-
tures of all isolates were obtained by streaking repetitively in
the same media. The colonies showing visible growth were
selected for further screening. The acidophilic strains obtained
were used for identication and molecular characterization.
DNA extraction and amplication
About 1 ml of bacterial suspensions was subjected to DNA
extraction by phenolchloroform method (Cullings 1992). The
isolated DNA samples were amplied with primer set of 8 F
(5-AGAGTTTGATCCTGGCTCAG-3) and 1492R (5-
GGTTACCTTGTTACGACTT-3) using the following prole
for the amplication: 94 C, 3 min; 94 C, 1 min; 55 C, 45 sec
and 72 C, 1 min; 72 C, 10 min. Thirty ve cycles were
performed for the amplication. Adequate controls were
included to rule out the presence of any contaminants. The
amplied products were separated on 1.2% agarose gel was run
and visualized for the presence of bands.
Evolutionary relationship
The 16S rRNA amplied gene products were sequenced using
the Sangers sequencing method. The sequences were checked
for quality and trimmed accordingly. The taxonomical classi-
cation of the sequences was accomplished using BLASTN algo-
rithm. An alignment was created using 10 closest matches of
the sequences with the query sequence using Clustal Omega.
Considering the resulting alignments, phylogenetic trees were
constructed in order to study the evolutionary relationship
with the closely related species. The nucleotide sequences of
four isolates were aligned and cladograms were created based
on neighbor-joining algorithm. The nucleotide distance was
measured by Kimura 80 model using CLC Sequence Viewer 7.6
software.
Mn solubilization
The solubilization of Mn was performed using agar plates sup-
plemented with varying concentrations of MnO2 ranging from
50 to 200 mM and within a varying pH of 24. The bacterial
isolates were inoculated in agar plates and were incubated at
30 C for 48 h. The plates were then checked for the presence of
Mn solubilization halos formed by the lightening of the
medium around the growing colonies on Mn supplemented
plates.
Biochemical characterization
The biochemical characterization for the isolates was carried
out in accordance with the Bergeys Manual of Determinative
Bacteriology. Gram staining, mannitol fermentation, citrate uti-
lization, starch hydrolysis, triple sugar iron and motility tests
were used (Breed et al. 1948).
Mannitol, motility test
Mannitol fermentation was checked by stabbing a single colony
into the mannitol-motility medium and incubated for 24 h.
Fermentation of mannitol results in change in color of the
medium from red to yellow and is interpreted as a positive
result. Motility test was carried out in mannitol-motility
medium. Upon 24-h incubation of a single colony stabbed into
the medium, the dispersion of the stabbed line is interpreted as
positive result.
Citrate utilization test
Simmons citrate was used to study the ability of the microor-
ganism to utilize citrate as the sole source of the carbon. Appear-
ance of blue coloration upon 24 h incubation was interpreted as
positive result.
 GEOMICROBIOLOGY JOURNAL 3
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Figure 1. Geographical location of sample collection site.

Triple sugar iron test Table 1. Physicochemical parameters of the pooled sample.

This test is conducted for Gram negative enteric bacteria. A Sample Water sample
single colony was stab-streaked into the medium and incu- pH 6
bated for 1824 h. Change in color of slant, butt, blackening Conductivity 31.5 mS
of the medium for H2S production and production of gas was Suspended solids 8.53 gm/l
Dissolved solids 0.8 gm/l
interpreted as positive.
 4 A. S. SANKET ET AL.

Table 2. The closest match of isolated microbes from BLASTN search results.

Isolates Accession no. Closest match Accession no. Query coverage (%) E value Identity (%)

AMSB1 KT165379 Enterobacter sp. SJZ6 LC014955 98 0.0 99

AMSB3 KT165380 Bacillus cereus strain P241 KM406406 99 0.0 99
AMSB4 KT165381 Bacillus nealsonii isolate 0511TES26Z1 LN477311 99 0.0 99
AMSB5 KT165382 Staphylococcus hominis strain ZJY-960 KP282765 99 0.0 99

Chemicals and instruments used Isolation of acidophilic bacteria

Isolation of bacterial colonies was carried out using nutrient agar
medium. MnO2 (HiMedia) was supplemented in agar plates as a
source of Mn for medium enrichment. Luria-Bertani broth (pH
optimized) was used for the isolation of bacterial DNA. For incu-
bation with appropriate culture conditions a shaker incubator
from RemiRS-24BL was used. 16S rRNA gene amplication was
performed using thermal cycler from Applied Biosystems. Boro-
sil glassware sets were used for experimental processes.
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Four acidophilic bacterial strains were isolated after 48 h of
incubation at 30 C. The media optimized with pH 4 and pH 3
resulted in growth of microorganisms; however, no growth at
pH 2 was observed. The plates with pH 3 supported the growth
of three bacterial isolates while only one isolate could grow on
plates of pH 4. The colonies were white and round on agar
plates and were 0.71.0 mm in diameter after 48 h of incuba-
tion. All the isolates were preserved in nutrient agar slants for
further experimental work.

Results
Sample characterization
The samples collected from different mining deposits and
pooled together to form one representative sample (Figure 1)
were studied for its physicochemical parameters. The sample
had a pH around 6 which is supported by the fact that mining
efuents are known to be contaminated with different metals
which tend to lower the overall pH. The sample also contained
dissolved and suspended solids (Ghosh et al. 2015; Das and
Mishra 2010), the details of which are listed in Table 1.
Molecular identication of isolated bacterial stains
The purity of extracted DNA was interpreted from the ratio of 260/
280 and the ratio ranging between 1.8 and 2. Molecular identica-
tion revealed the isolates to be Enterobacter sp. AMSB1, Bacillus
cereus AMSB3, Bacillus nealsonii AMSB4 and Staphylococcus homi-
nis AMSB5 with GenBank accession numbers KT165379,
KT165380, KT165381 and KT165382, respectively. The isolated
bacterial strains had a high percentage of similarity in BLAST
search to the available corresponding sequences (Table 2).

Figure 2. Neighbor-joining phylogenetic trees for AMSB1 with its 10 closest matches.

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Figure 3. Neighbor-joining phylogenetic trees for AMSB3 with its 10 closest matches.
Phylogenetic analysis
Studying an evolutionary timeline is used to inform about
the convergence/divergence of the organism from its ances-
tors. Phylogenetic trees are diagrams showing similarities
and dissimilarities in the morphological, physiological and
genetic characteristics for the concerned entity. A single cri-
terion is taken into consideration at a time. The microor-
ganism AMSB1 (Figure 2) was found to be closely matched
Figure 4. Neighbor-joining phylogenetic trees for AMSB4 with its 10 closest matches.
GEOMICROBIOLOGY JOURNAL 5
to Enterobacter sp. SJZ6 with a max identity of 99% whereas
the isolate AMSB3 (Figure 3) was closely related to B.
cereus strain P241 having a max identity of 99%. Isolate
AMSB4 (Figure 4) is closely related to B. nealsonii isolate
0511TES26Z1. Isolate AMSB5 (Figure 5) was found to be
closely related to S. hominis strain ZYJ-960.
AMSB1 Enterobacter sp. are a member of the Enterobacter-
iaceae family. The genus Enterobacter is a member of the
 6 A. S. SANKET ET AL.
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Figure 5. Neighbor-joining phylogenetic trees for AMSB5 with its 10 closest matches.

Figure 6. Evolutionary timeline for isolated microorganisms AMSB1, AMSB3, AMSB4 and AMSB5.
 GEOMICROBIOLOGY JOURNAL 7
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Figure 7. Nutrient agar plates supplemented with Mn showing lightening of the medium appearing around the growing colonies.

coliform group which has pathogenic strains. However several
non-pathogenic strains have been isolated and utilized in
industrial processing such as solubilization of phosphate
(Kumar et al. 2010). The isolate formed a separate branch thus
giving an interpretation of unrelatedness to any of the other
members of the genus. The isolates AMSB3 and AMSB4
showed a closer relationship to the genus Bacillus from the
family Bacillaceae. Members of this family produce endospores.
The phylogenetic tree for AMSB3 shows the divergence from
any other members of its genus. It shares the same node but
separated from its nearby branch representing B. cereus strain
B.Pat.40 as its closest member. B. cereus has been reported to
affect bioleaching studies of kaolin and quartz sands (Styriak
et al. 2003). However strains of B. cereus have also been utilized
for the leaching of copper, nickel and zinc from black shale
(Farbiszewska-Kiczma et al. 2004). The isolate AMSB4 also
diverged since its evolution even though it shares the same
node, but formed a separate clade from its neighbor B. nealsonii
isolate 0511TES26Z1. AMSB5 S. hominis is a Gram positive
bacterium having the morphology of cocci clusters and
exhibits a positive reaction for catalase. All the species of
Staphylococcaceae are coagulase negative with the exception of
Staphylococcus aureus. However AMSB5 showed divergence
from its close matched neighbor Staphylococcus hominis subsp.

Table 3. Biochemical characteristics of the isolates.

Tests AMSB1 AMSB3 AMSB4 AMSB5

Gram status C C C
Shape Round Irregular Irregular Round
Size Small Large Medium Small
Color White Cream Cream White
Margin Smooth Undulate Smooth
Opacity Translucent Opaque Translucent Opaque
Cell shape Rod Rod Rod Spherical
Motility
H2S NA NA NA
Gas C NA NA NA
Triple sugar iron A/A NA NA NA
Starch hydrolysis C C
Mannitol C C
Citrate C

Note. NA, not applicable.

 8 A. S. SANKET ET AL.
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Figure 8. Possible mechanisms of Mn tolerance and solubilization by acidophiles.
novobiosepticus isolate 0511MAR2B4, sharing the same node
but forming a different clade. Staphylococcus hominis subsp.
hominis strain NS5 shares the same node but forms a different
branch.
The phylogenetic tree for all the isolated microorganisms
shows the divergence of isolates from its root ancestor. How-
ever the isolates AMSB3 and AMSB4 share the same node thus
forming a single clade. The isolate AMSB5 S. hominis forms a
separate node thus giving an idea about its complete divergence
from all other isolated microorganisms. The isolate AMSB1
Enterobacter sp. is however connected to the node with both
AMSB3 and AMSB4 (Figure 6).
Mn solubilization
The capability of bacterial strains to solubilize insoluble metal
compounds and minerals is of biotechnological signicance.
Halo production or lightening of the medium around the grow-
ing colonies of an active strain demonstrates its Mn-solubiliz-
ing ability (Figure 7) (Acharya et al. 2002).
Biochemical performance
Biochemical characteristics studied for the isolates are listed in
Table 3. The results were compared with data discussed in Ber-
geys manual of determinative bacteriology.
Possible mechanism of Mn solubilization
Biotransformation of Mn (II) is known to be elicited by diverse
bacterial strains but whether the transformation is by direct or
indirect mechanism is a topic of lengthy debate. Some research-
ers believe that solubilization of Mn is due to the changes of the
mineral composition as a result of direct attack by the bacterial
strains while some believe that the process is a result of indirect
mechanism in which there is no contact between the mineral
surface and the bacterial strains (Das et al. 2011). Mn plays
indispensable roles as catalysts and enzyme cofactors in micro-
organisms and also takes part in several redox processes
(Bruins et al. 2000). Mn accumulation beyond its normal physi-
ological concentrations results in cellular toxicity. Toxic effect
can manifest by functional group blockage of enzymes, trans-
port systems inhibition, displacement of essential metals from
their native binding sites, and disruption of cellular membrane
integrity (Dopson et al. 2003). The ability of acidophilic
microorganisms to tolerate and thus solubilize heavy metals is
supported by ve basic mechanisms which include enzymatic
conversion, metal efuxing and reduction in sensitivity of
cellular targets, intra- or extracellular sequestration, and per-
meability barrier exclusion (Figure 8). Acidophiles thus possess
a variety of active and intrinsic metal resistance systems that
enable them to grow in very high metal concentrations besides
solubilizing the metals. These Mn solubilization mechanisms
can occur by both direct and indirect processes. Direct solubili-
zation occurs with the utilization of MnO2 as a nal acceptor of
electrons in the bacterial respiratory chain, instead of oxygen.
Indirect solubilization occurs due to the formation of metabolic
reductive compounds. Solubilization of metals such as Mn can
occur due to either one or a combinatorial effect of the mecha-
nisms. namely metal anion protonation, soluble Mnligand
complex formation and production of biogenerated organic
acids (Gadd et al. 2012; Ghosh et al. 2015).
Discussion and conclusion
The present investigation reports the isolation, screening and
molecular identication of acidophilic Mn solubilizing micro-
organisms from Mn mining deposits. Four isolates that are acid
tolerant at pH 3 and 4 were isolated suggesting the possibility
of existence of extremophiles having the potentiality to be

tested in various sectors of industrial processes. It can however
be mentioned that there are no such old literatures reporting
the nding of acid-tolerant species like B. nealsonii and its sub-
sequent utilization for metal solubilization. However, the pres-
ence of Mn in the articial growth medium has been reported
to induce sporulation in B. nealsonii (Venkateswaran et al.
2003), which may be indicative of a plausible mechanism
against manganese for their growth. The fact is supported by
the studies for the increase of sporulation during subsurface
mineralization by B. cereus, discovered from mining areas
(Hongmei et al. 2002). Because of its heavy metal tolerance, the
presence of B. cereus is used as a biogeochemical prospecting
tool for gold deposits (Cannon 1960). Diversied metabolic
spectrum of members from the genus Staphylococcus is an obvi-
ous reason for their existence in mining niches. Efciency in
mobilizing Mn from low grade ores has put an impact on
industrial processes. Das et al. (2012) studies report the ability
of Staphylococcus epidermidis to solubilize manganese. Mn
associated superoxide dismutase enzymes are reported for their
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underlying ability to solubilize metals (Sujith et al. 2014).
We can expect that different strains of acidophiles will
emerge as important mineral oxidizers and reducers due to
their higher tolerance of extreme conditions. Reconsidering the
burden of economy in conventional techniques such as smelt-
ing, roasting to recover precious metals from mines and to
remediate toxic heavy metal from environment, discovery of
such microorganisms and their subsequent utilization will play
a major role in future. Biomolecular techniques will continue to
have a major impact on ecological research in acidophilic
microbiology and mineral processing. Therefore using a com-
bined approach of both biomolecular and conventional meth-
odologies will lead to bridging the gap between acidophilic
microbial ecology and their application in mineral processing.
Funding
The authors thank the Department of Biotechnology Technology (DBT),
Government of India for funding the project entitled Biomineralization of
Mn from wastes and natural resources [BT/PR7454/BCE/8/949/2012].
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