Mineral Processing and Extractive Metallurgy Review

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Microbes Assisted Mineral Flotation a Future

Prospective for Mineral Processing Industries: A
Review

Sunil Kumar Behera & Antoine Floribert Mulaba-Bafubiandi

To cite this article: Sunil Kumar Behera & Antoine Floribert Mulaba-Bafubiandi (2016):
Microbes Assisted Mineral Flotation a Future Prospective for Mineral Processing
Industries: A Review, Mineral Processing and Extractive Metallurgy Review, DOI:
10.1080/08827508.2016.1262861

To link to this article: http://dx.doi.org/10.1080/08827508.2016.1262861

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MINERAL PROCESSING AND EXTRACTIVE METALLURGY REVIEW
http://dx.doi.org/10.1080/08827508.2016.1262861

Microbes Assisted Mineral Flotation a Future Prospective for Mineral Processing

Industries: A Review

Sunil Kumar Behera and Antoine Floribert Mulaba-Bafubiandi

Mineral Processing and Technology Research Center, Department of Metallurgy, Faculty of Engineering and the Built Environment, University of
Johannesburg, Johannesburg, South Africa

ABSTRACT KEYWORDS
The latest advancements in mineral biotechnology developed environmental benign microbial flotation Bioflotation;
processes for mineral beneficiation. Several studies were conducted to evaluate the microbes and microbial microorganisms;
metabolites as flotation reagents for separation of gangue materials from valuable minerals. The available biosurfactant; nucleic acid;
scientific literatures on microbial flotation process suggest that, interactions of minerals with cells of certain bio-collector
bacteria and microbial metabolites have a significant effect on their surface properties. As a consequence,
bacterial cells belong to genera Acidithiobacillus, Leptospirillum, Mycobacterium, Staphylococcus, and Bacillus,
etc., and microbial metabolites such as bio-surfactants, extracellular polymeric substances (EPS) and nucleic
acid have been used as collectors and surface modifiers in the mineral flotation process. The characteristic
properties of the microbial surfactants such as their abilities to lower surface tensions and foaming capacity
prompted their application as frother in mineral flotation process. To date, microbial flotations have primarily
studied in laboratory scale; however, there is ample scope to expand this eco-friendly process for mineral
processing industries. The current review addresses the recent developments and applications of micro-
organisms and bio-molecules in mineral flotation processes.

Introduction
The role of microorganisms and their metabolites in mineral
processing operations have been studied extensively. Currently,
the microbial processes are widely applied for bioleaching of
metals and bioremediation of mineral wastes (Behera et al.
2012, Behera and Mulaba-Bafubiandi 2015; Peng et al. 2013;
Rastegar et al. 2015; Watling 2008). The exponential rising
demand for metals and minerals coupled with the depletion of
high grade mineral resources accelerated the utilization of lean
grade ores and minerals in mineral processing industries to
extract metal values. Mineral beneficiation is an inevitable step
for processing of lean grade minerals in mineral processing
industries. Froth flotation is a well established vital process for
mineral beneficiation in mineral processing industries to remove
gangue materials from ores and minerals. The process is facili-
tated by the use of different chemical reagents namely frother,
collector, depressants, and activators, etc. (Bakalarz et al. 2015;
Sis and Chander 2003). Conventional chemical reagents such as
petroleum oils, xanthates, cyanides, and amines, etc., used in
mineral flotation are toxic, nondegradable and exorbitant in
nature. The stricter environmental legislations are intensifying
to curb on excessive utilization of hazardous and toxic chemical
reagents in mineral processing industries. The rising environ-
mental concerns as well as mineral resource considerations
encouraged the application of biotechnology in mineral proces-
sing industries. With the recent developments, microbial flota-
tion processes have been developed by using microbes and
microbial metabolites (proteins, surfactants and extracellular
polymeric substances [EPS]) as environment friendly flotation
reagents (Singh et al. 2006).
Microbial mineral flotation processes have been developed by
using bacterial strains of Rhodococcus opacus, Rhodococcus ruber,
Staphylococcus carnosus, Stenotrophomonas and Escherichia coli,
etc., as bio-collectors (Farahat et al. 2009; Kim et al. 2016; Ramos-
Escobedo et al. 2016; Yang et al. 2013) and Acidithiobacillus
ferrooxidans, Ferroplasma acidiphilum, Leptosririllum ferrooxi-
dans and Bacillus subtilis, etc., as depressant reagents (Abdel-
Khalek and El-MIdany 2013; Amini et al. 2009; Farahat and
Hirajima 2012; Vilinska and Rao 2008). The bacterial induced
mineral flotation process is critically governed by the interactions
of the bacterial cells to mineral surfaces. The adhesion of a
bacterial cell to mineral surface is mainly controlled by the bio-
chemical properties of constituent proteins and structural poly-
saccharides of bacterial cell membrane. The biochemical
properties of a bacterial cell membrane vary from species to
species, due to the difference in membrane protein and structural
polysaccharide compositions. Broadly, bacterial cells are classified
as Gram-positive and Gram-negative, according to their cell
membrane structure. The Gram positive bacterial cells have
thicker cell walls mainly composed of peptidoglycan and teichoic
acid. The peptidoglycan is a polymer containing sugars and amino
acids and the teichoic acid is a co-polymer of glycerol phosphate
and carbohydrates. On the other hand, the Gram negative bacter-
ium has a thin layer of peptidoglycan without teichoic acid and its

CONTACT Sunil Kumar Behera skbehera2020@gmail.com Mineral Processing and Technology Research Center, Department of Metallurgy, Faculty of
Engineering and the Built Environment, P. O. Box 17911, Doornfontein Campus, 2028, University of Johannesburg, Johannesburg, South Africa.
© 2016 Taylor & Francis
2 S. K. BEHERA AND A. F. MULABA-BAFUBIANDI
outer membrane contains lipopolysaccharides (Dwyer et al. 2012).
The presence of these polymeric substances and biomolecules on
the bacterial cell surface controls the physiochemical properties of
a bacterial cell and favors its selective adhesion to a mineral.
Conversely, the bacterial adhesion to a mineral surface alters its
surface properties.
The hydrophobic bacterial strains of Rhodococcus opacus,
Rhodococcus carnosus, Rhodococcus ruber, Stenotrophomonas,
etc., are used as bio-collector because the adhesion of these bac-
teria induced hydrophobic characters on mineral surfaces (Lopez
et al. 2015; Merma et al. 2013; Ramos-Escobedo et al. 2016; Yang
et al. 2013). Unlikely, the adhesion of bacterial strains like Bacillus
subtilis. Leptosririllum ferrooxidans, Acidithiobacillus ferrooxidans,
Ferroplasma acidiphilum, etc., are induced hydrophilic character-
istics on adhering to a mineral surface and as a result, they are
used as bio-depressants in mineral flotation process. In addition to
bacterial adhesion, the chemolithotrophic microorganisms
brought changes in surface properties of minerals by catalyzing
oxidation or reduction reactions on them. Iron-sulfur oxidising
bacterial strains of Leptosririllum ferrooxidans, Acidithiobacillus
ferrooxidans, Ferroplasma acidiphilum, etc., have been used as
pyrite depressant by inducing chemical changes on pyrite surface
by bacterial oxidation-reduction reaction process (Abdel-Khalek
and El-MIdany 2013; Amini et al. 2009; Farahat and Hirajima
2012; Hosseini et al. 2005; Vilinska and Rao 2008).
Furthermore, the modern bio-flotation includes the success-
ful application of microbial metabolites such as bio-surfactants
and extra-cellular polymeric substances to improve the separa-
tion process. Biosurfactants are surface-active amphiphillic com-
pounds synthesized by a variety of microorganisms. A
biosurfactant molecule has both hydrophobic and hydrophilic
domains and hence, it can decrease the surface tension of the
growth medium. The application of biosurfactant is gaining
importance because they are biodegradable and less toxic and
are highly effective at extreme temperatures and pH in compare
to their counterparts (Cameotra and Makkar 2010). Application
of biosurfactants as bioreagents in mineral flotation is a new
development. Adsorption of biosurfactant on mineral surface
alters its physiochemical properties and facilitates selective flota-
tion (Didyk and Sadowski 2012). Also, the recent studies
revealed that the biosurfactant molecules have better froth char-
acteristics in comparison to chemical frother used in mineral
flotation (Fazaelipoor et al. 2010). In addition to biosurfactant,
microbial biomolecules like EPS, nucleic acid, etc., have been
reported as alternative bioreagent for mineral flotation. The
current review presents the recent developments in the microbial
mineral flotation processes. An overview of microorganisms,
and microbial biomolecules (biosurfactants, EPS, and genetic)
with potential application in mineral flotation process is pre-
sented and discussed.
Microbial flotation process
The use of microbes and their metabolites in mineral flotation
is a spectacular development in the field of minerals
biotechnology. The use of microorganisms and their metabo-
lites in flotation process have been studied to selectively
separate gangue materials from valuable minerals. Microbes
and microbial metabolites have been reported as surface
-modifiers, collectors and depressants in mineral flotation
(Deo and Natarajan 1997, 1998; Chandraprabha et al. 2004,
2005; Chandraprabha and Natarajan 2006; Patra and Natarajan
2003, 2004, 2006, 2008). The fundamental scientific studies
explored that the adhesion of microorganisms and their bio-
molecules to mineral surfaces induce change in their surface
properties. Thus, there is a possibility to use microbes and
microbial metabolites as green and biodegradable reagents.
The attachment of bacteria onto mineral surfaces was found
to play an important role in improving the separation selectiv-
ity in flotation and flocculation (Elmahdy et al. 2013). The
presence of functional groups such as hydrocarbon chains
and polar groups (carboxyl, hydroxyl, phosphates) at the
microbial cellular surface or metabolic products can either
directly or indirectly modify the mineral surfaces, lending the
microbial culture similar characteristics of collector molecules.
Numerous research studies have been conducted for the pos-
sible application of microorganisms and microbial metabolites
as flotation reagents in selective separation of gangues from
minerals. Microbial strains of Mycobacterium phlei,
Paenibacillus polymyxa, Rodococcusopacus, Acidithiobacillus
ferrooxidans, Acidithiobacillus thiooxidans, Leptospirillium
ferrooxidans have been used as selective flotation or depression
reagents for different types of sulfides and oxide minerals
(deMesquita et al. 2003; Hosseini et al. 2005; Kolahdoozan
et al. 2004; Raichur et al. 1996; Santhiya et al. 2001; Vilinska
and Rao 2008; Yuce et al. 2006; Zheng et al. 1998). Of particular
interest are bioleaching bacteria, acidophilic metal sulfide
oxidizing species, which are already used in commercial bio-
hydrometallurgical processes have been elaborately studied
microbes for flotation process. These bacteria selectively attach
to sulfide surfaces forming a biofilm or an EPS layer. Compared
to conventional inorganic reagents, bacteria are nontoxic and
environmentally benign, potentially providing an alternative to
conventional flotation methods. Similarly, bio-molecules like
surfactants of microbial origin, EPS have been reported for
their effectiveness in their mineral bio-flotation.
Application of microorganisms in bio-flotation
Microorganism attachment to the mineral surface has an impor-
tant role during mineral bio-processing. These reactions can be
influenced by microbial activities, especially when the organisms
are in close contact with the metal surface forming a layer of
biofilm. Biofilms formed on the metal surface consist of micro-
bial cells, their EPS, inorganic precipitates derived from the
microbial environment (Beech and Gaylarde 1999). The EPS
facilitate attachment of microbial cells to the mineral surface.
Microbial EPS consists of polysaccharides, proteins, lipids and
nucleic acids. Formation of biofilm over the mineral surface
induces the change in surface properties like surface charge,
surface composition and hydrophobicity. The change in surface
properties of the mineral particles due to the biofilm formation
can alter their floatation properties. Hence, a large no of micro-
organisms have been studied for mineral bio-beneficiation or
flotation. For example, Mycobacterium phlei cells have been used
as flotation collectors for hematite (Misra and Smith 1991) and
as flotation depressants in the anionic flotation of apatite and
dolomite (Zheng et al. 1998). Staphylococcus carnosus and

Bacillus firmus were found to work as collectors for apatite
(Smith and Mittinen 2006). Acidithiobacillus ferrooxidans for-
merly known as Thiobacillus ferrooxidans was used in removing
pyrite from mixtures of sulfide minerals (Nagaoka et al. 1999).
Bacillus polymyxa was used in the separation of sulfide minerals
(Sharma et al. 2001). The attachment of microorganisms to the
minerals alters the surface properties of the minerals, facilitating
beneficiation of minerals of particular interest. The widely stu-
died microorganisms belong to chemolithotrophic group
(Acidithiobacillus, Leptospirillum, etc.) have been successfully
used for bioleaching of sulphidic minerals are also studied for
mineral bio-beneficiation.
According to Natarajan (2003), chemolythotrophic bacter-
ium Acidithiobacillus thiooxidans was successfully used for flota-
tion of sulfide minerals. The author studied the flotation
behavior of galena (PbS) and sphalerite ([Zn, Fe]S) mixture
treated with the bacterium. The flotation results revealed that
bacterial treatment enhanced the floatability of sphalerite and
depressed galena. The bacterial treatment with the sphalerite and
galena brought about an alteration of surface properties of these
minerals. This might be due to the adhesion of bacterial cells as
well as their metabolic products (proteins and EPS) significantly
altering the surface chemistry of these minerals. The author also
reported that the isoelectric points of both galena and sphalerite
were shifted toward higher pH values after interaction with the
bacterium. The shift in isoelectric points observed might be due
to specific adsorption of bacterial cells to the mineral surfaces.
The change in the surface chemical properties brought about by
bacterial interactions influenced upon the mineral flotation of
both galena and sphalerite.
Furthermore, Hosseini et al. (2005) reported the potential use
of bacterium Acidithiobacillus ferrooxidans in the froth flotation
of Sarcheshmeh copper ore of Iran. A pure strain of A. ferroox-
idans was used to influence surface properties of pyrite and
chalcopyrite, and subsequently flotation study was conducted.
The iron and sulfur metabolizing bacterium A. ferrooxidans is an
acidophilic bacterium (Behera et al. 2012). During the study the
bacterium was cultured in 9K medium with pH 1.9 and tem-
perature 32°C. Further, at the beginning of the stationary growth
phase, the bacterial cells are harvested in acidic water (pH 1.9)
for the flotation process. The bacterial cells harvested in acidic
water were used for the flotation of minerals. Al the flotation
tests were conducted at the natural pH of the respective minerals
in bacterial solution. In the flotation study, it was observed that
in the presence of A. ferrooxidans, and xanthate as a collector, the
pyrite flotation was depressed, whereas chalcopyrite and other
sulfide minerals were unaffected. A. ferrooxidans is a chemo-
lithotrophic bacterium and it has the ability to metabolize iron
and sulfur compounds. Selective adhesion of A. ferrooxidans,
cells on pyrite surface inhibited the attachment of collector
(xanthate) molecules to it; consequently the pyrites were
depressed during the process. The selective adhesion of the A.
ferrooxidance bacterium to pyrite surface attribute to the pre-
sence of various external structures like flagella, pili, fimbriae,
surface proteins and EPS on the bacterial cell surface favors its
adhesion to a pyrite surface. The chemolithotrophic nature of
the A. ferrooxidance bacterium generates Fe (III) via the bioox-
idation of the pyrite. The Fe (III) generated is engaged in the
degradation of pyrite molecules (Klingl et al. 2011). The
MINERAL PROCESSING AND EXTRACTIVE METALLURGY REVIEW 3
degradation of the pyrite surface brought about the changes in
surface properties and alters its surface properties. In other hand,
toxic nature of copper decreased the attachment affinity of
bacterium on chalcopyrite surface. As a result the surfaces of
chalcopyrites unoccupied by bacterium are covered by the col-
lector (xanthate) molecules and favored its selective floatation.
The adhesion of bacteria to pyrite surface induced hydrophilicity
and depression pyrite was observed. The changes in iso-electric
points and hydrophobicity of sulfur compounds like pyrite,
chalcopyrite surface were observed due to interaction with A.
ferrooxidans cells (Devasia et al. 1993).
Vilinska and Rao (2008), studied the use of bacterium
Leptosririllum ferrooxidans for flotation of chalcopyrite and pyr-
ite. They have conducted flotation experiments in Hallimond
tube by using the L. ferrooxidans as depressants and potassium
isopropyl xanthate as collector. The cells are adsorbed on
mineral surface through electrostatic interactions between the
functional groups on the bacterial cell surface and the metal ions
on the minerals. Due to the bacterial adhesion to the mineral
surfaces, the chalcopyrite recovery was depressed from 95% to
25%, while recovery of pyrite was depressed to 67% from 100%
in a flotation process conducted at pH 4 with a cell concentration
of 2.5×108 mL–1, collector concentration 0.5×10–4 Mol/L.
Bacteria strains of genus Leptospirillum are spiral shaped,
gram-negative and obligatory chemolithotrophic microorganism.
They metabolize carbon by the Benson-Calvin cycle through
oxidation of ferrous iron in the presence of oxygen as terminal
electron acceptor. The bacterium is considered as most metabo-
lically restricted microorganism, because, it uses only ferrous iron
as a source of electron for their metabolism. Therefore, they have a
high affinity for ferrous iron in compared to the cells of
Acidithiobacillus (Coram and Rawlings 2002). The higher L. fer-
rooxidans cell adsorption density on chalcopyrite in comparison
to pyrite attribute to its higher accessibility for ferrous iron atoms
present in the chalcopyrite molecules (Vilinska and Rao 2008).
The high surface area of chalcopyrite in comparison with pyrite
coupled with the ferrous iron as an exclusive energy source for the
bacterium is the favorable condition for selective adhesion of L.
ferrooxidans cells to chalcopyrite.
In another study by Farahat and Hirajima, (2012), has shown
utilization of Ferroplasma acidiphilum bacterium for pyrite
depression. The authors reported that under acidic environment
(pH 3.8) about 99% pyrite flotation was achieved in the presence
of xanthate as flotation collector. However, the pyrite flotation
was suppressed to 16% with the bacterial pre-treatment. The
study suggests that, the bacterial pretreatment induced hydro-
philic properties o pyrite surface and as a result it was suppressed
during its flotation. Analysis of F. acidiphilum cell surface
revealed that its outer cell membrane rich in phospholipids
and glycophospholipids. These lipid molecules are rich in hydro-
philic phosphate and OH groups and the adhesion of bacteria to
pyrite molecule induced hydrophilic nature on pyrite and thus
its flotation was suppressed.
Patra and Natarajan (2004), reported about the successful use
of cells and metabolic products of Bacillus polymyxa bacterium
in flocculation and flotation to remove chalcopyrite from quartz
and calcite. Adsorption studies revealed that the bacterial
adsorption was more on the chalcopyrite surface in comparison
to quartz and calcite. The electro kinetic properties of the
4 S. K. BEHERA AND A. F. MULABA-BAFUBIANDI
mineral surfaces before and after interaction with bacteria were
investigated. Bacterial by product such as extracellular bacterial
protein and extracellular bacterial polysaccharides were isolated
and their individual effects on minerals were studied through
flocculation and flotation experiments. Selective separation of
chalcopyrite from quartz and calcite was achieved through inter-
action with extracellular protein. Extracellular bacterial protein
flocculated chalcopyrite and quartz. Subsequently, an enhance-
ment in the hydrophobicity of quartz surfaces on interaction
with bacterial protein resulted in higher separation from chalco-
pyrite through flotation.
Sarvamangala et al., (2013), reported about the use of the
bacterium Bacillus subtilis in microbial flotation to remove pyrite
from galena. During the study the selective separation of pyrite
from galena was achieved through bacterial treatment. The
authors achieved galena flotation about 92% by bacterial pre-
treatment. The findings of the authors suggested that the bacter-
ium has a higher absorption affinity for pyrite in comparison to
galena. Adhesion of the bacterium to the pyrite surface induced
hydrophilic property on it. The gel electrophoresis revealed the
expression of mineral-specific stress proteins by the bacterium
due to stimulation of minerals. The toxic nature of the lead
associated with galena induced stress proteins by the bacteria.
However, no specific proteins are expressed by the bacterium in
the presence of pyrite. Adhesion of bacteria cells to the pyrite
surface induced hydrophilic characteristics due to the presence
of proteins, peptidoglycan and polysaccharides on the outer
membrane of the bacterium and consequently, the pyrite was
depressed and galena was floated by the process.
Further, the bacterium Bacillus subtilis was also used as
depressant for removal of sulfur and ash content of coal.
Abdel-Khalek and El-Midany, (2013), used Bacillus subtilis bac-
terium as a depressant for sulfur and mineral matters associated
with Egyptian coal. The authors conducted coal flotation in a
Denver cell with 50 g of bacterial pre-conditioned ground coal
sample and at pH 3 for 3 min at 1000 rpm. During the study,
more than 70% of sulfur and ash content was removed from the
coal. In a recent study by Lopez et al. (2015) has shown the
utilization of Rhodococcus ruber a hydrophobic bacterium as
bio-collector for hematite flotation. The bacterial adhesion and
zeta potential analysis revealed that the bacterium has strong
adsorption affinity for hematite molecules at pH 3. Thus adhe-
sion of the bacterium to hematite surface induced hydrophobi-
city and favored its flotation. In this study, about 84% of
hematite flotation was observed at a pH value around 3, by
using bacterial cell concentration 109 cells/ml.
Bacterial strain Rhodococcus opacus, was evaluated by Botero
et al. (2007), as a bio-collector for flotation of calcite and mag-
nesite. R. opacus is a unicellular gram positive and nonpatho-
genic bacterium. According to the authors, the microorganism
was used as a bio-collector in the mineral flotation test, the
magnesite showed better flotability than calcite. Further the
adhesion test and the study of the electrophoretic behavior of
the minerals are conducted by the authors confirmed that the
high affinity of R. opacus toward magnesite surfaces than calcite.
High adsorption of bacterial cell on magnesite is the reason
behind its better floatbility. Presence of polysaccharides,
carboxylic acids, lipid molecules and micolic acids in the cell
membrane of the R. opacus is responsible for its amphoteric
nature (Stratton et al. 2002). The interaction R. opacus cells
with magnesite and calcite modified their surface charge and
properties. Similarly, in a recent study R. opacus cells are used as
bio-collector for separation of malachite from silica (Kim et al.
2016). The authors conducted the flotation experiments con-
ducted in 1L Denver flotation cell with 10% pulp density of
malachite (particle size -150µm) and silica (particle size –250
µm) mixture with DowFroth-250 as frothing agent and R. opacus
as bio-collector. More than 90% malachite recovery was achieved
from the mixture of malachite and silica during the flotation
process conducted for 5 minutes with 10% pulp density at pH 7,
1mM ionic strength, 1200 RPM impeller speed, and 1010 cells/ml
cell contraction. The selective attachment of hydrophobic R.
opacus cells to the malachite surface favored its flotation.
Furthermore, the R. opacus bacterium was also used as bio-
collector for flotation of phosphate mineral apatite (Merma et al.
2013). In this study, about 90% of apatite flotation was achieved
by the authors in micro flotation experiments conducted in
Hallimond tube at pH about 5. The electrophoretic properties
of apatite after bacterial interactions indicate that, the R. opacus
cell surface showed negative charge over a wide range of pH and
its isoelectric point is about 2.8. The acidic isoelectric point of
bacterium indicates the bacterial cell surface is occupied by more
anionic groups in comparison to cationic groups. The zeta
potential analysis of apatite after bacterial interaction indicate
maximum number of bacterial cells absorbed to apatite at pH 5
and concurrently maximum flotability of the apatite achieved at
pH 5. Naturally the mineral surface is hydrophilic in nature.
However, hydrophobicity induced by the bacterial attachment to
the apatite surface favored its flotation. A brief summary of the
bacterial assisted flotation process for different minerals is pre-
sented in Table 1.
Bio-molecules in mineral flotation
Conventionally, synthetic chemicals of petroleum origin have
been used for mineral flotation. Synthetic reagents used in
froth flotation are now coming to a turning point on their
hazardous problems to living organisms. In the recent devel-
opments, metabolites of microbial origin have been used as
collectors, modifiers and frother for mineral flotation.
Amongst the microbial metabolites, biosurfactants are well
acknowledged as potentially constitute for their chemical
counterparts due to structural similarities with the synthetic
petroleum chemicals. Use of biosurfactants as reagents in the
bioflotation process of mineral processing operations is a new
way for upgrading minerals. In recent years, several studies
have been carried out on the use of biosurfactants as flotation
reagents. Adsorption of biosurfactant to a mineral surface
alters its surface properties (hydrophobicity and hydrophilli-
city) and facilitates the flotation process. Apart from the
biosurfactants, metabolites such as EPS, nucleic acids have
been reported for their application in mineral flotation.
Biosurfactants as mineral flotation reagent
Surfactants are those compounds which tend to reduce the sur-
face tension of liquid in which it dissolves (Didyk and Sadowski
2012). The profuse uses of chemical surfactant are toxic and have
 MINERAL PROCESSING AND EXTRACTIVE METALLURGY REVIEW 5

Table 1. Applications of microorganism in mineral flotation process.

Microorganisms
Rhodococcus opacus
Rhodococcus opacus
Rhodococcus opacus
Staphylococcus carnosus
Rhodococcus ruber
Bacillus subtilis
Leptosririllum ferrooxidans
Stenotrophomonas
Escherichia coli
Acidithiobacillus ferrooxidans
Ferroplasma acidiphilum
Flotation
Flotation of malachite-silica mixture was
conducted in Denver cell (1L) by using R.
opacus as bio-collector and DowFroth-250 as
frother.
Micro flotation of apatite and quartz mixture
conducted in by using R. opacus as bio-
collector and bio-frother
Micro flotation of Calcite and magnesite
conducted by using R. opacus as bio-collector
Staphylococcus carnosus bacterium used as
bio-collector for micro-flotation of coal.
Rhodococcus ruber used as bio-collector for
hematite flotation in Partridge–Smith
flotation cell
Bacillus subtilis used as depressant coal
flotation conducted at pH 3 in a Denver
flotation cell without any frother for 3 min.
Flotation of chalcopyrite and pyrite
conducted in Hallimond tube by using
Leptosririllum ferrooxidans as depressants and
potassium isopropyl xanthate collector.
Hematite flotation was conducted in 30ml
micro-flotation cell with pure hematite
(30µm) by using Stenotrophomonas as bio-
collector
Escherichia coli cell used as bio-collector for
quartz flotation in Hallimond tube.
Acidithiobacillus ferrooxidans, used as pyrite
depressant reagent along with kerosene as a
collector and MIBC as frother in coal flotation.
Ferroplasma acidiphilum used as depressant
for pyrite flotation in the presence of sodium
isopropyl xanthate as collector at pH 3.8
Highlights of Microbial Flotation process References
More than 90% malachite recovery was Kim et al. 2016
achieved from the binary mixture during the
flotation conducted for 5 minutes with 10%
pulp density at pH 7
About 90% apatite flotation was achieved in Merma et al. 2013
the micro flotation process conducted at pH 5
with bacterial biomass concentration 0.15g/L
for 5 min.
93% of magnesite flotation was achieved at Botero et al. 2007
pH 5 with R. opacus concentration 100 ppm
and 55% of calcite was floated at pH 7 with
bacterial concentration 220 ppm.
About 90% of organic matter recovered from Ramos-Escobedo et al. 2016
bacterium treated cola in the micro-flotation
conducted at pH 9, for 6 min.
84% hematite flotation was reported by the Lopez et al. 2015
process operated at pH 3, from hematite
sample feed 0.6 g/L with a particle size range
between- 53 to +38 µm.
More than 70% of sulfur and ash content of Abdel-Khalek and El-Midany 2013
the coal were removed from the feed
conditioning with 2×10–6 cells/cm3 B. subtilis
concentration.
Chalcopyrite recovery was depressed from Vilinska and Rao 2008
95% to 25%, while pyrite recovery was
depressed to 67% from 100% during the
flotation process conducted at pH 4 with cell
concentration of 2.5×108 mL–1, collector
concentration 0.5×10–4 mol/L
More than 75% hematite recovery was Yang et al. 2013
observed in the flotation process conducted
at pH 6 with bacterial cell concentration of 60
mg/L.
90% of the quartz floated at pH 2.5 with Farahat et al. 2009
bacterial cell concentration 1.5 × 109 cells/ml
in a flotation time of 3 min.
The bacterial treatment reduced the pyrite Amini et al. 2009
flotation by 14%
Flotation of pyrite was depressed from 99% Farahat and Hirajima 2012
to 16% by the bacterial treatment.

a hazardous effect on the environment. Hence, biodegradable
surfactants of microbial origin are gaining importance in indus-
trial applications (Cameotra and Makkar 2010). Biosurfactant
molecule is a single amphiphilic organic molecule which has a
hydrophilic head group and hydrophobic tail group. The hydro-
philic head is represented by sugar and sugar derivatives groups,
however the hydrophobic tail group is represented by fatty acid
groups. Hence, the biosurfactant molecules readily adsorb to a
solid–liquid interface. Adsorption of biosurfactant molecule is
facilitated by electrostatic interactions between its head group
with the oppositely charged mineral surface. Adsorption of
biosurfactant molecules brought changes in the properties of
the mineral surfaces. The rhamnolipids of microbial origin
have been widely studied biosurfactant in mineral bioflotation.
Chemically rhamnolipids have a hydrophilic glycosyl i.e. rham-
nose head group, and a hydrophobic fatty acid tail group.
Further, there are two main classes of rhamnolipids, mono-
rhamnolipids and di-rhamnolipids; consisting of one or two
rhamnose groups respectively [Figure 1a and b]. Due to such
chemical characteristic features of rhamnolipids it can be
absorbed into selective mineral surfaces and modifies the surface
property. Hence, these groups of biosurfactants have been used
as a bio-collector in mineral flotation process.
Didyk and Sadowski, (2012), studied the bio-collector effi-
ciency of bio-surfactants produced by Bacillus circulans and
Streptomyces sp., for separation of quartz and serpentinite.
Biosurfactants produced by these microbes were used as bio-
collector for micro flotation studies. The micro flotation
experiments conducted by using the biosurfactants as bio-col-
lector and nickel ions as activators. About 68% quartz recovery
was observed in the process conducted in the presence of
biosurfactant producing broth of Bacillus circulans and activa-
tor (Ni+). In the presence of the activator ions (Ni+) on quartz
and serpentinite surfaces develops a local positive charge which
favored electrostatic interaction with anionic surfactant mole-
cules and induced hydrophobic character on the minerals.
A variety of microorganisms, such as bacteria, yeasts and fungi,
are able to produce biosurfactants. Compared to synthetic surfac-
tants, biosurfactants have a higher surface-activity, lower toxicity,
higher biodegradability and better environmental compatibility;
therefore they have been widely applied in environmental reme-
diation, pharmaceutical, cosmetics, and food industries (Singh
et al. 2006). Biosurfactants are categorized according to their
chemical composition of hydrophobic tail and hydrophilic head
moieties. Among them, rhamnolipid a glycolipid-type biosurfac-
tant are the most important biosurfactant molecule studied as it
6 S. K. BEHERA AND A. F. MULABA-BAFUBIANDI
Hydrophilic head
HO
CH3
HO HO
Hydrophilic head
HO
CH3
HO
O (CH2)n = 4,6, 8
HO
CH3
HO HO
Figure 1. General structures of rhamnolipid biosurfactant molecules (a) mono-rhamnolipid, (b) di-rhamnolopid. The amphiphilic nature of the molecules are due to
the presence of hydrophilic rhamnose (sugar) head and hydrophobic fatty acid tail.
reduces surface tension of water (Mata-Sandoval et al. 1999; Wei
et al. 2008). Due to the amphiphilic nature as well as their
structural similarities with chemical frothers, the microbial sur-
factant rhamnolipids are widely studied reagent in flotation as
bio-collector and frother. Application of rhamnolipid biosurfac-
tants in mineral flotation have been reported by several research
groups. Among the various species of biosurfactants, much work
has been done on rhamnose containing microbial surfactants
produced by Pseudomonas aeruginosa strains. The effect of a
rhamnolipid biosurfactant produced by strains of Pseudomonas
aeruginosa in mineral beneficiation studies.
Khoshdast and Sam (2012), studied about the flotation per-
formance of a copper ore sample in the presence of a rhamno-
lipid bio-surfactant produced by Pseudomonas aeruginosa MA01
strain. The microbial rhamnolipid was first investigated with
respect to surface tension and frothing characteristics applied
to copper ore flotation as a frother and co-frother. The authors
studied frothing and surface properties of the rhamnolipid in
comparison to two conventional frother, such as, methyl isobu-
tyl carbinol (MIBC) and DF-250. In their study, the authors
observed the rhamnolopid molecule has higher frothability and
surface activity compared to the synthetic frothers. The observed
higher surface activity of the rhamnolipid molecule is attributed
to its higher molecular weight with the presence of multiple
oxygenated units in its structure.
Hydrophobic tail
O COOH
(CH2)6 (CH2)n = 4,6, 8
CH3 CH3
(a)
Hydrophobic tail
COOH
O
(CH2)6
CH3
CH3
(b)
Khoshdast and Shojaei (2012), studied the effect of a concen-
trated rhamnolipid biosurfactant on the ash removal performance
of a coal sample by froth flotation. The surface and frothing
properties of a rhamnolipid biosurfactant produced from a
Pseudomonas aeruginosa strain were characterized. Results
showed that rhamnolipid has high surface activity and frothing
power in comparison to pine oil (petroleum frothing agent). The
dynamic frothability index (FDI) measurement studies of pine oil
versus rhamnolipid biosurfactant revealed that the rhamnolipid
has high FDI value in comparison to pine oil. The Rhamnolipid
has a higher molecular weight compared to pine oil and due to,
the higher molecular weight it higher surface activity of rhamno-
lipid. Chemically pine oils are mainly composed of α-terpineol
(C10H17OH) containing only one –OH group which interact with
water molecules to form a monolayer on its surface. On the other
hand, the rhamnolipid contains multiple no hydroxyl groups and
these can interact with water molecules through hydrogen bond-
ing. Due to multiple hydrogen bonding with water, the rhamno-
lipid molecules closely packed and tend to lie on the water surface
as a cohesive layer. Thus, it was found that rhamnolipid has high
surface activity and frothing capacity in comparison to pine oil as
conventional frother.
Further, Fazaelipoor et al., (2012), studied the use of bio-
surfactant molecules isolated from Pseudomonas aeruginosa as
frother for flotation of coal. Flotation experiments performed

in recovery of combustible materials from the coal sample in a
1 L capacity Denver cell with tap water (pH 7). The process
was operated for 6 min with impeller speed 1000 RPM, by
using biosurfactant as frother and gas oil as a collector. About
72%–79% combustible matter recovery with 10%–15.5% ash
content was obtained in the flotation process. In this study,
the authors compared the frothing properties of the microbial
surfactant with a conventional frother methyl isobutyl carbi-
nol (MIBC). From the surface tension measurement studies, it
was observed that the biosurfactant showed higher surface
activity and froth heights in comparison to the MIBC at
equal concentrations.
EPS of microbial origin as flotation reagent
Govender and Mariekie (2011), reported about use of microbial
EPS extracted from of acidophilic chemolithotrophic bacteria
consortium for flotation of chalcopyrite and pyrite ores.
Particularly, the acidophilic chemolithotrophic bacteria are
widely studied microorganisms for bioleaching of sulfide
minerals. The prominent bacterial strains studied for bioleach-
ing process are concentrated on the strains of Acidithiobacillus
ferrooxidans, Acidithiobacillus thiooxidans and Leptospirillum
ferrooxidans. These bacteria selectively attach to sulfide mineral
surfaces forming a biofilm or an EPS layer (Sand et al. 2001;
Olson et al. 2003). Govender and Mariekie (2011), studied the
use of EPS extracted from bioleaching consortia, for chalcopyr-
ite flotation and compared the flotation efficiency of EPS with
the sodium isobutyl xanthate (SIBX). Laboratory scale flotation
studies conducted by using free EPS as collector recovered
about 77% chalcopyrite when chalcopyrite was floated alone
and about 70% of chalcopyrite recovery was achieved from the
flotation of chalcopyrite and pyrite mixture under the similar
conditions. However, in case of flotation process conducted by
using SIBX as flotation collector, the chalcopyrite recovery was
only 32%. These results indicated that EPS could be a green
reagent for mineral flotation process. Biochemical analysis of
the free EPS extracted from bacterial consortium mainly con-
stituted of carbohydrates, proteins and uronic acids. The
hydrophobic amino acids present in proteins present of EPS
impart hydrophobocity to the mineral surface while polysac-
charides present in EPS confer hydrophilicity (Vilinska and
Rao 2008). Therefore, the proteins present in the EPS aided
selective separation of chalcopyrite.
Deoxy-ribose nucleic acid as flotation reagent
Deoxy-ribose nucleic acid (DNA) is well known for carrying
genetic information. In the recent study the DNA molecules
have been used as a collector reagent in mineral flotation
process (Vasanthakumar et al. 2012). The authors investigated
the selective flotation of sphalerite ([Zn Fe] S) from a mixture
of sphalerite and galena (PbS) by using genetic materials of
Bacillus species. The DNA molecule of the bacterial cell was
primarily responsible for the flotation of sphalerite, which
functions as a bio-collector. In this study, the authors have
identified that DNA, or more specifically single stranded (SS)
DNA molecules, acts as a bio - collector for the selective
flotation of sphalerite from a sphalerite-galena mineral mixture
MINERAL PROCESSING AND EXTRACTIVE METALLURGY REVIEW 7
(Vasanthakumar et al. 2014). The amphipathic nature of
ssDNA facilitated it to play the role of collector in the flotation
process. The amphoteric nature of the ssDNA molecule is due
to the presence of hydrophilic phosphate backbone and hydro-
phobic aromatic nitrogenous bases (Figure 2). The hydropho-
bic face of the ssDNA represented by negatively charged
phosphate groups which electrostatically interact with the spha-
lerite and galena surface and the exposed hydrophobic surface
of ssDNA facilitated the flotation of minerals. The authors
found higher recovery of sphalerite in comparing to galena
during the flotation process performed with DNA as a bio-
collector. This might be due to the differential affinity of DNA
toward lead in galena (PbS) and Zn in sphalerite ([Zn, Fe] S)
(Duguid et al. 1993). In addition to it, the lattice structure of
sphalerite might be more amenable to interact with ssDNA in
comparing to galena. For these reasons, ssDNA may not induce
sufficient hydrophobicity to galena and hence its recovery was
low during the flotation process. In addition to the above
studies a brief summary of microbes and microbial metabolite
assisted flotation studies for different mineral is presented in
the Table 2.
Challenges and future prospective of minerals bio-
flotation
Environmental concerns regarding the use of chemical reagents in
the froth flotation of minerals have led investigators to explore
microbes and microbial reagents as potential substitutes. Despite
the fact that several microorganisms have been used in mineral
flotation process for a variety of mineral systems, a detailed
characterization of the bio-molecular mechanisms involved have
not been extensively reported so far. The growing demand of
mineral commodities with limited mineral resources available
across the world has led to the increased exploitation of lean
grade ores for the production of base metals, precious metals
and rare metals. Additionally, depletion of the high grade mineral
resources has resulted in the search of more advanced processes
for beneficiation of ores and minerals where conventional techni-
ques are not efficient. The conventional froth flotation process has
been industrially well accepted process for ore beneficiation.
However, the synthetic flotation reagents such as collector,
frother, and surface modifiers are toxic and hazardous in nature.
The rising demands for metals with, stricter environmental legis-
lation prompted for process with lower operating costs. In this
regard the use of microorganisms and their metabolites as envir-
onment friendly flotation reagents are the new developments
achieved in the area of bio hydrometallurgy. The microorganism
cell surface principally consists of functional groups derived from
phospholipids, proteins, and polysaccharides. Some of these
induce hydrophobic properties, since they can adhere selectively
to the mineral surface. Several studies have explored the interac-
tion between specific microbial cells and mineral particles bring
about significant changes in the chemistry of mineral surfaces.
The scientific studies conducted in the area of microbial flotation
gathered a wealth of information. However, a detailed under-
standing of the nature and characteristics of the bio reagent
responsible for a selective flotation of any given mineral from
the mineral mixture needs to study. Such development could
8 S. K. BEHERA AND A. F. MULABA-BAFUBIANDI

O
CH 2
H
O
+ O =P O CH 2 Thymine
+ +
Sphalerite +
+ (Zn, Fe)S
_O
+
+ +

O Guanine
O =P O CH 2
NH 2
_O
NH 2

+ O
+ +
Sphalerite + Cytosine
+ (Zn, Fe)S
O =P O CH 2
+ _O
+ +
NH 2

O
O =P O CH 2
+
+ + _O
Sphalerite + Adenine
+ (Zn, Fe)S
+
+ +
O

Figure 2. Schematic presentation of a single stranded DNA showing its amphiphilic nature. The hydrophilic surface represented by phosphate groups and
hydrophobic surface is represented by the aromatic nitrogenous bases. Phosphate ions in the hydrophilic surface of the ssDNA electrostatically interacted with
mineral surface (sphalerite [(Zn, Fe) S]) molecules and the free hydrophobic surface renders hydrophobic nature to mineral surface favours its flotation
(Vasanthakumar et al. 2012).

induce the first step in the large-scale generation and application
of bio-reagents in the flotation of minerals at industrial scale.
Compared to conventional inorganic reagents, microorgan-
isms and their metabolites used so far are biodegradable, non-
toxic and environmental friendly. The notable microbial strains
belong to the genera of Acidithiobacillus, Leptospirillium, etc.,
have been successfully used in mineral processing work are
nonpathogenic and human friendly in nature. In addition,
other nonpathogenic microbes such as strains of Bacillus and
Yeast, etc., are also used for flotation studies in laboratory scale
(Menezes et al. 2011; Vasanthakumar et al. 2014). Such
microbes are nonpathogenic and are omnipresent hence these
microbes can be effectively used for large scale application in
mineral industries. Furthermore, several bio-molecules such as
proteins, EPS, DNA, bio-surfactants have been reported as
potential mineral flotation reagents. In the last decades, bio-
surfactants have been used in several biomedical applications
such as anti-cancer, anti-bacterial agent, food preservatives, and
bioremediation of metals (Singh et al. 2006). The wider appli-
cations of microbial surfactants attribute to its human friendly,
nontoxic and biodegradable properties (Didyk and Sadowski
2012). Hence, industrial uses of surfactant molecules are gain-
ing momentum. Till date uses of microorganisms and their
metabolites for mineral flotation have been done mostly in
laboratory scale. Industrialization of microbial flotation relies
on the large scale production of microbial reagents. The large
scale propagation of microorganisms can be achieved by design
and fabrication of bioreactors. The advances of genetic
engineering and recombinant DNA technology can be used
to develop nonpathogenic, high yielding variety of microorgan-
isms suitable for industrial use.
Conclusion
From the aforesaid discussion, it might conclude that, the micro-
bial mineral flotation processing route can be a simple and
effective technology for mineral beneficiation. This article
focused on the previous studies which were concerned with the
flotation of different minerals by using microbes and microbial
produced surface active bio-molecules. The bioflotation process
may be defined as one in which microorganisms act as reagents,
collectors or modifiers, to facilitate the selective separation of
minerals. The use of bioreagents as collectors invokes several
interfacial aspects of the interacting biological and geological
materials, viz., the physicochemical properties of the mineral
surface, such as the atomic and electronic structure, the surface
charge, the acid–base properties, and the wettability of the sur-
face. Several microbes and microbial metabolites have been
identified for their possible use as bio-flotation reagents.
Microbial strains of Mycobacterium phlei, Paenibacillus poly-
myxa, Rodococcusopacus, Acidithiobacillus ferrooxidans,
Acidithiobacillus thiooxidans, Leptospirillium ferrooxidans have
been successfully demonstrated for mineral flotation uses. Use of
microbial metabolites as flotation reagent in industrial scale
relies upon their large scale production and purification in an
economic way. Application of advanced molecular biology and
 MINERAL PROCESSING AND EXTRACTIVE METALLURGY REVIEW 9

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