Int. J. Miner. Process.

74 (2004) 143 – 155

www.elsevier.com/locate/ijminpro

Microbially induced flocculation and flotation for separation of

chalcopyrite from quartz and calcite
Partha Patra, K.A. Natarajan *
Department of Metallurgy, Indian Institute of Science, Bangalore 560 012, India

Received 16 August 2003; received in revised form 26 October 2003; accepted 26 October 2003

Abstract

Cells and metabolic products of Bacillus polymyxa were successfully used in flocculation and flotation to remove
chalcopyrite from quartz and calcite with a view to environmental protection and ore beneficiation. Adsorption studies revealed
that the magnitude of adsorption density of bacterial cells onto chalcopyrite was the highest in comparison to quartz and calcite.
Electrokinetic behavior of the mineral surfaces before and after interaction with bacteria was investigated. Bacterial byproduct
such as extracellular bacterial protein and extracellular bacterial polysaccharides were isolated and their individual effects on
minerals were studied through flocculation and flotation. Selective separation of chalcopyrite from quartz and calcite was
achieved through interaction with extracellular protein. Extracellular bacterial protein flocculated chalcopyrite and dispersed
quartz. Subsequently, enhancement in the hydrophobicity of quartz surfaces on interaction with bio-protein resulted in higher
separation from chalcopyrite through flotation.
D 2003 Elsevier B.V. All rights reserved.

Keywords: Bacillus polymyxa; chalcopyrite; microbially induced flocculation; flotation; environment

1. Introduction grade ores. Serpentinitic low-grade copper ores have

Chalcopyrite (CuFeS2) is the most abundant cop-
per bearing mineral and is consequently extremely
important to most of the copper producing industries.
Besides other sulfides like pyrite, chalcopyrite is
associated with oxide gangue minerals such as quartz
and calcite. The gradual exhaustion and/or impover-
ishment of available mineral resources will probably
lead to the search for more advanced solution to the
problem in processing some refractory ores and lean
* Corresponding author. Tel.: +91-80-3600120; fax: +91-80-
3600472.
E-mail address: kan@metalrg.iisc.ernet.in (K.A. Natarajan).
proved difficult to concentrate by conventional means,
e.g. by froth flotation, as these ores are often finely
disseminated, which necessitates very fine grinding
(Carta et al., 1980). In some cases, selective floccu-
lation (Yarar and Kitchener, 1970) may become a
useful method for the separation of minerals from
the gangue, especially when the particles are too fine
for successful flotation.
Utility of microorganisms in beneficiation and
bioremediation was understood and developed only
in the last decade. Considering the above scenario, a
system of chalcopyrite along with quartz and calcite
was chosen in order to separate chalcopyrite from
rest of the oxide gangue materials. Natarajan et al.
(1997) have recently carried out beneficiation stud-

0301-7516/$ - see front matter D 2003 Elsevier B.V. All rights reserved.
doi:10.1016/j.minpro.2003.10.006
144 P. Patra, K.A. Natarajan / Int. J. Miner. Process. 74 (2004) 143–155
ies on bauxite and iron ores using Bacillus polymyxa
(B. polymyxa). B. polymyxa is a Gram-positive,
neutrophilic, periflagellated heterotroph indigenously
associated with many mineral deposits. Exo-poly-
saccharides, proteins and organic acids such as
oxalic acid, formic acid and acetic acid are the
principal components of the metabolic product
obtained from B. polymyxa (Prescott and Dunn,
1987). In order to utilize the beneficial properties
of the microorganisms in microbe –mineral interac-
tion, surface chemical studies of both bacteria and
minerals along with the mutual attachment behavior
among them becomes imperative.
In the present investigation, surface chemical
changes brought about on minerals and cells of B.
polymyxa before and after interaction with each other
were studied through electrokinetic and adsorption
studies. Interaction with the concerned individual
products of the biomass (supernatant of B. polymyxa
culture containing mostly extracellular protein and
extracellular polysaccharides) was also carried out.
Settling behavior of various minerals were also carried
out before and after bacterial interaction. Similar tests
were also performed using bio-protein and extra
cellular polysaccharides secreted by the bacteria.
The adsorption of bacterial cells onto the mineral
surface was also studied to optimize the efficiency
of selective separation of chalcopyrite from a binary
mixture of chalcopyrite –quartz and chalcopyrite– cal-
cite and also to understand probable mechanisms.
Both flotation and flocculation studies were carried
out to establish the selective separation of minerals
after interaction with bacterial cells and bio-proteins.
The bioprocess so developed will be practically useful
in the removal of sulphide-bearing minerals such as
pyrite and chalcopyrite from waste ore burden as well
as flotation tailings with a view to environmental
protection.
2. Materials and methods
2.1. Minerals
Handpicked highly pure mineral samples of chal-
copyrite, quartz and calcite were obtained from
Alminrock, Indscer Fabriks, Bangalore, India. Chem-
ical, X-ray and mineralogical analyses were carried
out to ascertain the purity of the minerals. The purity
of the mineral was ascertained as chalcopyrite
99.9%, quartz 99.5% and calcite 99.98%. The above
samples were ground in a porcelain ball mill, sieved
and fractioned to obtain different size fractions
[( 105 + 74) and 37 Am]. The 37 Am fraction
was further ground and samples of f 5 Am size
fractions were obtained through sedimentation. Par-
ticle size analysis determined using a Malvern Zeta-
sizer gave an average particle size of f 3 Am for
chalcopyrite, quartz, and calcite. Mineral samples of
the above size fractions were used in electrokinetic
and adsorption studies. The surface areas of the
samples used in adsorption studies were determined
by BET nitrogen-specific surface area method. The
surface areas of the samples mineral samples were
established to be:
2.659 m2/g for quartz,
1.93 m2/g for chalcopyrite and
1.773 m2/g for calcite.
The ( 105 + 63) Am size fraction was used in
flotation studies.
2.2. Bacterial culture
Strains of B. polymyxa (NCIM 2539) used in this
study were obtained from National Collection of
Industrial Microorganisms, National Chemical Labo-
ratory, Pune, India. They were subcultured in the
laboratory using a modified Bromfield medium
(Bromfield, 1954).
Potassium nitrate was used to maintain the ionic
strength, while nitric acid and potassium hydroxide
were used as pH modifiers. All reagents used in the
present studies were of analytical grade. Deionised
double distilled water with a specific conductivity of
< 1.5 Amhos was used in all the tests.
The bacteria were cultured by inoculating 10 ml of
pure strain of the bacterial cells to the Bromfield
medium. This was incubated at 30 jC on a Remi
rotary shaker maintained at 240 rpm. A Petroff –
Hausser counter under a Leitz phase contrast micro-
scope (Laborlux K Wild MPS 12) was used to
determine the bacterial cell count. The change in pH
was monitored at regular time intervals (30 min) using
a Systronics digital pH meter.
 P. Patra, K.A. Natarajan / Int. J. Miner. Process. 74 (2004) 143–155
2.3. Preparation of cell pellet and cell-free metabolite
The fully grown bacterial culture (48 h) was
centrifuged (Sorvall RC-5B) at 10,000 rpm for 15
min at 5 jC. The supernatant was decanted and
filtered through sterile Millipore (0.2 Am) filter paper
to remove all insoluble materials and any bacterial
cells still left out. The cell pellet was washed with
deionised double distilled water and again centri-
fuged. This process was repeated twice to obtain pure
cell pellet.
2.4. Protein isolation from metabolite
One liter of batch culture of B. polymyxa obtained
after a growth period of 48 h was centrifuged. The
supernatant was filtered through sterile Millipore (0.2
Am) filter paper. Analytical grade, extra pure and fine
powdered ammonium sulfate was added slowly to a
saturation level of 90% (600.16 g/l) in cold condition
(4 jC) with constant shaking. The solution was
allowed to stay in refrigeration for 12 h at 4 jC.
The protein precipitate was dissolved in a minimum
volume of 1 M Tris hydrochloride buffer of pH 7. It
was dialyzed against the same buffer for over 18 h at 4
jC. The precipitate, which was formed during dialy-
sis, was removed by centrifugation and discarded. The
clear supernatant was lyophilised, weighed and kept at
4 jC for further use (Deutscher, 1990). The total
protein content was determined by the Lowry method
(Lowry et al., 1951). Bovine serum albumin was used
as a standard.
2.5. Isolation of extracellular polysaccharide (ECP)
from culture supernatant
One liter of completely grown (48 h) bacterial
culture of B. polymyxa was centrifuged to remove
cells. The supernatant containing the ECP was filtered
through sterile Milipore filter paper. It was then
lyophilised using Virtis Freezemobile 12EL lyophil-
iser at 80 jC at a vacuum of 100 mTorr. The
dehydrated solid substance was dissolved in 10 ml
of distilled millipore water and cooled to below 10
jC. A 20 ml of double distilled ethanol was added to
precipitate ECP from other components of the bacte-
rial supernatant. It was then kept stationary for 8 h at 4
jC in a refrigerator. The precipitate was washed with
145
double distilled water. The ethanol precipitation was
repeated two to three times for further purification to
separate polysaccharides. This polysaccharide solu-
tion was dialyzed with double distilled water. Before
dialysis tubes were boiled in a solution containing
0.01 M EDTA and 2% sodium bicarbonate for 10 to
15 min in a water bath. After dialysis ECP was stored
at a low temperature. The concentration of ECP
utilized was determined by the phenol-sulphuric acid
method (Dubois et al., 1956).
2.6. Adsorption studies
For adsorption tests, 1 g each of the individual
mineral sample was taken and pulped in 100 ml of
10 3 M KNO3 solution at the desired pH maintaining
a known concentration of bacterial cells in 250 ml
Erlenmeyer flasks. The suspension was agitated for 15
min on a Remi orbital shaking incubator at 250 rpm
and 30 jC. After equilibration, the slurry pH was
again recorded. The suspension was then centrifuged
at 2000 rpm for 5 min to separate the mineral particles
containing the cells. The supernatant solution contain-
ing the unabsorbed cells was further filtered through
Whatman No. 42 filter paper and the residual cell
population in the supernatant estimated.
2.7. Electrokinetic measurements
Zeta-potentials of chalcopyrite, quartz and calcite
suspensions before and after interaction with the
bacterial cells were measured as a function of pH
using a Malvern Zetasizer 3000 instrument. A 1 g of
the mineral sample was interacted with 5  108 cells/
ml at pH 7 and at a temperature of 30 jC for different
time intervals. After interaction, the mineral particles
were separated by centrifugation followed by filtration
and the mineral surface was washed two to three times
to remove any entrapped bacterial cells. The mineral
particles and cells were conditioned in 10 3 M KNO3
solution at the required pH in the range of 3 – 12 for 30
min prior to zeta-potential measurements.
2.8. Flocculation tests
For the flocculation tests 1 g of the mineral sample
was added to 100 ml cell suspension in a measuring
cylinder. The bacterial cells were obtained for the
146 P. Patra, K.A. Natarajan / Int. J. Miner. Process. 74 (2004) 143–155
purpose after centrifugation followed by washing the
cells in deionised double distilled water. Cell count
was adjusted before adding to the mineral mixture.
The bacterial cell count was maintained at 1  108
cells/ml. The cylinder was tumbled 10 times and kept
still for 2 min. The supernatant (90 ml) was carefully
decanted, filtered and weighted. Experiments were
carried out at different ranges of pH (6– 12) and time.
2.9. Selective flocculation tests
For these experiments, 2.5 g of each of the mineral
(d50 < 5 Am) was taken in a stoppered cylinder and
thoroughly mixed with 100 ml of cell suspension. The
flocculation test was performed as mentioned above
and the supernatant decanted, filtered and weighed as
before. Desliming was done five more times each 2
min duration. In each case, 90 ml of cell suspension
was added each time. Similar studies were performed
with metabolic products. Chalcopyrite – quartz and
chalcopyrite – calcite systems were studied separately.
Amounts of chalcopyrite, quartz and calcite in the
settled and dispersed fractions were determined
through chemical analysis using ICP spectrometry.
2.10. Microflotation tests
Initially, 1 g of desired mineral was pulped in 100
ml of deionised double distilled water in a conical
flask containing 5  108 cells/ml of bacterial cells at
neutral pH. The flask was incubated on a rotary shaker
for 30 min. After interaction with the bacterial cells,
the mineral particles were separated from free cells as
mentioned earlier. Flotation of the mineral was carried
out using a modified Hallimond tube (Fuerstenau et
al., 1957) using nitrogen at a flow rate of 40 ml/min
for 3 min. The settled and floated fractions were
separated, dried and weighed. Flotation studies were
also carried out after sequential interaction of the
mineral with bacterial cells and collector (Hexamine).
3. Results and discussion
3.1. Adsorption studies
The adsorption behavior of bacterial cells onto
chalcopyrite, quartz and calcite as a function of time,
different pH values and equilibrium concentration was
investigated and the results obtained are portrayed in
Figs. 1, 2, and 3.
Kinetics of bacterial cell adsorption was obtained
by determining the adsorption density of cells onto the
mineral with respect to time. Due to solubility of
calcite in acidic pH, the adsorption behavior of
bacterial cells was studied only beyond pH 7, whereas
for other minerals such as chalcopyrite and quartz, the
entire pH range was covered. Adsorption behavior as
a function of time was studied in the pH range of 7.5–
8.5 for chalcopyrite, quartz and calcite in 10 3 M
KNO3 electrolyte solution. The initial cell concentra-
tion was 5  109 cells/ml. Fig. 1 shows that the
adsorption density of bacterial cells onto chalcopyrite
was 8  108 cells/m2 in less than 5 min, 1  108 cells/
m2 in almost 20 min onto quartz and 3  108 cells/m2
in 10 min onto calcite. This suggests that chalcopyrite
has a very high affinity for the cells compared to
quartz. All subsequent adsorption experiments were
carried out at 20 min equilibration time since all the
three minerals reached saturation in adsorption density
within that period.
The adsorption of cells onto minerals as a function
of pH is given in Fig. 2. It was observed that there
was a decrease in adsorption density for bacterial cells
in the alkaline range of pH for chalcopyrite. Higher
affinity for bacterial cells onto the surface of chalco-
pyrite was observed at acidic pH values while it was
Fig. 1. Adsorption density of B. polymyxa cells onto quartz, calcite
and chalcopyrite as a function of time.
 P. Patra, K.A. Natarajan / Int. J. Miner. Process. 74 (2004) 143–155 147

Fig. 2. Adsorption density of B. polymyxa cells onto quartz, calcite

and chalcopyrite as a function of pH.

lower at higher alkaline pH values. This can be

attributed to formation of unstable Cu(OH)2 dissoci-
ating to Cu ions into the solution from the surface of
chalcopyrite. Adsorption density of bacterial cells
onto chalcopyrite was observed to be almost 10 times
higher than that onto quartz in the acidic pH range.
But for calcite no significant change in the adsorption
of bacterial cells was observed as a function of pH.
For chalcopyrite and quartz, the small decrease in cell
adsorption density at the initial stage could be attrib-
uted to the increase in surface negative charge with
increase in pH resulting in electrostatic repulsion. For
calcite, the interaction between calcite and bacterial
cells may be totally chemical in nature, as the
coulombic force does not play a major role (Deo,
1998).
Adsorption isotherms for bacterial cells onto chal-
copyrite, quartz and calcite were then established.
From Fig. 3(a) it can be observed that the amount of
bacterial cells adsorbed onto quartz initially increases
with corresponding increase in equilibrium cell con-
centration and then attains a steady state at all pH
values. The bacterial cells adsorb onto the mineral
surfaces until the viable sites get saturated after which
the attachment stops. An inhomogeneous but steady
monolayer formation can be assumed. The adsorption
isotherm can be categorized as L-2 type following
Giles classification (Giles et al., 1960). The adsorption Fig. 3. Adsorption isotherms of B. polymyxa cells onto (a) quartz,
is essentially Langmuirian. (b) calcite and (c) chalcopyrite.
148 P. Patra, K.A. Natarajan / Int. J. Miner. Process. 74 (2004) 143–155

From Fig. 3(b) it can be observed that, adsorption also observed that the surface becomes increasingly
of bacterial cells increases with corresponding in- negative as the pH of the solution increases and
crease in equilibrium cell concentration for calcite becomes almost stable in the alkaline pH range. After
also attaining a steady value there after. The adsorp-
tion behavior was similar to that on quartz but the
number of cells adsorbed at saturation point is higher
than that in case of quartz. A monolayer formation can
be assumed but the adsorption density is higher
compared to that on quartz. This also can be classified
as L-2 type (Giles et al., 1960) and conform to
Langmuirian behavior.
From Fig. 3(c) it can be readily observed that the
adsorption density of the bacterial cells onto chalco-
pyrite is much lower at alkaline pH. In the acidic pH
range of 4 – 6 the adsorption density of cells steadily
increases with equilibrium number of cells. Since in
the acidic pH the adsorption density does not have a
plateau we can conclude that the surface of chalco-
pyrite can accommodate higher bacterial cell adsorp-
tion. This can be classified as L-1 type by Giles
classification (Giles et al., 1960) and conforms to
Langmuirian behavior.
In the alkaline pH range the increase in adsorption
density was not significant and a steady state value
was observed with increase in equilibrium cell con-
centration. This phenomenon can be attributed to the
unstable copper hydroxide in alkaline pH range. At
alkaline pH range copper hydroxide formed on chal-
copyrite surface dissociates and release copper ions
into the solution leading to subsequent detachment of
bacterial cells.

3.2. Electrokinetic studies

A prior knowledge of the surface charges on both

the cells and the minerals is important to establish the
possibility of separation of chalcopyrite from quartz
and calcite. Zeta-potentials were measured to assess
the changes in the surface chemical characteristics of
both cells and minerals before and after mutual
interaction. The cell density used for these experi-
ments was 1  109 cells/ml and the interaction period
was varied from 1 to 24 h (1, 12, and 24 h). Fig. 4
shows the zeta-potential of the cells before and after
interaction with the minerals as a function of pH.
Bacterial cells possess a net negative surface charge Fig. 4. Measured zeta-potentials as a function of pH for B. polymyxa
over a wide range of pH values and the isoelectric cells before and after interaction with (a) quartz (b) calcite and (c)
point (IEP) was observed to be at about pH 2. It is chalcopyrite.
 P. Patra, K.A. Natarajan / Int. J. Miner. Process. 74 (2004) 143–155 149

interaction with the minerals the IEP shift for the concentrations (higher than 1  107 cells/ml), chalco-
bacterial cells was in a positive direction and a pyrite gets flocculated in less than 5 min. So in this
reduction in surface negative charge was noticed. experiment chalcopyrite was reacted with low con-
However, the positive shift was found to be quite
less with quartz (Fig. 4(a)) compared to calcite (Fig.
4(b)). This may be due to the secretion of proteina-
ceous compounds or possibly a chemisorption phe-
nomenon. Large positive shifts in the measured zeta-
potentials of cells after interaction with chalcopyrite
as shown in Fig. 4(c) can be attributed to cationic
copper and iron species or the secretion of a protein-
aceous compound by the bacteria due to interaction
with the minerals.
Surface chemical changes on mineral surfaces after
interaction with bacterial cells are illustrated in Fig.
5(a,b,c).
Surface chemical changes on quartz surface be-
fore and after interactions with bacterial cells are
depicted in Fig. 5(a). The IEP of quartz was ob-
served to be at pH 1.5. This value is in agreement
with that reported by other workers (Healy and
Moignard, 1976). The measured zeta-potentials were
found to shift to a more positive direction after
interaction with the cells. The surface became more
negative as a function of increase in pH values. The
IEP was also observed to be shifted to more positive
pH values after bacterial interaction. Both bacterial
cell surfaces and quartz surfaces possess a net
negative charge beyond a pH value of about 2.
Under these conditions electrostatic forces may not
play any role in bacterial adhesion. Deo and Natar-
ajan (1997) have shown that there is a significant
shift in the IEP of quartz (1.5 – 4.7 pH) after inter-
action with cells of B. polymyxa and their metabolic
products, mainly bio-proteins.
Zeta-potentials of calcite as a function of pH before
and after interaction with bacterial cells are shown in
Fig. 5(b). Zeta-potential of calcite gradually shifts to a
more negative direction with pH. Deo and Natarajan
(1998) have shown that the change is due to both the
extra cellular protein and polysaccharides, and is
chemical in nature.
Zeta-potential of chalcopyrite as a function of pH
before and after interaction with bacterial cells is
shown in Fig. 5(c). There is neither a significant
change in surface chemical charge nor in the IEP Fig. 5. Measured zeta-potential as a function of pH for (a) quartz,
value. Unreacted pure chalcopyrite is observed to (b) calcite and (c) chalcopyrite before and after interaction with cells
have an IEP in the pH range of 3 – 3.5. At higher cell of B. polymyxa.
150 P. Patra, K.A. Natarajan / Int. J. Miner. Process. 74 (2004) 143–155

centration of cells and hence the effect on the surface

charge was negligible even after long interaction
periods.

3.3. Flocculation tests

The settling behavior of chalcopyrite, quartz and

calcite mineral fines were established under different
experimental conditions such as in presence of bacte-
rial cells, extracellular bacterial protein, ECP and both
as a function of time and pH.
Fig. 6 shows the settling behavior of minerals in
the presence and absence of cells as a function of time
and pH. Adsorption studies discussed in the previous
section showed that the bacterial cells exhibited a
higher affinity towards chalcopyrite compared to
quartz. Adsorption density of bacterial cells followed
the trend:

chalcopyriteHcalcite > quartz

The settling rates of chalcopyrite and calcite were

observed to be enhanced in presence of bacterial cells
unlike the case with quartz suspensions.
From the results in Fig. 6(a), it is observed that in
absence of bacterial cells and at pH 6, settling rate of
quartz increased from 20% in 5 min to 70% in 20
min and at pH 8 it increased from 20% in 5 min to
55% in 20 min. In the presence of bacterial cells and
at pH 6, settling rate of quartz increased from 20%
in 5 min to 50% in 20 min and at pH 8, it increased
from 20% in 5 min to 45% in 20 min. It could
readily be concluded that interaction with bacterial
cells promoted dispersion of quartz rather than
flocculation.
From Fig. 6(b), it could be observed that at pH 8,
settling rate of calcite increased from 40% in 5 min
to 72% in 20 min and at pH 12, it increased from
40% in 5 min to 80% in 20 min. However, in the
presence of bacterial cells and at pH 8, settling rate
of calcite increased from 70% in 5 min to 90% in
20 min and at pH 12, it increased from 80% in 5
min to 95% in 20 min. This indicates that in
presence of bacterial cells settling rate of calcite as
a function of time was significantly higher compared
to quartz. Fig. 6. Settling behavior of minerals in absence and presence of
From Fig. 6(c), it is observed that in the absence of cells of B. polymyxa as a function of time and at selected pH, (a)
bacterial cells and at pH 6, the percentage of chalco- quartz, (b) calcite, and (c) chalcopyrite.
 P. Patra, K.A. Natarajan / Int. J. Miner. Process. 74 (2004) 143–155 151

pyrite settled increased from 20% in 5 min to 85% in

20 min, while at pH 8, the percentage of chalcopyrite
settled increased from 20% in 5 min to 55% in 20
min. Whereas in presence of bacterial cells percentage
of chalcopyrite settled at pH 6 was 90% in 5 min and
it increased to almost 95% in 20 min. At pH 8 chal-
copyrite settled was 90% in 5 min and increased to
almost 95% in 20 min. It could readily be concluded
that presence of bacterial cells significantly enhanced
the settling rate of chalcopyrite compared to quartz
and calcite.
Both the bacterial cell wall as well as their metabolic
products comprises of proteins and polysaccharides.
Hence to understand the bacterial cell activity towards
the minerals as observed in the previous sections the
flocculation behavior was studied in presence of these
metabolic products as well. The results are illustrated in
Fig. 7. Since calcite gets dissolved in the acidic pH, the
pH of investigation was restricted to a pH value of 8.
From the results in Fig. 7(a), it is observed that in
absence of any bacterial by-product and at pH 8 the
settling rate of quartz increased from 20% to 55% in 20
min and in presence of cells it increased from 20% to
40% in 20 min. When quartz was interacted with
extracellular bacterial protein and subjected to floccu-
lation, the settling rate of quartz was found to increase
from 20% at 5 min to less than 40% at 20 min. On
interaction with ECP the amount of quartz settled was
unchanged compared to the settling of pure quartz
alone. It then becomes clear that an increase in surface
hydrophobicity of quartz after bacterial interaction
could only be due to extracellular bacterial protein.
Presence of bacterial proteins promoted dispersion of
quartz, while that of exopolysaccharrides promoted
flocculation.
From Fig. 7(b), it could be observed that at pH 8,
the settling rate of calcite increased from 40% in 5 min
to 93% in 20 min. However, in presence of bacterial
cells and at pH 8, settling rate of calcite increased from
40% at 5 min to around 55% at 20 min. When calcite
was treated with extracellular bacterial protein and
subjected to flocculation, the settling rate of calcite
increased from 25% at 5 min to less than 30% in 20

Fig. 7. Settling behavior of minerals in absence and presence of

cells of B. polymyxa, extracellular bacterial protein and extracellular
polysaccharides as a function of time and at pH 8, (a) quartz, (b)
calcite, and (c) chalcopyrite.
152 P. Patra, K.A. Natarajan / Int. J. Miner. Process. 74 (2004) 143–155

20 min. The settling behavior in presence of extra-

cellular bacterial protein showed that, 90% of chalco-
pyrite settled in 5 min and 95% at 20 min. When
chalcopyrite was interacted with ECP and then sub-
jected to flocculation, the amount settled was not
significant as compared to that of fresh chalcopyrite.
The presence of bio-proteins significantly enhanced
chalcopyrite flocculation.
In order to estimate the optimum amount of protein
required for selective separation, settling studies were
carried out in presence of different concentrations of
extracellular bacterial protein at various pH values.
From Fig. 8(a), it can be observed that at 25 mg/l of
protein concentration, more than 90% of chalcopyrite
could be settled in less than 10 min, at a pH of 6– 8.
To achieve maximum settling rates for chalcopyrite,
concentration levels of bio-proteins needed to be
maintained beyond 25 mg/l.

3.4. Selective flocculation

From the results of individual flocculation tests it

Fig. 8. Settling behavior of chalcopyrite in the presence of
extracellular bacterial protein of B. polymyxa as a function time
with respect to (a) pH, and (b) protein concentration.
is clear that quartz can be separated from chalcopyrite
after interaction with cells of B. polymyxa. Selective
flocculation tests were carried out at pH 8 using a
mixture of chalcopyrite and quartz (1:1, wt.%). Six
desliming stages of 2 min duration each were used.
The wt.% separation is shown in Table 1. At pH 6 the
cumulative removal of quartz was found to be 57% in
absence of any bioreagent, which can attributed to the
diference in the specific gravity of chalcopyrite and
quartz (chalcopyrite 4.28; quartz 2.65). But at pH
8 and in presence of bacterial cells, quartz separation

min. On interaction of calcite with ECP followed by Table 1

flocculation, it was observed that at pH 8 the settling Selective flocculation of chalcopyrite from a mixture of chalcopyrite
rate of calcite was 80% at 5 min and it increased to and quartz (1:1, wt.%) in the presence of bacterial cells (1  108
90% in 20 min. This indicated that the dispersion cells/ml) of B. polymyxa and extracellular bacterial protein (25 mg/l)
behavior of calcite on interaction with bacterial cells No. of %Quartz (cumulative) removal at different pH
was due to extracellular bacterial protein and similar to desliming Without Bacterial Bacterial
stages
quartz, presence of bacterial proteins promoted its bio-treatment cells protein
(2 min each)
dispersion, while exopolysaccharides aided in calcite 6 6 8 8
flocculation.
1 4.6 14.2 16.1 14.2
From Fig. 7(c), it is observed that in the absence of 2 15.6 29.2 32.0 39.4
bacterial cells and at pH 8, the percentage of chalco- 3 27.8 40.0 57.6 71.2
pyrite settled increased from 20% in 5 min to 55% at 4 35.9 76.3 81.2 92.6
20 min, while in presence of bacterial cells and at pH 5 47.8 89.0 91.3 94.2
6 57.0 90.1 91.9 94.6
8 chalcopyrite settled was 90% in 5 min and 95% in
 P. Patra, K.A. Natarajan / Int. J. Miner. Process. 74 (2004) 143–155 153

Table 2 in selective separation of chalcopyrite from a binary

Selective flocculation of chalcopyrite from a mixture of chalcopyrite mixture of chalcopyrite and quartz was obtained at pH
and calcite (1:1, wt.%) in the presence of bacterial cells (1  108
cells/ml) of B. polymyxa and extracellular bacterial protein (25 mg/l) 8. Hence all the flotation studies were also carried out
at this pH. From Table 3 it can be observed that about
No. of %Calcite (cumulative) removal at different pH
desliming 60 wt.% of quartz and 26.4 wt.% of chalcopyrite
Without Bacterial Bacterial
stages could be floated in presence of bacterial cells. Hydro-
bio-treatment cells protein
(2 min each) phobic surfaces of quartz after interaction with bacte-
8 8 8 rial cells required only a small amount of amine
1 8.9 10.9 16.2 (Hexaamine, 1  10 4 M) collector to further enhance
2 17.6 24.2 29.4 its hydrophobicity. Significant increase in the flotation
3 24.4 33.0 34.1
efficiency was observed when small quantity of amine
4 31.0 55.2 50.2
5 38.6 67.4 73.4 collector (Hexaamine) was used to float quartz. The
6 46.9 70.1 81.1 recovery of quartz in this case as observed from the
Table 3 was about 90% while that chalcopyrite was
only less than 30%. Similar flotation tests were also
was observed to be 91.9% and 90.1% at pH 6. As it carried out after interaction with extracellular bacterial
was observed from prior experiments that extracellu- protein. After interaction of a mixture of chalcopyrite
lar bacterial protein was mainly responsible for the and quartz with 25 mg/l of extracellular bacterial
selective separation, subsequent investigations were
also carried out in presence of bacterial protein. From Table 3
Table 1 it can also be observed that up to about 95% Selective microflotation test results on a mixture of quartz and
of quartz could be removed from 1:1 mixtures con- chalcopyrite (1:1, wt.%)
taining chalcopyrite and quartz at pH 8 in the Experimental conditions Quartz Chalcopyrite
(wt.%) (wt.%)
presence of 25 mg/l of bio-proteins separated from
B. polymyxa. Float Sink Float Sink
From Fig. 6(b) and (c) it was observed that there Flotation of a mixture of 57.68 42.32 31.3 58.6
was a significant difference in the settling rates of chalcopyrite and quartz
(1:1, wt.%) in absence
calcite and chalcopyrite as a function of time and pH.
of collector (pH 8)
Selective flocculation tests were performed in the Flotation in absence of 59.6 40.4 26.4 73.5
presence of bacterial cells at pH 8. It could be seen collector and after
from Table 2 that after six desliming stages and in the interaction with cells
absence of any bioreagent, the cumulative removal of of B. polymyxa for
15 min (pH 8)
calcite was 46.9%, whereas in the presence of bacte-
Flotation of the above 89.9 10 29.6 70.3
rial cells more than 70 wt.% of calcite could be mixture after
separated from a binary mixture of 1:1 wt.% of calcite conditioning
and chalcopyrite. Selective flocculation tests were with Hexaamine
also carried out in the presence of 25 mg/l of extra- (1  10 4 M) for 15
min (pH 8)
cellular bacterial protein, when it was observed that
Flotation of the mixture 96.1 3.9 15.9 84
more than 81% of calcite could be removed under after interaction with
similar conditions. extracellular bacterial
protein (25 mg/l) for
3.5. Microflotation 15 min (pH 8)
Flotation of the mixture 95.1 4.9 10.4 89.6
after interaction with
A mixture (1:1, wt.%) of chalcopyrite and quartz extracellular bacterial
was treated with bacterial cells for 15 min. The treated protein (25 mg/l)
mixture was then floated in a Halimond tube. It was followed by conditioning
observed from the previous section that through with Hexaamine for 15
min (pH 8)
selective flocculation studies, the maximum efficiency
154 P. Patra, K.A. Natarajan / Int. J. Miner. Process. 74 (2004) 143–155
protein for 15 min, the recovery of quartz was found to
be 96.1% while that of chalcopyrite was significantly
lower at about 16%. This again can be attributed to the
fact that bio-proteins depress chalcopyrite and float
quartz by making the surface of quartz preferably more
hydrophobic. This mixture was subsequently treated
with hexamine to enhance their separation through
flotation of quartz. More than 95% of quartz could be
selectively floated, while about 90% of chalcopyrite
got depressed under the above conditions.
4. Conclusions
The following major conclusions could be drawn
based on this study:
1. Cells of B. polymyxa exhibited higher affinity
towards chalcopyrite compared to quartz in acidic
pH range. Bacterial cell adsorption onto various
minerals at neutral pH follows the order: chalco-
pyriteHcalciteHquartz.
2. Electrophoretic mobility of bacterial cells after
interaction with the minerals was not significantly
altered. However, the IEP of cells shifted to slightly
higher pH after interaction with chalcopyrite and
calcite. Significant surface chemical changes were
observed on quartz and calcite after bacterial
interaction.
3. Flocculation behavior of various minerals after
bacterial interaction was found to be quite different
from each other. Chalcopyrite settled rapidly
compared to calcite in the neutral pH, whereas in
case of quartz the settling rate was reduced. Similar
behavior was observed with extracellular bacterial
proteins as well.
4. Through selective flocculation chalcopyrite could
be efficiently separated from binary mixtures
containing quartz and calcite after interaction with
bacterial cells as well as extracellular bacterial
protein.
5. Chalcopyrite could be efficiently separated from
quartz after interaction with bacterial cells or
extracellular bacterial protein, through flotation. It
was observed that there was further increase in the
efficiency of selective separation on addition of
small amount of amine collector to enhance higher
recovery of quartz.
6. In conventional processes, collector-assisted flo-
tation is used for recovery of chalcopyrite from
lean grade resources, waste ore burden as well
as tailings. Such a process could be uneconom-
ical considering the enormous amounts of such
low value materials. The results reported in this
paper could lead to the development of a very
economical process utilising native microorgan-
isms available in the abandoned ore piles and
tailing dams. Through microbially induced floc-
culation and flotation, both pyrite and chalcopy-
rite could be removed from oxide gangue
minerals such as silicates and calcite, leading
to desulphurisation and thus environmental
protection.
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