Int. J. Miner. Process.

72 (2003) 175 – 188

www.elsevier.com/locate/ijminpro

Surface modification studies on sulphide minerals

using bioreagents
S. Subramanian *, D. Santhiya, K.A. Natarajan
Department of Metallurgy, Indian Institute of Science, Bangalore 560 012, India
Received 28 October 2002; received in revised form 28 October 2002; accepted 30 June 2003

Abstract

The interaction of galena and sphalerite minerals with the metabolite obtained from Bacillus polymyxa has been examined
through adsorption, electrokinetic, microflotation and flocculation tests. The adsorption density of the carbohydrate
component of the metabolite for sphalerite exhibits a characteristic maximum in the pH range of 6 – 7, while in the case of
galena the amount adsorbed increases with increase of pH. On the other hand, the adsorption density of the bacterial protein
shows a continuous decrease with increase of pH, for both the minerals. The adsorption affinity of both the metabolic
components is higher for galena vis-à-vis sphalerite. The electrophoretic mobility of the chosen minerals becomes less
negative after interaction with the metabolite, in proportion with the time of interaction. Interestingly, the isoelectric point of
sphalerite is shifted to less acidic values after treatment with the metabolite, but that of galena is unaltered. Bioflotation and
bioflocculation studies on a synthetic mixture of galena and sphalerite demonstrate that galena can be selectively depressed or
flocculated from sphalerite under appropriate conditions. Co-precipitation tests confirm complexation of lead and zinc species
with the metabolic products, in the bulk solution. Possible mechanisms of interaction between the chosen sulphide minerals
and the bioreagents are discussed.
D 2003 Elsevier B.V. All rights reserved.

Keywords: galena; sphalerite; Bacillus polymyxa metabolite; carbohydrate; protein; bioflotation and bioflocculation

1. Introduction of ores and discharge of solid wastes and effluents,

The necessity to process and beneficiate ores with
lower grades, which are more refractory and finely
disseminated has resulted in the conventional mineral
processing and hydrometallurgical operations becom-
ing inadequate and inefficient. Further, stringent envi-
ronmental regulations have constrained the processing
* Corresponding author. Tel.: +91-80-2932261; fax: +91-80-
3600472.
E-mail address: ssmani@metalrg.iisc.ernet.in (S. Subramanian).
from the mining operations. It thus becomes impera-
tive to develop novel technologies for mineral process-
ing and waste remediation. Recent developments in
biotechnology hold promise to process such difficult-
to-treat ores as well as to safeguard the environment.
The growing importance of mineral bioprocessing can
be gauged from the conferences exclusively dedicated
to this topic (Smith and Misra, 1991a; Holmes and
Smith, 1995; Hanumantha Rao and Forssberg, 2001).
An excellent overview of mineral bioprocessing has
been compiled by Smith and Misra (1991b). Typical
examples of the potential applications of microbes in

0301-7516/$ - see front matter D 2003 Elsevier B.V. All rights reserved.
doi:10.1016/S0301-7516(03)00097-8
176 S. Subramanian et al. / Int. J. Miner. Process. 72 (2003) 175–188
mineral beneficiation and coal preparation, include
their utilisation as flocculants (Schneider et al., 1994;
Dubel et al., 1992; Attia, 1987), flotation collectors
(Smith et al., 1993) and flotation modifiers (Elzeky
and Attia, 1987; Atkins et al., 1987; Rao et al., 1992;
Zheng et al., 2001). Detailed biobeneficiation studies
carried out using Bacillus polymyxa with respect to
iron ore and bauxite processing have been reported
(Deo and Natarajan, 1997, 1998; Natarajan and Deo,
2001). The utility of Thiobacillus thiooxidans and
B. polymyxa in the selective depression and floccula-
tion of galena from its mixture with sphalerite has been
recently brought out by us (Santhiya et al., 2001a,b,c).
It is well known that both organic and inorganic
reagents like different types of mineral acids, fatty
acids, polysaccharides, proteins and chelating agents
are generated by microorganisms. These metabolic
products have advantages over their chemical counter-
parts in biodegradability and effectiveness at extreme
temperatures or pH and in having lower toxicity.
Motivated by this, the present investigation was taken
up to study the surface chemical changes brought
about on galena and sphalerite minerals using the
metabolite secreted by B. polymyxa during its growth.
The feasibility of selective flotation and flocculation
in the galena –sphalerite system has been examined in
the presence of the bacterial carbohydrate and protein
components, and the interaction mechanisms have
been ascertained.
2. Experimental materials and methods
Mineral samples of sphalerite and galena were
obtained from Gregory, Bottley and Lloyd, UK.
Mineralogical studies as well as X-ray powder dif-
fraction data indicated that the samples were of high
purity. The above samples were dry ground using a
porcelain ball mill and then sieved through 105, 63
and 37 Am BSS sieves. The 37 Am size fraction
was further ground finer in a Retsch mortar grinder.
The mean (d50) particle size of this fraction was found
to be c 5 Am using a Malvern Mastersizer model
3000 particle size analyser. This fraction was used for
the adsorption, electrokinetic and flocculation studies.
For the microflotation experiments, 105 + 63 Am
fraction of galena and sphalerite was used. The BET
nitrogen specific surface areas of c 5 Am fraction of
sphalerite and galena were respectively found to be
1.43 and 0.99 m2/g.
The strain of B. polymyxa (NCIM 2539) used in
this study was obtained from the National Collection
of Industrial Microorganisms (NCIM), National
Chemical Laboratory, Pune. The bacteria were sub-
cultured in the laboratory using the modified Brom-
field medium as per the procedure previously
described (Anand et al., 1996).
The starch sample used in this study for the
calibration of the extracellular polysaccharide fraction
was obtained from BDH Limited, Poole, England.
Potassium nitrate was used to maintain the ionic
strength, while nitric acid and potassium hydroxide
were used as pH modifiers. All the reagents used in
this study were of analytical grade. De-ionised double
distilled water with a final conductivity of < 1.5
Amhos was used for all the tests.
2.1. Separation of metabolite from the bacterial
culture
The bacterium B. polymyxa was grown by adding
10% v/v of an active inoculum to the Bromfield
medium and incubated at 30 jC on a Remi rotary
shaker at 240 rpm for 48 h. The growth conditions
and the composition of the medium have been kept
fixed in all experiments. Precautions were taken to
prevent contamination by undesirable microorganisms
by maintaining aseptic conditions. This fully grown
culture was centrifuged at 10,000 rpm for 10 min at
10 jC to separate the cells from the extracellular
materials. The supernatant (metabolite) obtained was
filtered through a sterile Millipore membrane filter
paper of pore size 0.2 Am using a Tarsons membrane
filter holder.
2.2. Adsorption studies
For the adsorption tests, 1 g of the mineral powder
of mean (d50) size c 5 Am was taken and pulped to
50 ml. This was followed by the addition of metab-
olite of desired concentration and pre-adjusted pH
equivalent to that of the suspension pH. The ionic
strength was kept at 10 3 M using KNO3 and the
total volume was made up to 100 ml. The suspension
was agitated for 4 h at 200 rpm in a Remi orbital
shaking incubator at 27 F 2 jC. Subsequently, the
 S. Subramanian et al. / Int. J. Miner. Process. 72 (2003) 175–188
suspension was centrifuged at 5000 rpm using a
Sorvall RC-5B refrigerated high speed centrifuge
for 10 min and filtered through a Whatman 42 filter
paper. The clear supernatant solution was analysed
for the residual carbohydrate and protein concentra-
tions using a Shimadzu UV-260 UV-vis spectropho-
tometer as per standard procedures (Dubois et al.,
1956; Bradford, 1976).
2.3. Electrokinetic measurements
Electrophoretic mobilities of sphalerite as well as
galena were determined in the presence and absence
of metabolite using a Malvern 3000 model Zetasizer.
A known concentration of the metabolite solution at
a desired pH was added slowly to the suspension
containing 10 mg of the solids under agitated
conditions at the same pH and made up to 100
ml. In all these experiments, the pH of the suspen-
sion was adjusted to a desired value using AR grade
nitric acid or potassium hydroxide and the ionic
strength was maintained at 10 3 M KNO3. The
suspension was allowed to equilibrate for 1 h and
ultrasonicated for 30 s before measuring its electro-
phoretic mobility. The effects of pH and interaction
time on the electrophoretic mobilities of the minerals
were determined.
2.4. Microflotation studies
Microflotation tests were carried out using a mod-
ified Hallimond tube (Fuerstenau et al., 1957). Potas-
sium isopropyl xanthate (10 4M) was used as a
collector for both sphalerite and galena, while
10 6M CuSO4 was used as an activator for sphalerite.
For the flotation tests, 1 g of ( 105 + 63) Am size
mineral sample was pulped to a final volume of 200 ml
with double distilled water after conditioning with the
chosen reagents at a desired pH for 15 min using a
magnetic stirrer. The sample was then transferred to the
Hallimond tube and nitrogen gas was passed at the rate
of 40 ml per minute. After flotation for 3 min, the
concentrate and the tailing fractions were separately
filtered, dried and weighted. The discoveries are ex-
pressed on a weight % basis. In experiments where the
metabolite was added, the sample was further condi-
tioned for 15 min at a given pH with it, prior to
flotation.
177
2.5. Differential flotation tests
In these experiments, 0.5 g each of galena and
sphalerite (1:1) mixture, of particle size ( 105 + 63)
Am was interacted with the metabolite at its natural pH
of 3.2– 3.4 and at pH 9– 9.5 for 15 min prior to
flotation. The same procedure as adopted for the
flotation of the individual minerals was followed.
The lead and zinc contents in the concentrate and
tailing fractions were separately analysed using a
Jobin Yvon JY 24 inductively coupled plasma emis-
sion spectrometer.
2.6. Flocculation tests
The flocculation test procedure was standardised as
follows: 0.5 g of galena or sphalerite of c 5Am size
was suspended in a volume of 100 ml of the metab-
olite of pre-adjusted pH in the range of 3 –11 in a 100-
ml measuring jar. The measuring jar was gently
tumbled 10 times and allowed to stand for 2 min.
Exactly 90 ml of the supernatant was siphoned out
into a clean, 100 ml beaker, filtered through a What-
man 42 filter paper and the residue was dried and
weighed. The percentage weights of the solids dis-
persed as well as those settled were determined.
2.7. Selective flocculation tests
In these experiments, 0.25 g each of galena and
sphalerite (1:1) of particle size c 5 Am was initially
mixed and interacted with 100 ml of the metabolite at
pH 3 –3.5 and at pH 9 –9.5 for 2 min in a 100-ml
measuring jar. The same procedure as described for
the flocculation of the individual minerals was adop-
ted. The lead and zinc contents in the dispersed and
flocculated portions were analysed using a Jobin
Yvon JY24 inductively coupled plasma emission
spectrometer.
2.8. Co-precipitation studies
In these studies, a known amount of Pb(NO3)2/
Zn(NO3)2 solution was mixed with 100 ml of the
metabolite in a such a way that the final concentration
of lead or zinc was 50 ppm in 100 ml solution. The pH
was adjusted to a desired value in the range of 3 –11 by
adding either nitric acid or potassium hydroxide solu-
178 S. Subramanian et al. / Int. J. Miner. Process. 72 (2003) 175–188
tion. After agitation for 1 h in a Remi orbital shaker,
the solution was centrifuged and filtered. The clear
supernatant solution was then analysed for lead or zinc
content using a Jobin Yvon JY24 inductively coupled
plasma emission spectrometer. The residual carbohy-
drate and protein contents of the metabolite were also
determined by adopting the methods described earlier.
Additionally, control experiments were also carried out
by agitating 100 ml of the metabolite solution or the
desired metal ion concentration for 1 h as a function of
pH and the residual concentrations of carbohydrate
and protein or the metal ions were analysed.
3. Results and discussion
3.1. Adsorption studies
The metabolite obtained from the fully grown
culture of B. polymyxa after 48 h of growth was
separated from the bacterial cells and interacted with
the minerals. The adsorption of the protein and
exopolysaccharide (carbohydrate) components of the
metabolite was studied as a function of pH and the
adsorption isotherms were also determined.
3.1.1. Effect of pH
The adsorption density of carbohydrate from B.
polymyxa metabolite for sphalerite and galena as a
function of pH is portrayed is Fig. 1. The amount
Fig. 1. Adsorption density of carbohydrate from metabolite of B.
polymyxa for sphalerite and galena as a function of pH.
Fig. 2. Adsorption density of protein from metabolite of B.
polymyxa for sphalerite and galena as a function of pH.
adsorbed increases with increase of pH and attains a
region of higher adsorption density in the pH range
of 6 – 8.5 and thereafter decreases for sphalerite.
Adsorption maxima around pH 7.5 – 8 have been
observed for the adsorption of polysaccharides such
as dextrin and guar gum onto sphalerite (Rath and
Subramanian, 1999a,b). The adsorption density of
the bacterial carbohydrate continuously increases
with increase of pH in the case of galena, although
a distinct maximum is not apparent. Earlier inves-
tigations had revealed a maximum around pH 11.5
for the adsorption of dextrin and guar gum onto
galena (Liu and Laskowski, 1989a; Rath and Sub-
ramanian, 1999a,b). It is noteworthy that the maxi-
mum adsorption density of carbohydrate for galena
(160 mg/m2) is about 50 times higher than that for
sphalerite (3.2 mg/m2).
The adsorption of protein from B. polymyxa me-
tabolite onto galena and sphalerite as a function of pH
is shown in Fig. 2. The adsorption density decreases
with increase of pH for both galena and sphalerite.
Additionally, the amount of bacterial protein adsorbed
onto galena is slightly higher than that onto sphalerite.
The functional groups present in the protein, their
charge characteristics and conformation govern the
adsorption process. Most proteins have isoelectric
points in the pH range of 3 – 6 and the reduced
adsorption in the alkaline pH range may be attributed
to electrostatic repulsion between the protein and the
sulphide mineral.
 S. Subramanian et al. / Int. J. Miner. Process. 72 (2003) 175–188
3.1.2. Adsorption isotherms
The adsorption isotherms of carbohydrate, from the
metabolite of B. polymyxa, for sphalerite at three
different pH values are depicted in Fig. 3(a). For the
isotherm at pH 7.1 –7.2, the adsorption density ini-
tially increases sharply up to 1000 ppm concentration
and thereafter tends to attain a plateau level of
adsorption. At pH 3.4 –3.6, the adsorption density
marginally increases with increase in equilibrium
concentration up to about 3000 ppm followed by a
sharp increase. The isotherm at pH 11.1 –11.2 indi-
cates a very feeble increase in the adsorption density
up to about 2000 ppm and thereafter attains a satura-
tion value. These results parallel the pH trend ob-
served earlier (Fig. 1). The adsorption density at pH
Fig. 3. (a) Adsorption isotherms of carbohydrate from metabolite of
B. polymyxa for sphalerite. (b) Adsorption isotherms of carbohy-
drate from metabolite of B. polymyxa for galena.
179
7.1– 7.2 is the highest followed by that at pH 3.4 –3.6,
while the isotherm at pH 11.1– 11.2 has the lowest
adsorption density. The isotherms at pH 7.1 and 11.1
resemble the S-2 type of the Giles classification (Giles
et al., 1960), while that at pH 3.4 may be categorised
as L-1 type.
Fig. 3(b) shows the adsorption isotherms of bacte-
rial carbohydrate for galena at different pH values.
The isotherm at pH 11 shows the maximum adsorp-
tion density followed by those at pH 7 and 3 in that
order. The isotherm at c pH 11 shows an increase in
the adsorption density up to about 1600 ppm equilib-
rium concentration, and subsequently tends to attain a
plateau level. The isotherms at about pH 7 and 3 show
a similar trend, except that the plateau level of
adsorption is attained around 2000 ppm concentration.
All the isotherms resemble the S-2 type of the Giles
classification (Giles et al., 1960).
A comparison of Fig. 3(a) and (b) unambiguously
reveals that the adsorption density of bacterial carbo-
hydrate for galena is significantly higher compared to
that for sphalerite. This is in agreement with that
observed in Fig. 1.
The adsorption isotherms of protein from B. poly-
myxa metabolite for sphalerite and galena are shown
in Fig. 4(a) and (b), respectively. In the case of
sphalerite, the adsorption density decreases with in-
crease of pH as observed earlier (Fig. 2). From Fig.
4a, it is evident that the adsorption density initially
increases steeply for the isotherms at c pH 3.5 and 7
and thereafter a decrease in the slope of the adsorp-
tion density takes place. The amount of protein
adsorbed at pH 11 is significantly less. The isotherms
at pH 3.5 and 7 may be classified as belonging to the
S-3 type, while those at pH 11 resemble the low
affinity S-2 type of the Giles category (Giles et al.,
1960). With respect to galena (Fig. 4b), the adsorp-
tion density at pH 3.5 is higher than that at pH 7.1
and 11.1. The isotherms at pH 3.5 and 7.1 show a
steep rise in the adsorption density at lower concen-
trations followed by reduced adsorption at higher
concentrations. On the other hand, at pH 11 the
amount adsorbed is substantially less, presumably
due to electrostatic repulsion between the negatively
charged mineral surface and anionic component of
the bacterial protein. The isotherms for galena resem-
ble those obtained for sphalerite and can be categor-
ised in a similar manner. The magnitude of protein
180 S. Subramanian et al. / Int. J. Miner. Process. 72 (2003) 175–188

metabolite. The reduction of the negative electropho-

retic mobilities and the shift in the iep may be
attributed to the adsorption of the positively charged
amino group of the protein component of the metab-
olite, onto the negatively charged sphalerite surface.
Since the adsorption of the cationic protein component
increases with time of interaction till it reaches the
saturation level, the shift in the iep as well as a
decrease in the electronegative character of sphalerite,
also increases with increase in the interaction time.
It is apparent that specific adsorption is occurring
at the pH of isoelectric point of sphalerite, since

Fig. 4. (a) Adsorption isotherms of protein from metabolite of B.

polymyxa for sphalerite. (b) Adsorption isotherms of protein from
metabolite of B. polymyxa for galena.

adsorbed is marginally higher for galena compared to

that for sphalerite.

3.2. Electrokinetic studies

The electrophoretic mobilities of sphalerite, before

and after interaction with B. polymyxa metabolite at
various time intervals, as a function of pH are shown in
Fig. 5(a). It is interesting to note that the isoelectric
point of sphalerite is shifted from pH 2.2 towards less
acidic pH values in proportional magnitude to the
Fig. 5. (a) Electrophoretic mobility of sphalerite as a function of pH
interaction time. Additionally, the electronegative before and after interaction with B. polymyxa metabolite. (b)
character of the sphalerite particles also decreases with Electrophoretic mobility of galena as a function of pH before and
increase in the time of interaction with the bacterial after interaction with B. polymyxa metabolite.
 S. Subramanian et al. / Int. J. Miner. Process. 72 (2003) 175–188 181

positive electrophoretic mobilities are observed at this

pH. At pH values above the isoelectric point, electro-
static attraction favours the adsorption of the positive-
ly charged protein component onto the negatively
charged sphalerite sites. However, at and below the
isoelectric point of sphalerite, the non-electrostatic
forces such as hydrogen bonding and chemical forces,
govern the adsorption process, as these interactions
take place at charged or neutral sites.
Fig. 5(b) portrays the electrophoretic mobilities of
galena as a function of pH before and after interaction
with B. polymyxa metabolite at different time inter-
vals. It is evident from the figure that the electroki-
netic potential of galena after interaction with the
bacterial metabolite, becomes slightly more negative
up to about pH 4.2, and then more or less becomes
independent of pH at c 0.6 Am/sec/V/cm, between 1
and 24 h of interaction. In contrast to the sphalerite–
B. polymyxa metabolite system (Fig. 5a), the magni-
tude of the negative electrophoretic mobilities of
galena are found to be more or less independent of
the interaction time, especially between 1 and 24 h,
presumably without any significant shift in the iso-
electric point value, resembling the influence of an
indifferent electrolyte. These results may be attributed
to the higher adsorption of the polysaccharide com-
ponent of the bacterial metabolite than the protein
component onto the galena surface.
The results of the electrokinetic tests exhibit con-
trasting trends with respect to the two minerals stud-
ied, the protein component of the bacterial metabolite Fig. 6. (a) Effect of pH on the flotation recovery of sphalerite before
influencing the surface charge characteristics of sphal- and after interaction with B. polymyxa metabolite. (b) Effect of pH
erite, whereas in the case of galena, the electrokinetic on the flotation recovery of galena before and after interaction with
B. polymyxa metabolite.
behaviour resembles that of the carbohydrate fraction.
Such a behaviour has been independently verified
using the isolated protein and polysaccharide fractions sphalerite shows a steep decrease with increase in pH,
of the metabolite, with respect to the electrokinetics of and becomes less than 20% in the pH range of 8.3 –
galena and sphalerite (Santhiya, 2001). 11.5. The addition of CuSO4 and PIPX to the sphalerite
sample initially interacted with B. polymyxa metabolite
3.3. Microflotation tests drastically improves the recovery of sphalerite to
around 90%, over a wide pH range of 4.3– 10.3.
The flotation recovery of sphalerite as a function of The effect of pH on the floatability of galena before
pH before and after interaction with B. polymyxa and after interaction with B. polymyxa metabolite
metabolite under different conditions is portrayed in under different test conditions is shown in Fig. 6(b).
Fig. 6(a). It can be observed from the figure that after It is interesting to note that galena is completely
interaction with the metabolite for 15 min, 95% flota- depressed in the pH range investigated, both in the
tion recovery of sphalerite is obtained at the natural pH absence and presence of PIPX, after interaction with
of the metabolite (pH 3.3). Thereafter, the recovery of B. polymyxa metabolite for 15 min.
182 S. Subramanian et al. / Int. J. Miner. Process. 72 (2003) 175–188

A closer scrutiny of Fig. 6(a) and (b) clearly Table 1

reveals that the trends observed with respect to the Differential flotation test results on sphalerite – galena system using
B. polymyxa metabolite
flotation of sphalerite and galena in the presence of
Experimental conditions PbS (%) ZnS (%) S.I.
the metabolite complement the adsorption results
(Figs. 1 and 2). Additionally, it is noteworthy that Float Sink Float Sink
sphalerite can be selectively floated from galena at the Flotation in the presence 98.6 1.2 94.1 2.0 0.76
natural pH of the metabolite (3.3) without any acti- of 10 4 M PIPX and 98.2 1.4 96.3 2.4
10 6 M CuSO4
vator/collector, and in the pH range of 5 –10.5 in the
at pH 9 – 9.5
presence of the metabolite, CuSO4 as well as PIPX. It Flotation after interaction 3.1 96.1 90.4 8.6 18.12
also becomes evident that while galena is completely with B. polymyxa 2.9 96.6 90.6 9.1
depressed by the metabolite in the entire pH range, metabolite at pH 3.2 – 3.4
sphalerite is appreciably depressed only in the pH (no collector/activator)
Flotation after interaction 1.3 98.6 8.6 90.3 2.58
range of 8 –11. However, the flotation recovery of
with B. polymyxa 0.8 98.9 9.2 89.1
sphalerite, pretreated with the metabolite, is restored metabolite (at pH 9 – 9.5)
after the addition of collector/activator. (no collector/activator)
Flotation in the presence 3.7 95.7 94.6 4.9 22.76
3.4. Differential flotation tests of 10 4 M PIPX and 3.9 95.2 95.2 4.3
10 6 M CuSO4 at
pH 9 – 9.5 after
In these investigations, the differential flotation of interaction with
the synthetic mixture (1:1) of galena and sphalerite B. polymyxa metabolite
was carried out using B. polymyxa metabolite at its Flotation in the presence 8.6 90.8 93.5 6.2 13.24
natural pH of 3.2 – 3.4 and at pH 9 – 9.5, under of 10 6 M PIPX and 7.2 92.3 92.6 6.1
10 8 M CuSO4 at
different conditions and the results are shown in Table
pH 9 – 9.5 after
1. Here again, the minerals were interacted with B. interaction with
polymyxa metabolite for 15 min. Based on the results B. polymyxa metabolite
highlighted in Table 1, the following observations can
be made:
galena from sphalerite is feasible but with a
1. It is interesting to note that the differential flotation reduced SI ( c 13).
tests carried out in the presence of B. polymyxa
metabolite alone at its natural pH of 3.2– 3.4 show From these results, it is clearly evident that sphal-
that about 90% of sphalerite is floated, while over erite is floated and galena is depressed either in the
96% of galena is depressed. The selectivity index is presence of bacterial metabolite alone or along with
found to be around 18, attesting to good separation. collector and activator.
2. The interaction of the synthetic mixture with B.
polymyxa metabolite at pH 9 – 9.5 in the absence of 3.5. Flocculation studies
the collector and activator reveals that 98% galena
and 90% sphalerite are depressed, indicating 3.5.1. Effect of time
inadequate selectivity. The effect of time on the percentage galena and
3. The addition of 10 6 M CuSO4 and 10 4 M PIPX sphalerite settled in the absence and in the presence of
to the synthetic mixture initially interacted with B. B. polymyxa metabolite at pH 3.2 –3.4 is portrayed in
polymyxa metabolite at pH 9– 9.5, demonstrates Fig. 7(a). From the figure, it is evident that in the
that around 95% of galena is selectively depressed, absence of the metabolite, the amounts of sphalerite
whereas about 95% of sphalerite is floated. A high and galena settled are not very different. The percent-
selectivity index (SI = 22.8) is achieved. age galena or sphalerite settled gradually increases
4. It is also evident that by reducing the concentration from about 28% in 2 min to 40% in 5 min and then
of CuSO4 and PIPX from 10 6 and 10 4 M to slightly increases to around 45% in 15 min. It is
10 8 and 10 6 M, respectively, separation of noteworthy that in the presence of B. polymyxa
 S. Subramanian et al. / Int. J. Miner. Process. 72 (2003) 175–188 183

erite is settled at 2 min. The amount settled remains

more or less at that value up to 4 min. Beyond 4 min,
the amount of sphalerite settled, increases with in-
crease in time and becomes around 20% in 15 min. In
contrast, after interaction with the metabolite over
90% of galena is settled in the time period investigat-
ed. As the differences in the amount of galena and
sphalerite settled are more at shorter time of settling,
further tests were carried out keeping the time of
settling at 2 min.

3.5.2. Effect of pH
The effect of pH on the settling behaviour of
sphalerite and galena in the absence and presence of
B. polymyxa metabolite is depicted in Fig. 8. In the
absence of the metabolite, the amount of sphalerite
settled decreases from about 35% at pH 3 to 17% at
pH 11, while the amount of galena settled increases
from 30% at pH 3 to about 55% at pH 11. In the
presence of the metabolite, the percentage sphalerite
settled is about 10% in the pH range investigated. On
the other hand, the percentage galena flocculated
steeply increases from about 67% at pH 3.4 to about
98% at pH 11.2, in the presence of the metabolite. A
careful scrutiny of Fig. 8 indicates that in the presence
of the metabolite, galena can be selectively flocculat-
ed from sphalerite in the pH range of 9 –11 using B.
polymyxa metabolite.

Fig. 7. (a) Effect of time on the settling of sphalerite and galena in

the absence and presence of B. polymyxa metabolite at pH 3.2 – 3.4.
(b) Effect of time on the settling of sphalerite and galena in the
absence and presence of B. polymyxa metabolite at pH 9.2 – 9.6.

metabolite, the amount of sphalerite settled is de-

creased below 10% after 2 min and remains more or
less at that value up to 15 min. On the other hand,
after interaction with B. polymyxa metabolite, the
amount of galena settled increases from 60% in 2
min to around 83% in 5 min and thereafter marginally
increases to 87% at 15 min.
The percentage solids settled in the absence and
presence of B. polymyxa metabolite at pH 9.2 – 9.6 as
a function of time is shown in Fig. 7(b). When
sphalerite is interacted with the bacterial metabolite Fig. 8. Effect of pH on the settling of sphalerite and galena in the
and subjected to flocculation tests, around 10% sphal- absence and presence of B. polymyxa metabolite.
184 S. Subramanian et al. / Int. J. Miner. Process. 72 (2003) 175–188

Table 2 The effect of pH on the residual concentration of

Selective flocculation test results on sphalerite – galena system using carbohydrate of B. polymyxa metabolite and zinc
B. polymyxa metabolite
species is shown in Fig. 9(a). In this experiment, the
Experimental ZnS (%) PbS (%)
initial concentration of zinc was fixed at 50 ppm, while
conditions Dispersed Flocculated Dispersed Flocculated the carbohydrate concentration was 3600 ppm. The
Blank at 90.5 7.8 82.5 17.2 zinc concentration decreases from about 50 ppm at pH
pH 3 – 3.5 90.2 7.1 83.1 16.8 3 to about 33 ppm at pH 5.5, and significantly drops to
In the presence 93.7 6.1 52.8 47.0
of B. polymyxa 94.2 5.7 52.3 47.6
metabolite at
pH 3.2 – 3.5
Blank at 70.5 27.8 27.0 71.7
pH 9 – 9.5 70.3 29.5 27.0 72.3
In the presence 91.8 5.4 6.8 92.6
of B. polymyxa 91.8 4.7 6.3 92.8
metabolite at
pH 9 – 9.5

3.6. Selective flocculation tests

Table 2 shows the selective flocculation results of a

1:1 synthetic mixture of sphalerite and galena, in the
absence and presence of B. polymyxa metabolite,
under different conditions. From the results, the fol-
lowing facts emerge:

1. In the control experiment carried out at pH 3 –3.5

in the absence of B. polymyxa metabolite, about
90% of sphalerite and 83% of galena are dispersed.
2. The addition of B. polymyxa metabolite at pH 3 –
3.5, marginally enhances the dispersion of sphalerite
to 94%. On the other hand, the metabolite
flocculates 47% of galena, indicating partial selec-
tivity of separation between galena and sphalerite.
3. It is noteworthy that in the presence of the
metabolite at pH 9 – 9.5, about 92% of sphalerite
is dispersed while 93% galena is selectively
flocculated.

3.7. Co-precipitation studies

In addition to the extensive surface chemical

studies carried out on the chosen minerals, to eluci-
date the processes taking place at the solid –solution
interface, the interactions between the lead or zinc
species and the metabolite in the bulk solution have
Fig. 9. (a) Effect of pH on the residual concentration of
also been examined. Towards this, co-precipitation carbohydrate of B. polymyxa metabolite and zinc species. (b) Effect
studies have been carried out and the results are of pH on the residual concentration of carbohydrate of B. polymyxa
highlighted below. metabolite and lead species.
 S. Subramanian et al. / Int. J. Miner. Process. 72 (2003) 175–188 185

below 2 ppm in the pH range of 6 – 7. The zinc

concentration steadily increases beyond pH 7. It has
been reported that precipitation of zinc commences in
the neutral region and in the basic media Zn(OH)42
and Zn2(OH)62 are presumably formed (Baes and
Mesmer, 1986). The increase in zinc concentration in
the alkaline pH range may be attributed to the forma-
tion of zincates. In the presence of the metabolite, there
is a marginal decrease in the zinc concentration,
although there is no change in the trend with respect
to pH. On the other hand, there is a distinct decrease in
the carbohydrate concentration between pH 6 and 7 in
the presence of zinc nitrate. It may be noted that, in the
absence of zinc ions, the carbohydrate concentration
remains unaltered in the entire pH range studied. It is
pertinent to highlight that the adsorption maximum of
carbohydrate for sphalerite was observed between pH
6 and 7 (Fig. 1). The interaction of polysaccharides
such as dextrin and guar gum with zinc species also
revealed maximum co-precipitation around pH 7 (Rath
and Subramanian, 1999a,b).
Fig. 9(b) depicts the residual concentration of lead
species and carbohydrate as a function of pH. In the
absence of the metabolite, the concentration of lead
species decreases from 50 ppm at pH 3 to about 45
ppm at pH 5.5, and is then drastically reduced to below
3 ppm in the pH range of 7 –9. Beyond pH 9, there is
an increase in the concentration of lead species and at
pH 11 about 45 ppm is obtained, due to the formation
of poly- and mono-nuclear lead species such as
Pb4(OH)44 +, Pb6(OH)84 + and Pb(OH)+ (Baes and Mes-
mer, 1986). In the presence of the metabolite, the lead
concentration is marginally reduced in the pH range of
3 –5.5 and is significantly decreased between pH 10
and 11.5. The carbohydrate concentration, in the
presence of lead ions decreases steadily with increase
of pH up to about 8, and remains almost constant
beyond that pH. It may be recalled that the adsorption
of carbohydrate onto galena was found to continuously
increase with increase of pH (Fig. 1). These results Fig. 10. (a) Effect of pH on the residual concentration of protein of
B. polymyxa metabolite and zinc species. (b) Effect of pH on the
attest to strong interaction between lead species and residual concentration of protein of B. polymyxa metabolite and lead
carbohydrate in the alkaline pH range. The concentra- species.
tion of carbohydrate, in the absence of lead, remains
unaltered in the entire pH range studied.
The effect of pH on the residual concentration of the metabolite, especially in the pH range of 3– 5. It is
the protein component of metabolite and zinc species noteworthy that the protein concentration is signifi-
is portrayed in Fig. 10(a). A marginal decrease in the cantly reduced in the pH range of 6– 8 and to a lesser
zinc concentration can be observed in the presence of extent between pH 3 and 5.5, after interaction with
186 S. Subramanian et al. / Int. J. Miner. Process. 72 (2003) 175–188
zinc nitrate. The protein concentration in the absence
of zinc remains at 13 ppm in the entire pH range
studied. It may be recalled that the protein adsorption
onto sphalerite was found to be higher below pH 6
and very little adsorption was observed beyond pH 10
(Fig. 2). Thus, the results of the co-precipitation tests
are in agreement with the adsorption data.
Fig. 10(b) shows the variation in the residual
concentration of lead species and protein as a function
of pH. The lead concentration is marginally reduced
in the presence of the metabolite below pH 4, and
appreciably in the pH range of 10.5 – 11.5, compared
to that in the absence of the metabolite. The protein
concentration in the presence of lead nitrate is signif-
icantly reduced in the pH range of 2– 9, attesting to
strong interaction. Such a trend corroborates the
adsorption results, wherein higher adsorption of the
protein onto galena was observed below pH 7 (Fig. 2).
3.8. Adsorption mechanisms of carbohydrate from B.
polymyxa metabolite at mineral surfaces
From the results of the adsorption, flotation, floc-
culation and co-precipitation experiments carried out
on sphalerite and galena, using the bacterial carbohy-
drate, the following facts emerge:
1. The adsorption density of carbohydrate onto galena
is higher than that onto sphalerite. Additionally, the
amount adsorbed onto galena continuously in-
creases with increase of pH. However, in the case
of sphalerite, an adsorption maximum is observed
around pH 7.
2. Flotation and flocculation tests reveal depression of
galena and dispersion of sphalerite respectively, in
the presence of the bacterial metabolite.
3. Co-precipitation tests confirm strong interaction
between Zn species and carbohydrate at around pH
7, while in the case of Pb species significant
interaction is observed in the pH range of 7– 11. On
a comparative basis, the interaction between lead
species and the carbohydrate is significantly higher.
4. It is noteworthy that the pH of maximum
adsorption of carbohydrate onto sphalerite coin-
cides with the pH of maximum co-precipitation for
the corresponding zinc –polymer system. Similarly,
a close parallelism exists between the pH of
maximum adsorption and significant co-precipita-
tion in the case of lead –carbohydrate system,
although a distinct adsorption maximum is not
observed in this case.
It is well known that sulphide minerals get hy-
droxylated as a function of pH. In this context, it is
pertinent to recall that the solubility product of lead
hydroxide is over three orders of magnitude less than
that of zinc hydroxide (Lide, 1999). Further, zinc
hydroxide is amphoteric while lead hydroxide is
highly basic (Phillips and Williams, 1965). Thus in
the alkaline pH range, zinc hydroxide decomposes to
ZnO22 ions, while lead hydroxide is stable. The
interaction of polysaccharides with various sulphides
and oxides has been extensively studied by Laskowski
and co-workers, and a chemical complexation mech-
anism has been proposed (Liu and Laskowski,
1989a,b,c). More recently, an acid – base interaction
model has been suggested by them (Liu et al., 2000).
These mechanisms have been corroborated by the
work of Rath and Subramanian (1999a,b), Rath et
al. (2000) for the interaction of polysaccharides such
as dextrin and guar gum with several sulphides.
In line with the proposed mechanisms, the hydrox-
yl groups in polysaccharides interact with the mineral
surface metal hydroxide both by hydrogen bonding
and chemical forces.
According to the acid – base interaction model the
hydroxylated mineral surface would behave as a
Bronsted base with the hydroxyl groups in the poly-
saccharides behaving as a Bronsted acid (Liu et al.,
2000). It can be expected that stronger the basicity, the
greater will be the interaction with the polysacchar-
ides. It is thus logical to expect that lead hydroxide
being strongly basic, will have a higher affinity for the
bacterial carbohydrate, compared to hydroxylated
sphalerite, which is amphoteric. The basicity of the
surface hydroxyl groups will be dependent on the
valence states, ionic radii and coordination numbers of
the lead and zinc ions on the corresponding mineral
surfaces. For equivalent valence states and coordina-
tion numbers, ionic radii of zinc are less than that of
lead (Lide, 1999). Consequently, the surface hydrox-
ide groups on galena exhibit more basicity resulting in
stronger interaction with the carbohydrate. This facil-
itates selective depression and flocculation of galena
from sphalerite especially in the alkaline pH range. In
the case of bacterial carbohydrate, apart from the
 S. Subramanian et al. / Int. J. Miner. Process. 72 (2003) 175–188
hydroxyl groups, the carboxylate ions, which are
present in uronic acid could partake in the adsorption
process. It is understandable that the carboxylate ions
will be relatively much less compared to the hydroxyl
groups. The presence of carboxyl groups in the
metabolite has been confirmed by FTIR spectroscopic
studies detailed elsewhere (Santhiya, 2001).
3.9. Adsorption mechanisms of B. polymyxa protein at
mineral surfaces
Proteins are copolymers of some 22 different
amino acids linked together in a linear polypeptide
chain. The sequence of the amino acids in the poly-
peptide chain ultimately determines the folded, spatial
architecture, that is the 3D structure of the protein
molecule. By far, the greatest proportion of the protein
species contains different structural elements, which
are folded together into a compact dense globule, the
globular proteins.
The major interactions for a protein molecule in an
aqueous medium include the following:
1. Hydrophobic interaction
2. Coulombic interaction
3. Lifshift – van der Waals interaction
4. Hydrogen bonding
The results of the adsorption tests carried out on
galena and sphalerite minerals indicate that the ad-
sorption density of the protein component of the
metabolite from B. polymyxa, for both the minerals
is found to be higher in the acidic pH region, and is
considerably reduced in the alkaline pH range. Other
researchers (Norde, 2000) have found that proteins
often exhibit a maximum of adsorption on electrically
charged surfaces near their isoelectric point. It must be
emphasized that characterization of the protein com-
ponent of the metabolite revealed a number of frac-
tions of different molecular weights (Santhiya, 2001).
Thus, it becomes difficult to precisely delineate the
isoelectric point of the protein secreted from the
metabolite. However, the isoelectric points of most
proteins lie in the pH range of 3 – 6. Thus, the higher
affinity of the bacterial protein from the metabolite, in
this pH region, is to be expected. The significant
decrease in the adsorption density in the high alkaline
pH range may be attributed to electrostatic repulsion
187
between the negatively charged protein molecules and
the anionic mineral surfaces. Thus, electrostatic, hy-
drogen bonding, hydrophobic and van der Waals
interactions contribute to the adsorption mechanisms,
in addition to factors leading to conformational
changes of the protein molecules.
4. Conclusions
From the results of the present investigation, the
following major conclusions can be arrived at:
1. The adsorption density of carbohydrate for spha-
lerite exhibits a characteristic maximum in the pH
range of 6– 7, while the amount adsorbed increases
with increase in pH in the case of galena.
2. For both the minerals, the adsorption density of the
protein component of the metabolite decreases with
increase of pH.
3. The adsorption affinity of both carbohydrate and
protein is higher onto galena vis-à-vis sphalerite.
4. Electrokinetic measurements indicate a reduction in
the negative electrophoretic mobility values of the
two minerals consequent to interaction with the
metabolite, in proportion with the time of inter-
action. A shift in the isoelectric point of sphalerite to
less acidic pH values is observed after treatment
with the metabolite. On the contrary, the isoelectric
point of galena is not altered after interaction with
the metabolite.
5. Bioflotation tests demonstrate selective depression
of galena from its synthetic mixture with sphalerite.
6. Bioflocculation studies confirm selective floc-
culation of galena especially in the pH range of
9 – 9.5.
7. Co-precipitation tests reveal complexation of lead
and zinc species with the bioreagents.
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