Minerals Engineering 75 (2015) 63–69

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Minerals Engineering
journal homepage: www.elsevier.com/locate/mineng

Fundamental aspects of hematite flotation using the bacterial strain

Rhodococcus ruber as bioreagent
Leslie Y. Lopez, Antonio G. Merma, Mauricio L. Torem ⇑, Gabriela H. Pino
Department of Materials Engineering, Pontifical Catholic University of Rio de Janeiro, Rua Marquês de São Vicente 225, Gávea, Rio de Janeiro, RJ 22453-900, Brazil

a r t i c l e i n f o a b s t r a c t

Article history: Previous research showed the effectiveness of bacterial strains as flotation reagents on Hematite benefi-
Received 30 July 2014 ciation. The aim of this work is to study and evaluate Rhodococcus ruber as a biocollector. The sample was
Revised 4 December 2014 conditioned with the biomass suspension by stirring under specific conditions as particle size, biomass
Accepted 12 December 2014
concentration, pH solution and conditioning time. The results showed a change in hematite zeta potential
Available online 17 January 2015
profile after interaction with R. ruber, and its adhesion onto the mineral surface was higher at pH 3 and at
concentration of 0.60 g/L (109 cells/mL). Flotation studies were carried out in a 0.23 L modified
Keywords:
Partridge–Smith cell flotation, and the highest floatability (84%) was achieved at size fraction 53 lm
Hematite
Flotation bioreagents
+38 lm under the conditions mentioned before. Complementary floatability studies were performed
Rhodococcus ruber using the conventional frother Flotanol D24 combined with the R. ruber biomass, finding interesting
results for the bigger particle size range. Thus, this research aims to evaluate the efficiency of bioflotation
of minerals, particularly hematite, and the potential use of R. ruber as biocollector, projecting its future
application in mineral flotation industry.
Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction
The application of microorganisms in mineral beneficiation is
not relatively new. For more than 20 years, considerable research
has been carried out. It started as a solution to actual problems
in mineral flotation, such as the depletion of high-grade ores and
the consequent generation of fine and ultrafine particles due to
the processing of low grade ores, and to the constant quest of envi-
ronmental friendly reagents. Therefore, the term bioflotation
turned out attractive not only as the solution of the problems men-
tioned before but to its technological potential and mineral selec-
tivity (Dwyer et al., 2012; Díaz-Lópes et al., 2012; Kuyumcu
et al., 2009).
Upon its promising future application in conventional flotation,
it is further needed to remark how mineral bioflotation works,
which could be attained by fundamental studies. A fundamental flo-
tation study can be developed by using pure minerals. Moreover,
one may consider that the adhesion of the microorganisms onto
the mineral surface is mainly related to an attractive and repulsive
⇑ Corresponding author. Tel.: +55 21 3527 1723.
E-mail address: torem@puc-rio.br (M.L. Torem).
forces between the cell wall and the mineral surface. Therefore, the
separation of the desirable mineral depends on the selectiveness of
the bacterial strains for a mineral surface, conferring to them hydro-
phobic or hydrophilic properties (Natarajan, 2006; Rao and
Subramanian, 2007).
Previous studies indicate that the hematite bioflotation suc-
ceeded using other hydrophobic non-pathogenic bacteria such as
Mycobacterium phlei (Dubel et al., 1992; Yang et al., 2007),
Paenibacillus polymyxa (Deo et al., 2001), Rhodococcus opacus
(Mesquita et al., 2003), Rhodococcus erythropolis (Yang et al.,
2013) and Bacillus subtilis (Sarvamangala and Natarajan, 2011).
Rhodococcus ruber (R. ruber), a gram positive non-pathogenic
strain, can be found widely in nature. It was used as biomass
for the removal of cobalt and nickel from liquid solutions
(Borges, 2011). The R. ruber strain is being recently studied in
mineral biotechnology and its interaction with hematite will be
assessed for the first time in this work. The aim of the present
research is to study and evaluate the strain R. ruber before and
after interaction with hematite particles. Therefore, the funda-
mental aspects of hematite flotation using R. ruber as a bacterial
strain are presented through the study and evaluation of the sys-
tem hematite–R. ruber, as hematite and R. ruber separately, by
zeta potential and adsorption measurements, flotation studies
and SEM micrographs.

http://dx.doi.org/10.1016/j.mineng.2014.12.022
0892-6875/Ó 2014 Elsevier Ltd. All rights reserved.
64 L.Y. Lopez et al. / Minerals Engineering 75 (2015) 63–69
2. Materials and methods
2.1. Sample mineral preparation
Pure hematite was used in this study. The sample was provided
by a local supplier, Estrada Mining, Belo Horizonte, Minas Gerais
State. Originally, the sample was delivered in a medium particle
size of 3 mm. After dry grinding (in a porcelain mortar) and wet
screening (in Tyler mesh sieves), were obtained the desired size
fractions (Table 1). The ground hematite was washed with HCl
10 4 M, and later with double-distilled water until the pH of the
mineral suspension was the same as initially. Finally, all samples
were stored in a desiccator.
2.2. Biomass obtaining from bacterial strain growth
R. ruber was used as the bacterial strain from which was
obtained the flotation reagent, and it was supplied by the Culture
collection of Fundação Tropical de Pesquisa e Tecnologia AndréTo-
sello – Campinas, São Paulo.
R. ruber is a non-pathogenic and gram-positive microorganism,
obtained by isolation from the ground. This bacteria was sub-culti-
vated on the laboratory using the culture medium TSB (Tryptic Soy
Broth) from HimediaÒ, composed from 15 g/L de tryptone, 5 g/L
digesting enzyme of soybean meal e 5 g/L of sodium chloride.
Stocks of the bacteria were prepared and renewed periodically
using this medium in Petri plates and saving them for 48 h at
20 °C in a bacteriological stove. After the bacteria cultivation in
the liquid medium (pH 7.2), the flasks were disposed on a rotary
shaker, maintained at 200 rpm and 28 °C, for 24 h. After this time
the bacterial suspension was centrifuged, twice washed in deion-
ized water and finally concentrate in 100 mL of 10 3 M NaCl solu-
tion. To avoid further bacterial development, the concentrate was
inactivated in the autoclave.
From now on, the cellular suspension is named as bioreagent or
biomass, and its concentration was quantified first by dry weight
on an oven at 60 °C for 24 h, and by optical density in UV-1800 Shi-
madatzu Spectrophotometer, at a wavelength of 620 nm.
2.3. Zeta potential measurements
Zeta potential measurements were made using the Zeta Meter
4.0+ apparatus (Zeta-Meter, Inc., Staunton-USA), using the electro-
phoresis technique. The evaluation of the zeta potential profiles of
pure hematite was carried out before and after interaction with the
R. ruber biomass suspension and the pH was an adjusted using
diluted HCl and NaOH solution. It was used as aqueous medium
the indifferent electrolyte 10 3 M NaCl, for concentrations of
0.2 g/L for hematite and 0.1 g/L for the bioreagent for electropho-
retic measurements carried separately.
2.4. Adsorption measurements
In the adhesion experiments, 0.25 g of mineral sample was
added to 25 mL of cellular suspension with a fixed initial concen-
tration of 0.6 g/L, and conditioned for 10 min after adjusting the
Table 1
Particle size range for each experiment.
Test Particle size (lm)
Microflotation ( 90+75)
( 75+53)
Microflotation and adsorption ( 53+38)
Zeta potential ( 38+20)
pH values. It was observed that the adhesion between the bacteria
and both minerals was complete after 5 min. An additional time of
1 min was allowed for settling of the mineral particles, after which
5 mL of the supernatant was collected for absorbance measure-
ments to estimate the amount of adsorbed cells. The tests were
performed varying pH, biomass concentration, and contact time,
at 25 °C.
2.5. Microflotation experiments
Microflotation experiments were performed in a modified
Partridge–Smith cell flotation. For each of the evaluations were
varied the pH of the solution, the biomass concentration, and the
particle size range. An amount of 0.5 g of hematite was added to
0.25 L total volume suspension of known bacterial concentration,
with pH adjusted with diluted HCl and NaOH solutions. The min-
eral was conditioned with the biomass suspension inside the
Partridge–Smith cell under constant stirring with a magnetic agita-
tor for 5 min, and then the mineral flotation tests were carried out
using air at a flow rate (supplied by a Vacuum pump) of 15 mL/min
for 5 min. The floatability was calculated as the ratio of the weights
of floated related to the total weighted sample.
2.6. SEM micrographic analysis
A sample of hematite after interaction with the biomass sus-
pension was centrifuged at 4000 rpm for 5 min. After that, water
remains were removed and later were dehydrated in graded series
of ethanol or acetone, and air dried under vacuum. Dry samples
were loaded for SEM studies in a Quantax 70 TM-3000 by Hitachi.
3. Results and discussion
3.1. Zeta potential studies
Zeta potential (ZP) measurements were carried out with the
aim of evaluate a possible variation of the electrokinetic properties
of hematite after interaction with R. ruber cells. Fig. 1 presents the
zeta potential profiles of the R. ruber biomass, hematite and of the
system R. ruber–hematite in function of pH. The zeta potential pro-
file of the microorganism and the location of the isoelectric point
(IEP), at around pH 3, go in concordance with the results presented
in a previous study of R. ruber as a metal removal biomass (Borges,
2011). The values of ZP reveal the stability of cells suspension at
25
R.ruber
20
Hematite
Hematite - R.ruber
15
10
Zeta potential (mV)
5
0
-5
-10
-15
-20
-25
Tyler mesh -30
1,7 2,4 2,8 3,5 5,5 7 8
170/200
200/270 pH
270/400
400/635 Fig. 1. Zeta potential of hematite before and after biomass R. ruber interaction, with
NaCl 10 3 M as background electrolyte (biomass concentration 0.20 g/L).
 L.Y. Lopez et al. / Minerals Engineering 75 (2015) 63–69
15 mV in the slightly alkaline range, whilst in acidic range, the
stability is reduced and after 2 min the bacteria cells started to
agglomerate and settle.
The IEP of hematite was found at pH 5.5 approximately.
According to literature, the IEP point of hematite fluctuates
between a pH range of 4.8–6.4 (Dubel et al., 1992; Yang et al.,
2007, 2013; Deo et al., 2001; Mesquita et al., 2003; Sarvamangala
and Natarajan, 2011). The result of the interaction of hematite
and R. ruber cells can be seen in Fig. 1 as a modification of ZP profile
of hematite. The electrokinetic behaviour of hematite changed
utterly and adopted a ZP profile similar to that of the bacteria; this
can be a result of cells interaction at the mineral surface (Vilinska
and Rao, 2008; Dubel et al., 1992; Raichur et al., 1996; Faharat
et al., 2008; Botero et al., 2007; Mesquita et al., 2003; Deo et al.,
2001; Subramanian and Santhiya, 2003 and Chandraprabha and
Natarajan, 2006). According to previous researches, in acid med-
ium, the surface of the bacteria and of the mineral have a contrary
charge, which makes possible for electrostatic interaction to occur
between both surfaces and forming a biofilm onto the mineral sur-
face and therefore the value of the ZP of the mineral surface is close
to that of the bacteria (Merma et al., 2013; Faharat et al., 2008).
The literature shows that the zeta potential profile of a mineral
can change after interaction with bacterial cells. This modification
is achieved through adhesion/adsorption of bacterial cells and/or
metabolic products onto the mineral surface. The zeta potential
tests also help to elucidate the mechanisms involved in the inter-
action between bacterial cells and mineral surface. According to
several authors (Dubel et al., 1992; Raichur et al., 1996; Faharat
et al., 2008) there are values in the range of pH that would repre-
sent a potential area for electrostatic attraction between the bacte-
rial cells and a negative mineral surface. It corresponds to the pH
values where the bacteria and the mineral present contrary charge.
It is only possible for pH values smaller than the IEP of the bacteria,
in the case of R. ruber this track correspond to values lower than pH
2.8. On the other hand, at pH values above the IEP of the bacteria,
besides to electrostatic interactions may also exist specific interac-
tions (Merma et al., 2013).
3.2. Adsorption studies
Studies of bacterial adhesion provide important information
about the affinity of the microorganism towards the mineral sur-
face. This ability depends on the functional groups of the bacteria
cell wall and of the mineral surface. Deo et al. (2001) and Faharat
et al. (2008) expressed their adsorption/adhesion tests respect of
the number of bacterial cells adsorbed onto hematite surface.
Moreover, Mesquita et al. (2003) accomplished bacterial cells
adhesion studies expressed in the milligrams of R. opacus adhered
per gram of hematite (mg R. opacus/g hematite), represented by Q,
or adsorption capacity of bacterial cells onto hematite surface. Sim-
ilarly, Botero et al. (2007) also performed adhesion experiments for
the mineral system R. opacus – calcite, magnesite, and barite.
The 53 lm +38 lm size fraction of hematite was used for
adsorption studies because it was the size fraction of higher sur-
face area among the other fractions used for flotation experiments.
Contact time, biomass concentration and pH solution were evalu-
ated to determine the adsorption capacity of R. ruber among the
surface of hematite.
R. ruber adsorption onto hematite surface was studied, first, on
function of conditioning time. For concentrations 0.45 g/L and
0.60 g/L, the adhesion time remained constant in the first five min-
utes. Therefore, the optimum adhesion time considered for later
studies was five minutes, not depending of the biomass concentra-
tion (López, 2014).
In order to evaluate the effect of R. ruber biomass concentration, it
was selected the values of pH where it presented a better adsorption
65
of the bacterial cells onto the hematite surface. Fig. 2 shows that at a
pH 3, the profile of Q vs. biomass concentration achieves its top val-
ues. At this pH is found the IEP of R. ruber where adsorption of the
cells onto hematite is more favourable. Results showed that at
higher biomass concentration, higher is its capacity of adsorption,
from concentration 0.15 g/L until 0.60 g/L. From that value, adsorp-
tion capacity of R. ruber remains constant for upper concentrations.
R. ruber cells adhered onto hematite particles as a function of
pH values was also studied and it is presented in Fig. 3. The
adhered quantity of microorganism is high at the acid pH range,
especially at pH around 3. The uppermost quantity of R. ruber
biomass per gram of hematite was around at a concentration of
bacterial cells of 0.60 g/L, as previously found by Mesquita et al.
(2003), for the hematite–R. opaccus system. For a higher concentra-
tion (0.75 g/L), the quantity adsorbed remained the same as 0.60 g/
L. These results reaffirmed the previous studies of the influence of
biomass concentration in the adsorption capacity of R. ruber cells.
Deo et al. (2001) found out a marked decrease in cell adsorption
of P. polymyxa on hematite above pH 9. A high adsorption at low
pH may be due to the fact that in the acidic pH range electrostatic
forces of attraction between the negative bacterial cell surfaces and
positive hematite surface is significant. At pH values higher than
the isoelectric point, both cell surfaces and the hematite surface
is negatively charged resulting in electrostatic repulsion between
the mineral and bacterial cells. Although the adsorption of bacte-
rial cells decreased on hematite in the alkaline pH range, a good
number of cells still adsorbed suggesting that other non-electro-
static forces such as chemical interaction, hydrogen bonding and/
or hydrophobic interaction also play an important role in the
adsorption processes.
Mesquita et al. (2003) studied the effect of pH for the adhesion
of R. opacus cells onto hematite and quartz particles microorgan-
ism was high at the acid pH range. Even though the bacterial cells
tended to adhere on both mineral samples, the adhesion was
higher on hematite than on quartz, for which almost negligible
adhesion was observed above pH 5.5.
3.3. Microflotation studies
Microflotation studies were carried out in a modified Partridge–
Smith cell. For the flotation experiments, using R. ruber strain as
bioreagent, hematite particles were initially contained in a column
of the test solution closed at the bottom by a glass frit of fine 40 lm
30
pH 7
25 pH 2
pH 3
pH 5
Q (mg R.ruber . g HZ)
20 pH 9
-1
15
10
5
0
0,0 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8
Biomass concentration (g.L-1)
Fig. 2. Adsorption measurements for R. ruber adsorb onto hematite surface in
function of biomass concentration, for a conditioning time of 5 min, particle size
range ( 53+38) lm.
66 L.Y. Lopez et al. / Minerals Engineering 75 (2015) 63–69

28 100
-1
26
B.concent. 0.15g.L
-1 90
24 B.concent. 0.30g.L
-1
22 B.concent. 0.45g.L 80
-1
20 B.concent. 0.60g.L
70
Q (mg R.ruber. g HZ)

-1
18 B.concent. 0.75g.L
-1

60

% Floatability
16
14 50
12
40
10
8 30 (-90+75 um)
6 (-75+53 um)
20 (-53+38 um)
4
2 10
0
0
1 2 3 4 5 6 7 8 9 10 0,0 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8
pH -1
Biomass concentration (g.L )
Fig. 3. Adsorption measurements for R. ruber adsorb onto hematite surface in
function of pH, for a conditioning time of 5 min, particle size range ( 53+38) lm. Fig. 4. Hematite microflotation as function of biomass concentration; particle size
range of ( 90+75) lm, ( 75+53) lm and ( 53+38) lm; flotation time 5 min; pH 7.

pore size. The bed was maintained in a gently moving suspension

by the rotating magnetic stirrer and a controlled flow of air was
passed through the porous base. The volume of the cell was of
0.2 L (López, 2014).
Particles exhibiting hydrophobic surface properties became
attached to the bubbles. Depending upon the properties of the
solution the bubbles either formed froth or burst. Floated particles
were retained in both cases, either in the froth or on the annular
floor of the upper section, and were collected at the end of the test
by washing through the side tube. The gas flow rate was standard-
ized at 15 mL/min (at atmospheric pressure). Flotation was contin-
ued for 5 min after the first appearance of bubbles.
R. ruber froth was formed after 2 min of air released at the
Partridge–Smith flotation cell. The bigger amount of bubbles was
formed at a value of pH of 3. A lower amount of bubbles was
observed at pH 4 and 5, decreasing until its complete depression
at pH range from 6 to 11. In resume, R. ruber cell suspension
achieves a higher froth formation from a pH range of 3–5, produc-
ing the highest amount at the pH of its isoelectric point (pH 3). This
effect could be explained due to the fact that at this pH the cells
have zero charge on their surface, which let the cells to coagulate
forming cell flocs. Moreover, the interactions between the bacterial
cells and the air bubbles would increase since the electrostatic
repulsion would be reduced (Merma et al., 2013; Okada et al.,
1990).
The flotation studies were done for different particle sizes:
90 lm +75 lm, 75 lm +53 lm, 53 lm +38 lm. The solution
was conditioned with NaCl 10 3 M, at a pH of 5, at 25 °C, and under
constant agitation to keep the particles suspended.
Before flotation experiments were performed, blank assays
were made to know how much of the initial mineral sample was
carried just by air bubbles (no addition of biomass or frother),
3.3.1. Effect of biomass concentration on mineral floatability
Biomass concentration was varied in order to find an optimal
concentration of R. ruber cells for the performance of further flota-
tion studies. It was found that the highest hematite floatability, an
84%, corresponded for a concentration value of 0.60 g/L, in concor-
dance with previous results of zeta potential and adsorption stud-
ies (Fig. 4).
According to Dubel et al. (1992), hematite flotation in a
Hallimond tube for a particle size range of +53 lm 20 lm, at
pH 7 and with a flotation time of 3 min, the floatability of hematite
increases as M. phlei concentration does.
The effect of P. polymyxa onto hematite was studied by Deo
et al. (2001), and they showed that the flotation recovery of hema-
tite was unaffected by increasing the cell number. At a cell density
of 1.5  109 cells/mL, only 9% of the hematite sample was recov-
ered in the float fraction.
According to Yang et al. (2013), the hematite recovery increased
rapidly as concentration of R. erythropolis increased from 0 to
75 mg/L, but levelled off at above 75 mg/L, therefor an optimal
hematite recovery was at that concentration (89.67% recovery).
The recoveries of other three minerals increased slightly with
increasing concentrations of R. erythropolis.
80
-90+75um
-75+53um
70 -53+38um
60
%Floatabilty

50
expressed as % floatability, or how much was burst to the froth
by agitation, expressed as % entrainment. Table 2 presents these
40
previous results.
As particle size decreases, the percentage of entrainment also
30
decreases, whilst % floatability achieved higher values.
20

Table 2
10
% Entrainment and % floatability for each particle size range.
2 4 6 8 10
Particle size range (lm) % Entrainment % Floatability pH
( 90+75) 5 8
( 75+53) 3 9 Fig. 5. Hematite microflotation as function of pH; particle size range of
( 53+38) 2 10 ( 90+75) lm, ( 75+53) lm and ( 53+38) lm; flotation time 5 min; biomass
concentration 0.15 g/L.
 L.Y. Lopez et al. / Minerals Engineering 75 (2015) 63–69 67

Fig. 6. Images of blank floatability experiments: (a) biomass + flotanol and (b) only biomass, both at biomass concentration 0.60 g/L, at pH 3.

100 100
Biomass+Flotanol pH 3 -1
90 90 Biomass+Flotanol B.concent 0.15g.L
Biomass pH 3 -1
Biomass+Flotanol pH 7 Biomass B.concent 0.15g.L
80 80 Biomass+Flotanol B.concent 0.60g.L
-1
Biomass pH 7
-1
70 Biomass B.concent 0.60g.L
70
%Floatability

60 60
%Floatability

50 50

40 40

30 30

20 20

10 10

0 0
1 2 3 1 2 3
Particle size Particle size

Fig. 7. Effect of Flotanol in the bioflotation of hematite with R. ruber in function of Fig. 8. Effect of Flotanol in the bioflotation of hematite with R. ruber in function of
pH and particle size (biomass concentration 0.15 g/L). biomass concentration and particle size.

3.3.2. Effect of pH on mineral floatability

In the flotation process, an important variable, perhaps the most
important, is pH of the suspension. This statement is supported by
the fact that mineral surface or the bacterial cell wall is activated
through dissolution or hydrolysis reactions.
For this stage of the work, floatability studies, were performed
at a constant particle size range and biomass concentration
0.15 g/L, and in the pH range between 2 and 9. The solution con-
taining hematite and R. ruber biomass was conditioned with NaCl
10 3 M, at 25 °C and carried out for 5 min.
As it can be seen in Fig. 5, for all the size ranges, their highest
floatability values are at a pH around 3, pretty much similar to
the pH of the IEP of R. ruber. This result goes in concordance with
was found out as stated lines above, and in similarity with
Merma et al. (2013), Mesquita et al. (2003) and Botero et al.
(2007). Also, as seen in Fig. 5, higher floatability values are
achieved for the smallest particle size range, 53 lm +38 lm. This
was expected because smaller particles are easier to be attached to
R. ruber biomass and less heavy to be carried by gas microbubbles.
The previous flotation results are in good accordance with the
former zeta potential and adsorption results, where it was
presented a higher interaction between R. ruber cells and hematite,
mainly for pH values below 3. Despite the high influence of pH, the
strong affinity between hematite and microbial cells is clearly
evident.
Some studies reported a wide floatable pH range, well below
the IEP of hematite. Strong flotation of hematite from pH 3 to 11
was reported, using octadecylammonium chloride C18H37NH4Cl
as a collector (Iwasaki et al., 1960). The IEP of the hematite sample
was reported to be pH 6.7. Along the same line, it was found that
hematite could be floated using dodecylamine as collector over
the pH range from 0.8 to 2.0 (Shergold et al., 1968), from pH
3–10 (Shergold and Mellgren, 1971), and from pH 2–12
(Partridge and Smith, 1971). Therefore, it seems that the mecha-
nism of electrostatic adsorption stabilized by hydrophobic interac-
tions alone cannot satisfactorily explain the floatability of hematite
well below its IEP.
According to Mesquita et al. (2003) the R. opacus bacteria pre-
sented a higher preference for hematite than for quartz, achieving
a floatability around 90% and using 600 mg/L of the bacteria. It is
possible to observe that this floatability value was presented next
68 L.Y. Lopez et al. / Minerals Engineering 75 (2015) 63–69
Fig. 9. Scanning electron images of hematite particles, showing the R. ruber cells adhered onto the mineral surfaces (magnification 3000 and 10000).
to the IEP of the bacteria, which is in accordance with the present
work. Likewise, Yang et al. (2013) noticed a floatability of hematite
around 90% when the R. erythropolis bacterium was used, using
75 mg/L and at pH 6. However, according to the authors, electro-
static attraction is unlikely between this bacterium and hematite,
so, specific interactions (hydrogen bonding, chemical interaction
forces) should also be involved in the system, which is also in
accordance with the present work.
Finally, according to Faharat et al. (2008) the Bacillus polymyxa
can act as a depressant of hematite. The Bacillus polymyxa rendered
quartz surfaces more hydrophobic whilst simultaneously confer-
ring hematite, corundum and calcite surfaces enhanced hydrophi-
licity. The authors observed that is possible to float quartz at
pH < 4.3 with a recovery of 58% under these experimental condi-
tions. Whilst, hematite did not float at any pH value and its flota-
tion recovery was less than 10%. Finally, the results presented in
this work show that R. ruber strain presents a great potential as a
biocollector of hematite.
3.3.3. Use of flotanol as frother
Depending on the pH of the suspension, some flotation
reagents, as amines, can act as collector and as frother (El-Shall
et al., 2000). However, the partial replacement of the amine by
ordinary frothers has been investigated to be an attractive route.
According to Soares (2012) the use of the frother Flotanol M
(75 mg/L) together with the collector Flotigam EDA (10 6 M)
increases the floatability of hematite at the pH range between 8
and 10. Araujo et al. (2004) reported that replacing approximately
10% of the total amine dose with polyglycol-type frothers, such as
Flotanol D14 and Flotanol C7 increased the quartz recovery for the
laboratory scale single mineral and Brazilian iron ore flotation.
As mentioned in the Literature review, some microorganisms
currently used as bioreagents can also behave as biofrothers. The
property of reduce surface tension of water was conferred natu-
rally to these microorganisms due to the presence of amides in
their cell walls, which could make them behave as frothers. How-
ever, the use of an ordinary frother was not reported in the
literature.
Thus, the flotation experiments using flotanol, as frother, fol-
lowed the same experimental procedure. The mineral was condi-
tioned with the biomass suspension inside the Partridge–Smith
cell under constant stirring with a magnetic agitator for 5 min in
aqueous medium using the indifferent electrolyte NaCl 10 3 M,
and a concentration of flotanol of 75 mg/L. The value of the concen-
tration of flotanol was taken from the literature and chose for eval-
uation in the hematite–R. ruber system after previous blank tests.
Fig. 6 shows the images of blank floatability experiments, with
no hematite particles present in the flotation device. As can be seen
there is a significant difference between both images. Fig. 6a shows
the mixture of R. ruber biomass (0.60 g/L) with flotanol (0.75 g/L),
and the high of the froth formed is taller than the froth formed only
by the biomass (Fig. 6b). This behaviour is supported by the fact
the R. ruber strain only form a stable froth at a pH value of 3. There-
fore, even when this strain works effectively as a biocollector of
hematite, it strongly depends on the pH of pulp solution and of
concentration. Furthermore, flotation experiments were carried
out using flotanol mixture with biomass suspension inside the
Partridge–Smith cell in aqueous medium using the indifferent elec-
trolyte NaCl 10 3 M, under same experiment conditions.
The effect of flotanol in function of pH and biomass is shown in
Figs. 7 and 8. In both figures the particle size range is represented
by numbers 1 ( 90 lm +75 lm), 2 ( 75 lm +53 lm) to 3 ( 53 lm
+38 lm).
Results of hematite bioflotation demonstrated the affinity of R.
ruber for hematite working successfully as a collector, especially
for pH 3 and for a biomass concentration of 0.60 g/L. When adding
75 mg/L of flotanol to the aqueous medium, flotation results var-
ied, as Figs. 7 and 8 shows.
For smaller particles size ( 53 lm +38 lm), the floatability of
hematite with R. ruber increases whilst floatability with R. ruber
and flotanol presents no major influence in hematite floatability
by achieving almost the same values, even when the best condi-
tions for bioflotation of hematite are presented (pH 3 and 0.60 g/
L). Therefore, use a conventional frother as flotanol can be effective
for a size particle minus than 90 lm and bigger than 53 lm.
Mesquita et al. (2003), Botero et al. (2007) and Merma et al.
(2013) showed that R. opacus strain can work not also as a selective
collector but also as a biofrother. More recently Merma et al.
(2013) studied the behaviour of R. opacus as water surface tension
(70 m N/m) reducer. The greatest reduction in surface tension was
with 0.15 g/L biomass. Froth was higher in between pH 3 and 7,
with surface tension values between 54 m N/m and 56 m N/m.
Moreover, the adaptation of the bacteria to a mineral substrate
caused an increase in tension surface of pH values 3, 5 and 7 and
a reduction in alkaline medium.
3.4. SEM results
Fig. 9 shows the results of SEM of hematite particles after inter-
action with R. ruber biomass. The concentration of biomass was of
0.60 g/L or 109 cells/mL. The detailed experimental procedure for
bacteria cell counting is explained elsewhere (López, 2014). The
micrographs revealed the presence of R. ruber rod cells adhere onto
hematite surface (particle size fraction +38 lm 53 lm). R. ruber
cells tend to gather at the moment of adhesion onto the mineral
surface, forming a bond colony of cells.
For the B. subtilis cells adsorption onto hematite, adsorption
density of cells was found to be significantly higher on hematite
compared to that on corundum, calcite, and quartz respectively.
Compared to quartz, adsorption density of bacterial cells was
almost ten times higher on hematite surfaces. Adsorption density
of bacterial cells on calcite was two times higher than that on
 L.Y. Lopez et al. / Minerals Engineering 75 (2015) 63–69
quartz. Among the above minerals, quartz exhibited the least sur-
face adsorption of B. subtilis followed by calcite (Sarvamangala and
Natarajan, 2011).
Mesquita et al. (2003) showed that scanning electron micro-
graphs of quartz and hematite particles were obtained of the best
microflotation test results, at pH 4.5 for hematite and pH 3.5 for
quartz. SEM examination of the interacted mineral surfaces
revealed the presence of R. opacus cells adhered on the mineral sur-
faces. Even though the bacterial cells tended to adhere on both
mineral surfaces, for hematite particles, more density of cells
adhered is pronounced.
Moreover, Yang et al. (2013) found out in the SEM images of
adsorption of R. erythropolis onto hematite surfaces, that the bacte-
ria on hematite surface were attracted to each other. The individual
hematite particles were transformed into agglomerates of hema-
tite through bridging of the bacteria.
4. Conclusions
The results presented in this paper showed the strong affiliation
between R. ruber cells and hematite. For a constant ionic strength
(10 3 M NaCl), the zeta potential evaluation of hematite particles
before and after interaction with bacterial cells showed the modi-
fication of hematite zeta potential profile. The biomass adhesion
was found more strongly attached for a pH around 3, at a concen-
tration of 0.6 g/L (or 109 cells/mL). The bioflotation of hematite
using R. ruber as bioreagent depends on the pH value, bacterial
concentration and particle size. The higher floatability of hematite
was found at a pH value around 3, with a percentage of 84%, using
0.6 g/L and a particle size range between 53 lm and +38 lm.
Also, were performed bioflotation studies using a commercial
frother for iron ores. Flotanol can be effective for a size particle
minus than 90 lm and bigger than 53 lm. The scanning electron
images showed the presence of R. ruber cells adhered on hematite
surface.
Acknowledgements
The authors acknowledge CNPq (Conselho Nacional de
Desenvolvimento Científico e Tecnológico), VALE, CAPES
(Coordenação de Aperfeiçoamento de Pessoal de Nível Superior)
and FAPERJ (Fundação Carlos Chagas Filho de Amparo à Pesquisa
do Estado do Rio de Janeiro) for the financial support.
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