Hematite Flotation Using a Crude Biosurfactant

Extracted from a Gram Positive Bacteria

Jhonatan Puelles, Antonio Merma, Carlos Castaeda and Maurcio Torem
Pontificia Universidade Catlica do Rio de Janeiro, Brazil

ABSTRACT
Bioflotation is defined as a separation process by which the mineral of interest is floated or
depressed selectively using reagents of biologic origin, also known as bioreagents. These substances
are characterized by their green chemistry, selectivity and potential to treat fine particles. In that
sense, the researchs principal objective is the assessment of the hematite floatability using a crude
biosurfactant extracted from a Gram positive bacteria and consequently determine its potential as
an alternative against synthetic reagents or the bacteria itself.
Characterization by FTIR spectrophotometry identified alcohol (-OH), ketone (C=O) groups, and
saturated and unsaturated carbon chains, which may compose the mycolates that form the cellular
wall of the bacteria. Surface tension measurements of the crude biosurfactant determined a CMC of
92 ppm lowering the water surface tension from 72 to 52.5 mN.m-1.
Finally it was tested the crude biosurfactant against the bacteria itself in microflotation tests,
resulting in a faster process and improved hematite recovery. The maximum hematite floatability
obtained with the bacteria was around 43.5% at neutral pH whilst the optimum recovery with the
crude biosurfactant was 95% at pH 3. The results showed a high affinity of the crude biosurfactant
for hematite particles and relatively low reagent consumption.

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INTRODUCTION
Bioflotation has been extensively studied these late years, because it is an attractive alternative to
replace the common reagents by environmental friendly bioreagents (Dwyer et al., 2012). They are
characterized by having low toxicity and degrading easily, once discharged to the environment. In
addition, the raw material for its production is low cost, renewable and available (Salehizadeh et al.,
2014). Finally, they have potential to treat low grade ores, giving new alternatives for uneconomic
deposits (Mesquita et al., 2003).
On the other hand, even though there is plenty of evidence that bioflotation processes have good
recovery and selectivity (Natarajan, 2006; Mesquita et al., 2003; Merma et al., 2013; Sharma, 2001),
problems such as the low technology including a poor understanding of the mechanisms, kinetics
and thermodynamics of the process (Fomina et al., 2014) hinder a successful scale up. Because the
bacteria is a heterogeneous mixture of several compounds, it is complicated to know the specific
mechanism by which they selectively turn the minerals hydrophobic. Furthermore, it is interesting
to highlight that the theoretical models that describe the adhesion between the mineral and the
bacteria do not account for biological factors (Hermansson, 1999). The inclusion of a biological
frame in the field of bioflotation will have a great significance in understanding what occurs behind
the process.
Some biomolecules that are responsible of the adhesion and selective floatability of the minerals are
surface active substances which are excreted or cell bounded on the microorganism surface
(Kuyumcu et al., 2009). They are known as biosurfactants and have several functions such as
facilitate the growth of their producers by increasing the substrate availability, transporting
nutrients, and acting as biocide agents (Rodrigues et al., 2006). Therefore, in order to narrow the
heterogeneity and reduce the complexity of the bioflotation process; this study focuses on the
extraction, characterization, modeling and use of the biosurfactants as potential flotation reagents.

METHODOLOGY

Microorganism and culture media

A Gram positive bacteria obtained from the Brazilian Collection of Environmental and Industrial
Microorganisms (CBMAI-UNICAMP) was used in this study. The maintenance solid medium
consisted of 10.0 g dm-3 of glucose, 5.0 g dm-3 of peptone, 3.0 g dm-3 of malt extract, 3.0 g dm-3 of
yeast extract, 2.0 g dm-3 of CaCO3, and 12 g dm-3 of agar. In addition, the composition of the liquid
medium was the same as the solid media with the absence of agar.

Crude biosurfactant extraction and characterization by FTIR

The extraction method using hot ethanol was based on the work of Moreau et al. 2003. In order to
estimate the concentration of the crude biosurfactant the equation 3.1 was used. The crude
biosurfactant concentrate was stored at 4C for a maximum of 5 days. This assumption was based
on the biosurfactant biodegradability studies conducted by Pei et al. 2009.
In addition, in order to identify the functional groups presented in the crude biosurfactant, infrared
absorption spectra was carried out on a Nicolet FTIR 2000 spectrophotometer; a KBr matrix was
used as reference and a deuterated triglycine sulphate (DTGS) as detector. The sample was dried at
75 C and it was properly mixed with the KBr.

2
Surface tension measurements
The effect of crude biosurfactant concentration on the surface tension of distillated water was
studied at neutral pH and varying the biosurfactant concentration from zero until two hundred
fifty ppms, with intervals of fifty ppms. The surface tension measurements were performed using
the ring method in a Kruss K10 digital tensiometer.
In addition, to estimate the critical micelle concentration of the crude biosurfactant two tangents at
the minimum and maximum surface tension points were drew, corresponding the intersection to
the CMC. This estimation was based on Frana et al. (2015).

Microflotation studies
The flotation tests were carried out in a modified Hallimond tube, with 10-3 M NaCl as background
electrolyte, air rate of 35 dm3 min-1, hematite size fraction +75 -150 m, conditioning time 2 min. and
flotation time 1 min. A variable concentration of bioreagents was added, the flotability was then
calculated as the ratio of floated and the total weighed mineral.

RESULTS AND DISCUSSION

Crude biosurfactant extraction

The bacteria was incubated in a rotatory shaker at 125 rpm by 3 days. After the incubation time, the
biomass was centrifuged and washed two times with deionized water to remove the broth. The
crude biosurfactant recovery was 0.3 g per liter of broth. It is necessary highlight that the crude
biosurfactant may be composed of phospholipids, fatty acids, aminolipids in addition to the
glycolipids, due to the non-selectivity of the ethanol and the cell lysis as result of the relatively high
temperatures and pressures in the autoclaving.

Comparison of the crude biosurfactant and gram positive FTIR spectra

It can be seen in the fingerprint region of bacteria, below 1500 cm-1, a large number of absorptions
due to a variety of C-C, C-O and C-N single-bond vibrations may be occur; this region is unique for
every substance (McCurry, 2012). Additionally, it was found an intense peak between 1750 and
1620 cm-1 characteristic of aromatics, aldehydes, ketones and esters; Sharma (2001) reported the
same peak. The mycolates that form part of the cell envelope (Stratton et al., 1999) and are
responsible of the bacteria hydrophobicity (Sutcliffe, 1998) may be reflected by the alkane, ketone
and aldehyde peaks. The presence of amino groups and aromatic compounds, which may be part
of aromatic amino acids, indicate proteic substances which where reported to play a determinant
role in flocculation and flotation processes (Patra et al., 2008).
Regarding the crude biosurfactant, Table 1 shows the possible functional found in the crude
biosurfactant. The alcohol, alkane, alkene, and ketone groups at 3398, 2929 and 1629 cm -1
respectively, may indicate the presence of mycolic acids (Nishiuchi et al., 2000). The identification of
aromatic groups as well as amine groups at 1400, 1548 and 3350 cm -1, could indicate the presence of
polar amino acids such as tyrosine (Berg et al., 2012).

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Table 1 Possible functional groups identified by the FTIR spectra of the crude biosurfactant

Wavenumber(cm-1) Intensity Possible functional group Structures*

3397.94 High Alcohols

3350.00 Medium Amines

2929.27 Medium Alkanes

1629.44 High Alkenes, ketones ,

1400.41 Medium Aromatic

1047.35 Medium Alkanes

Finally, the similarity between both spectra as is show in Figure 1 indicate that the biosurfactant
was contained in the biomass, which also was used in microflotation tests. Although, it was
reported bacterial proteins play a determinant role in flocculation and flotation processes because
of its amphiphilic character (Patra, 2008). In the case of this bacteria evidence, suggest that proteic
compounds are not the active substances responsible of conferring mineral hydrophobicity, because
they would be denaturized by the temperature and pressure conditions as well as the solvent itself.

Figure 1 FTIR spectra of the bacteria (above) and the dried crude biosurfactant extracted by hot ethanol

Surface tension measurements

Figure 2 shows the surface tension in function of the crude BS concentration at neutral pH. The
estimated CMC is around 92 ppm and the surface tension decreases until 50.5 mN m-1. On the other
hand, most of the biosurfactants extracted from bacteria were reported to low the surface tension of
water from 72 mN m-1 to values between 19 and 43 at CMC values between 17 and 37 ppm after
refination processes (Christova et al., 2014).

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Figure 2 Effect of the crude biosurfactant concentration on the surface tension of deionized water at 23C and
neutral pH

Merma et al. (2013) studied the surface tension of a gram positive bacteria, which also had a
hydrophobic character. It was reported that at 300 ppm and pH 3, the surface tension of water was
lowered to 52.5 mN m-1. Therefore, the crude biosurfactant extracted has a moderate capacity to low
the surface tension of water but it is not as efficient as a pure biosurfactant. In addition, the extract
may be composed of polymeric biosurfactants, which do not necessarily reduce the surface tension,
but they may effectively reduce the interfacial tension between immiscible liquids and form stable
emulsions (Dhanarajan et al., 2014).

Hematite microflotation tests

Figure 3 shows the hematite floatability using the crude biosurfactant extracted from bacteria. The
optimum is reached at pH 3, around 96%. Vidyadhar et al. (2014) reported a hematite flotability of
90 % at 304 ppm of sodium oleate in microflotation tests carried out in a Hallimond tube. Literature
review suggest that most of the non-toxic biosurfactants are anionic (Christova et al., 2014); in
addition, based on electrophoretic studies reported by Mesquita et al. (2013), the hematite isoelectric
point was reported around 5.1. It is easy to see the correlation between the pH and the BS adhesion.
At acid pH there is going to be electrostatic attraction between the mineral surface and the anionic
biosurfactant, resulting in maximum adhesion and therefore maximum hematite recovery. On the
other hand at basic pH there is going to be minimal adhesion because of the electrostatic repulsion.

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 Figure 3 Hematite flotation test, NaCl 10-3 M as background electrolyte, size fraction +75-150, conditioning
time 2 min, flotation time 1 min and air rate of 35 dm3 min-1

Figure 4 shows the mineralized froth using the crude biosurfactant to float 1 g of hematite at pH 3.
The affinity of the biosurfactant for hematite is significant.

Figure 4 Biosurfactant froth with hematite particles (+75 -150 m), NaCl 10-3 M, and concentration of 125 ppm
and pH 3

Figure 5 shows a bar diagram comparing the hematite floatabilities using the biosurfactant and
bacteria itself. In order to evaluate this contrasting, both experiments were conducted at similar
conditions, airflow, conditioning time, flotation time, hematite sample, size fraction and
background electrolyte concentration.
It is worth to note that the hematite recovery using the biosurfactant as collector is much greater
than the bacteria itself. In addition, while the maximum hematite recovery using the bacteria
resulted at pH 7, the biosurfactant had an optimum recovery at pH 3. The high performance of the
bioreagent at acid pH is characteristic of most biosurfactants which are stable, even at extreme
temperatures, pH and salinity (Kosaric, 2001).

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 Figure 5 Bar diagram comparing hematite floatabilities using the crude biosurfactant (green) and bacteria
(blue)

CONCLUSIONS
The extraction method allowed the recovery of the cell associated and intracellular substances that
are responsible of conferring hydrophobicity to the hematite surface. The crude biosurfactant
recovery was around 0.3 g.dm-3.
The functional groups identified in the FTIR spectra of the crude biosurfactant suggest that it is
composed of mycolyc acids and polar aminoacids. The former is responsible of the bacteria
hydrophobicity. The similarity of both spectra, the bacteria and the crude biosurfactant, indicate
that the biosurfactant was contained in the biomass. Finally, it was discarded the presence of
proteins in the crude biosurfactant, because of its denaturation along the extraction process.
Throughout surface tension measurements of the crude biosurfactant, it was estimated a CMC
around 92 ppm, lowering the water surface tension from 72 to 52.5 mN.m -1.
Regarding hematite flotation tests with the bisurfactant showed that it required a relatively shorter
flotation time, requiring of 1 min to achieve its highest hematite recovery of 95%, at acid pH of 3
and crude biosurfactant concentration of 125 ppm. Finally, it was concluded a better floatability of
hematite in the presence of the crude biosurfactant than the bacteria itself. Such results show the
potential use of the crude biosurfactant as a low toxic and environmental friendly alternative
against synthetic reagents in hematite flotation.

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ACKNOWLEDGEMENTS
The authors acknowledge PUC-Rio (Pontifical Catholic University of Rio de Janeiro), CNPq
(Conselho Nacional de Desenvolvimento Cientfico e Tecnolgico), Fundao de Amparo
Pesquisa do Estado do Rio de Janeiro-FAPERJ and ITV-VALE for the financial support.

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