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Fundamental aspects of hematite flotation using the bacterial strain Rhodococcus ruber as bioreagent
Minerals Engineering xxx (2015) xxx–xxx
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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
Article history: Received 30 July 2014 Revised 4 December 2014 Accepted 12 December 2014 Available online xxxx Keywords: Hematite Flotation bioreagents Rhodococcus ruber
a b s t r a c t Previous research showed the effectiveness of bacterial strains as flotation reagents on Hematite beneficiation. The aim of this work is to study and evaluate Rhodococcus ruber as a biocollector. The sample was conditioned with the biomass suspension by stirring under specific conditions as particle size, biomass concentration, pH solution and conditioning time. The results showed a change in hematite zeta potential 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 Partridge–Smith cell flotation, and the highest floatability (84%) was achieved at size fraction 53 lm +38 lm under the conditions mentioned before. Complementary floatability studies were performed 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 environmental friendly reagents. Therefore, the term bioflotation turned out attractive not only as the solution of the problems mentioned before but to its technological potential and mineral selectivity (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 flotation 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: [email protected] (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 hydrophobic or hydrophilic properties (Natarajan, 2006; Rao and Subramanian, 2007). Previous studies indicate that the hematite bioflotation succeeded 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 fundamental aspects of hematite flotation using R. ruber as a bacterial strain are presented through the study and evaluation of the system 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.
Please cite this article in press as: Lopez, L.Y., et al. Fundamental aspects of hematite flotation using the bacterial strain Rhodococcus ruber as bioreagent. Miner. Eng. (2015), http://dx.doi.org/10.1016/j.mineng.2014.12.022
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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 AndréTosello – Campinas, São Paulo. R. ruber is a non-pathogenic and gram-positive microorganism, obtained by isolation from the ground. This bacteria was sub-cultivated 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 deionized water and finally concentrate in 100 mL of 10 3 M NaCl solution. 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 Shimadatzu 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 electrophoresis 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 electrophoretic measurements carried separately.
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 measurements 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 mineral was conditioned with the biomass suspension inside the Partridge–Smith cell under constant stirring with a magnetic agitator 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 suspension 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 profile 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 Hematite Hematite - R.ruber
20 15
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 concentration of 0.6 g/L, and conditioned for 10 min after adjusting the
Zeta potential (mV)
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Table 1 Particle size range for each experiment.
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Test
Particle size (lm)
Tyler mesh
Microflotation
( ( ( (
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Microflotation and adsorption Zeta potential
90+75) 75+53) 53+38) 38+20)
-30 1,7
2,4
2,8
3,5
5,5
7
8
pH 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).
Please cite this article in press as: Lopez, L.Y., et al. Fundamental aspects of hematite flotation using the bacterial strain Rhodococcus ruber as bioreagent. Miner. Eng. (2015), http://dx.doi.org/10.1016/j.mineng.2014.12.022
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Studies of bacterial adhesion provide important information about the affinity of the microorganism towards the mineral surface. 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. Similarly, 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 surface area among the other fractions used for flotation experiments. Contact time, biomass concentration and pH solution were evaluated 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 minutes. Therefore, the optimum adhesion time considered for later studies was five minutes, not depending of the biomass concentration (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
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 pH 2 pH 3 pH 5 pH 9
25
-1
3.2. Adsorption studies
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 values. 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, adsorption 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 concentration (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 bacterial cells decreased on hematite in the alkaline pH range, a good number of cells still adsorbed suggesting that other non-electrostatic 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 microorganism 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.
Q (mg R.ruber . g HZ)
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 medium, 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 surface 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 interaction 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 represent a potential area for electrostatic attraction between the bacterial 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 interactions (Merma et al., 2013).
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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.
Please cite this article in press as: Lopez, L.Y., et al. Fundamental aspects of hematite flotation using the bacterial strain Rhodococcus ruber as bioreagent. Miner. Eng. (2015), http://dx.doi.org/10.1016/j.mineng.2014.12.022
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-1
B.concent. 0.15g.L -1 B.concent. 0.30g.L -1 B.concent. 0.45g.L -1 B.concent. 0.60g.L -1 B.concent. 0.75g.L
26 24 20 18
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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 standardized at 15 mL/min (at atmospheric pressure). Flotation was continued 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, producing 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), expressed as % floatability, or how much was burst to the froth by agitation, expressed as % entrainment. Table 2 presents these previous results. As particle size decreases, the percentage of entrainment also decreases, whilst % floatability achieved higher values.
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.
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 flotation studies. It was found that the highest hematite floatability, an 84%, corresponded for a concentration value of 0.60 g/L, in concordance with previous results of zeta potential and adsorption studies (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 hematite was unaffected by increasing the cell number. At a cell density of 1.5 109 cells/mL, only 9% of the hematite sample was recovered 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 -53+38um
70
60
%Floatabilty
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.
50
40
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20
Table 2 % Entrainment and % floatability for each particle size range.
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Particle size range (lm)
% Entrainment
% Floatability
( 90+75) ( 75+53) ( 53+38)
5 3 2
8 9 10
4
6
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10
pH Fig. 5. Hematite microflotation as function of pH; particle size range of ( 90+75) lm, ( 75+53) lm and ( 53+38) lm; flotation time 5 min; biomass concentration 0.15 g/L.
Please cite this article in press as: Lopez, L.Y., et al. Fundamental aspects of hematite flotation using the bacterial strain Rhodococcus ruber as bioreagent. Miner. Eng. (2015), http://dx.doi.org/10.1016/j.mineng.2014.12.022
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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.
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Fig. 7. Effect of Flotanol in the bioflotation of hematite with R. ruber in function of pH and particle size (biomass concentration 0.15 g/L).
Fig. 8. Effect of Flotanol in the bioflotation of hematite with R. ruber in function of 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 containing 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 mechanism of electrostatic adsorption stabilized by hydrophobic interactions alone cannot satisfactorily explain the floatability of hematite well below its IEP. According to Mesquita et al. (2003) the R. opacus bacteria presented 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
Please cite this article in press as: Lopez, L.Y., et al. Fundamental aspects of hematite flotation using the bacterial strain Rhodococcus ruber as bioreagent. Miner. Eng. (2015), http://dx.doi.org/10.1016/j.mineng.2014.12.022
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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, electrostatic 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 conferring hematite, corundum and calcite surfaces enhanced hydrophilicity. The authors observed that is possible to float quartz at pH < 4.3 with a recovery of 58% under these experimental conditions. Whilst, hematite did not float at any pH value and its flotation 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 naturally to these microorganisms due to the presence of amides in their cell walls, which could make them behave as frothers. However, the use of an ordinary frother was not reported in the literature. Thus, the flotation experiments using flotanol, as frother, followed the same experimental procedure. The mineral was conditioned 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 concentration of flotanol was taken from the literature and chose for evaluation 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. Therefore, 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 electrolyte 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 varied, 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 conditions 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 interaction 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
Please cite this article in press as: Lopez, L.Y., et al. Fundamental aspects of hematite flotation using the bacterial strain Rhodococcus ruber as bioreagent. Miner. Eng. (2015), http://dx.doi.org/10.1016/j.mineng.2014.12.022
L.Y. Lopez et al. / Minerals Engineering xxx (2015) xxx–xxx
quartz. Among the above minerals, quartz exhibited the least surface adsorption of B. subtilis followed by calcite (Sarvamangala and Natarajan, 2011). Mesquita et al. (2003) showed that scanning electron micrographs 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 surfaces. 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 bacteria on hematite surface were attracted to each other. The individual hematite particles were transformed into agglomerates of hematite 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 modification of hematite zeta potential profile. The biomass adhesion was found more strongly attached for a pH around 3, at a concentration 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. References Araujo, A.C., Viana, P.R.M., Peres, A.E.C., 2004. Reagents in iron ores flotation. Miner. Eng. 18, 219–224. Borges, E., 2011. Biosorption of Co (II) and Ni (II) from aqueous solutions using Rhodococcus ruber strain. MSc. Dissertation – Department of Materials Engineering, Pontifical Catholic University of Rio de Janeiro, Rio de Janeiro, 136p. Botero, A.E.C., Torem, M.L., Mesquita, L.M.S., 2007. Fundamental studies of Rhodococcus opacus as a biocollector of calcite and magnesite. Miner. Eng. 20 (10), 1026–1032.
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Chandraprabha, M.N., Natarajan, K.A., 2006. Surface chemical and flotation behavior of chalcopyrite and pyrite in the presence of Acidithiobacillus thiooxidans. Hydrometallurgy 83, 146–152. Deo, N., Natarajan, K.A., Somasundaran, P., 2001. Mechanisms of adhesion of Paenibacillus polymyxa onto hematite, corundum and quartz. Int. J. Miner. Process. 62, 27–39. Díaz-Lópes, C.V., Pecina-Treviño, E.T., Orrantia-Borunda, E., 2012. A study of bioflotation of chalcopyrite and pyrrhotite mixtures in presence of L. ferrooxidans. Can. Metall. Q. 51 (2), 118–125. Dubel, J., Smith, R.W., Misra, M., Chen, S., 1992. Microorganisms as chemical reagents: the hematite system. Miner. Eng. 5 (3–5), 547–556. Dwyer, R., Bruckard, W.J., Rea, S., Holmes, R.J., 2012. Review, bioflotation and bioflocculation review: microorganisms relevant for mineral beneficiation. Miner. Process. Extractive Metall. (Trans. Inst. Miner. Metall. C) 121 (2), 65–71. El-Shall, H., Abdel-Khalek, N.A., Svoronos, S., 2000. Collector-frother interaction in column flotation of Florida phosphate. Int. J. Miner. Process. 58, 187–199. Faharat, M., Hirajima, T., Sasaki, K., Aiba, Y., Doi, K., 2008. Adsorption of SIP E. coli onto quartz and its applications in froth flotation. Miner. Eng. 21, 389–395. Iwasaki, I., Cooke, S.R.B., Choi, H.S., 1960. Flotation characteristics of hematite, goethite and activated quartz with 18-carbon aliphatic acids and related compounds. Trans. AIME 217, 237–244. Kuyumcu, H.Z., Bielig, T., Vilinska, A., Rao, H., 2009. Biocoagulation and its application potentials for mineral bioprocessing. Open Miner. Process. J. 2, 1– 11. López, L.Y., 2014. Fundamental aspects of hematite flotation using the bacterial strain Rhodococcus ruber as a bioreagent. MSc. Dissertation – Department of Materials Engineering, Pontifical Catholic University of Rio de Janeiro, Rio de Janeiro, 132p. Merma, A.G., Torem, M.L., Morán, J.J.V., Monte, M.B.M., 2013. On the fundamental aspects in the apatite–quartz flotation using a Gram positive strain as a bioreagent. Miner. Eng. 48, 61–67. Mesquita, L.M.S., Lins, F.A.F., Torem, M.L., 2003. Interaction of a hydrophobic bacterium strain in a hematite–quartz flotation system. Int. J. Miner. Process. 71, 31–44. Natarajan, K.A., 2006. Microbially-induced mineral flotation and flocculation: prospects and challenges. In: Proceedings of XXIII International Mineral Processing Congress, pp. 487–498. Okada, K., Akagi, Y., Kogure, M., Yoshioka, N., 1990. Effects on surface charges of bubbles and fine particles on air flotation process. Can. J. Chem. Eng. 68 (3), 393–399. Partridge, A.C., Smith, G.W., 1971. Flotation and adsorption characteristics of the hematite–dodecylamine–starch system. Can. Metall. Q. 10, 229–234. Raichur, A.M., Misra, M., Bukka, K., Smith, R.W., 1996. Flocculation and flotation of coal by adhesion of hydrophobic Mycobacterium phlei. Colloids Surf., B 8, 13–24. Rao, K.H., Subramanian, S., 2007. Bioflotation and bioflocculation of relevance to minerals bioprocessing. In: Donati, Edgardo R., Sand, Wolfgang (Eds.), Microbial Processing of Metal Sulfides. Springer, ISBN 978-1402055881, pp. 267–286. Sarvamangala, H., Natarajan, K.A., 2011. Microbially induced flotation of alumina, silica/calcite from haematite. Int. J. Miner. Process. 99, 70–77. Shergold, H.L., Mellgren, O., 1971. Concentration of hematite at the iso-octane– water interface with dodecylamine as a collector. Trans. Inst. Min. Met. 80, 60– 68. Shergold, H.L., Prosser, A.P., Mellgren, O., 1968. New region of floatability in the hematite-dodecylamine system. Trans. Inst. Min. Met 77, C166–C170. Soares, Vanessa Aparecida Alves de Paula, 2012. Characterization of frothers with different hydrocarbon chains and their influence on hematite surfaces. Master Dissertation, Federal University of Minas Gerais, Graduate Program on Metallurgical and Mines Engineering, 212p. Subramanian, S., Santhiya, D., Natarajan, K.A., 2003. Surface modification studies on sulphide minerals using bioreagents. Int. J. Miner. Process. 72, 175–188. Vilinska, A., Rao, K.H., 2008. Leptosririllum ferrooxidans-sulfide mineral interactions with reference to bioflotation and bioflocculation. Trans. Nonferrous Met. Soc. China 18, 1403–1409. Yang, H., Zhang, Q., Jiang, Z., 2007. Adsorbability of Mycobacterium phlei on hematite surface. J. Univ. Sci. Technol. Beijing 14 (2), 103–106. Yang, H., Tang, Q., Wang, C., Zhang, J., 2013. Flocculation and flotation response of Rhodococcus erythropolis to pure minerals in hematite ores. Miner. Eng. 45, 67– 72.
Please cite this article in press as: Lopez, L.Y., et al. Fundamental aspects of hematite flotation using the bacterial strain Rhodococcus ruber as bioreagent. Miner. Eng. (2015), http://dx.doi.org/10.1016/j.mineng.2014.12.022
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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
Article history: Received 30 July 2014 Revised 4 December 2014 Accepted 12 December 2014 Available online xxxx Keywords: Hematite Flotation bioreagents Rhodococcus ruber
a b s t r a c t Previous research showed the effectiveness of bacterial strains as flotation reagents on Hematite beneficiation. The aim of this work is to study and evaluate Rhodococcus ruber as a biocollector. The sample was conditioned with the biomass suspension by stirring under specific conditions as particle size, biomass concentration, pH solution and conditioning time. The results showed a change in hematite zeta potential 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 Partridge–Smith cell flotation, and the highest floatability (84%) was achieved at size fraction 53 lm +38 lm under the conditions mentioned before. Complementary floatability studies were performed 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 environmental friendly reagents. Therefore, the term bioflotation turned out attractive not only as the solution of the problems mentioned before but to its technological potential and mineral selectivity (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 flotation 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: [email protected] (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 hydrophobic or hydrophilic properties (Natarajan, 2006; Rao and Subramanian, 2007). Previous studies indicate that the hematite bioflotation succeeded 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 fundamental aspects of hematite flotation using R. ruber as a bacterial strain are presented through the study and evaluation of the system 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.
Please cite this article in press as: Lopez, L.Y., et al. Fundamental aspects of hematite flotation using the bacterial strain Rhodococcus ruber as bioreagent. Miner. Eng. (2015), http://dx.doi.org/10.1016/j.mineng.2014.12.022
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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 AndréTosello – Campinas, São Paulo. R. ruber is a non-pathogenic and gram-positive microorganism, obtained by isolation from the ground. This bacteria was sub-cultivated 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 deionized water and finally concentrate in 100 mL of 10 3 M NaCl solution. 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 Shimadatzu 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 electrophoresis 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 electrophoretic measurements carried separately.
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 measurements 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 mineral was conditioned with the biomass suspension inside the Partridge–Smith cell under constant stirring with a magnetic agitator 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 suspension 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 profile 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 Hematite Hematite - R.ruber
20 15
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 concentration of 0.6 g/L, and conditioned for 10 min after adjusting the
Zeta potential (mV)
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Table 1 Particle size range for each experiment.
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Tyler mesh
Microflotation
( ( ( (
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90+75) 75+53) 53+38) 38+20)
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2,4
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pH 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).
Please cite this article in press as: Lopez, L.Y., et al. Fundamental aspects of hematite flotation using the bacterial strain Rhodococcus ruber as bioreagent. Miner. Eng. (2015), http://dx.doi.org/10.1016/j.mineng.2014.12.022
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Studies of bacterial adhesion provide important information about the affinity of the microorganism towards the mineral surface. 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. Similarly, 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 surface area among the other fractions used for flotation experiments. Contact time, biomass concentration and pH solution were evaluated 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 minutes. Therefore, the optimum adhesion time considered for later studies was five minutes, not depending of the biomass concentration (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
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 pH 2 pH 3 pH 5 pH 9
25
-1
3.2. Adsorption studies
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 values. 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, adsorption 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 concentration (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 bacterial cells decreased on hematite in the alkaline pH range, a good number of cells still adsorbed suggesting that other non-electrostatic 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 microorganism 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.
Q (mg R.ruber . g HZ)
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 medium, 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 surface 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 interaction 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 represent a potential area for electrostatic attraction between the bacterial 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 interactions (Merma et al., 2013).
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0 0,0
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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.
Please cite this article in press as: Lopez, L.Y., et al. Fundamental aspects of hematite flotation using the bacterial strain Rhodococcus ruber as bioreagent. Miner. Eng. (2015), http://dx.doi.org/10.1016/j.mineng.2014.12.022
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B.concent. 0.15g.L -1 B.concent. 0.30g.L -1 B.concent. 0.45g.L -1 B.concent. 0.60g.L -1 B.concent. 0.75g.L
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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 standardized at 15 mL/min (at atmospheric pressure). Flotation was continued 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, producing 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), expressed as % floatability, or how much was burst to the froth by agitation, expressed as % entrainment. Table 2 presents these previous results. As particle size decreases, the percentage of entrainment also decreases, whilst % floatability achieved higher values.
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.
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 flotation studies. It was found that the highest hematite floatability, an 84%, corresponded for a concentration value of 0.60 g/L, in concordance with previous results of zeta potential and adsorption studies (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 hematite was unaffected by increasing the cell number. At a cell density of 1.5 109 cells/mL, only 9% of the hematite sample was recovered 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 -53+38um
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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.
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Table 2 % Entrainment and % floatability for each particle size range.
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Please cite this article in press as: Lopez, L.Y., et al. Fundamental aspects of hematite flotation using the bacterial strain Rhodococcus ruber as bioreagent. Miner. Eng. (2015), http://dx.doi.org/10.1016/j.mineng.2014.12.022
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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.
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Fig. 7. Effect of Flotanol in the bioflotation of hematite with R. ruber in function of pH and particle size (biomass concentration 0.15 g/L).
Fig. 8. Effect of Flotanol in the bioflotation of hematite with R. ruber in function of 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 containing 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 mechanism of electrostatic adsorption stabilized by hydrophobic interactions alone cannot satisfactorily explain the floatability of hematite well below its IEP. According to Mesquita et al. (2003) the R. opacus bacteria presented 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
Please cite this article in press as: Lopez, L.Y., et al. Fundamental aspects of hematite flotation using the bacterial strain Rhodococcus ruber as bioreagent. Miner. Eng. (2015), http://dx.doi.org/10.1016/j.mineng.2014.12.022
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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, electrostatic 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 conferring hematite, corundum and calcite surfaces enhanced hydrophilicity. The authors observed that is possible to float quartz at pH < 4.3 with a recovery of 58% under these experimental conditions. Whilst, hematite did not float at any pH value and its flotation 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 naturally to these microorganisms due to the presence of amides in their cell walls, which could make them behave as frothers. However, the use of an ordinary frother was not reported in the literature. Thus, the flotation experiments using flotanol, as frother, followed the same experimental procedure. The mineral was conditioned 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 concentration of flotanol was taken from the literature and chose for evaluation 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. Therefore, 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 electrolyte 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 varied, 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 conditions 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 interaction 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
Please cite this article in press as: Lopez, L.Y., et al. Fundamental aspects of hematite flotation using the bacterial strain Rhodococcus ruber as bioreagent. Miner. Eng. (2015), http://dx.doi.org/10.1016/j.mineng.2014.12.022
L.Y. Lopez et al. / Minerals Engineering xxx (2015) xxx–xxx
quartz. Among the above minerals, quartz exhibited the least surface adsorption of B. subtilis followed by calcite (Sarvamangala and Natarajan, 2011). Mesquita et al. (2003) showed that scanning electron micrographs 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 surfaces. 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 bacteria on hematite surface were attracted to each other. The individual hematite particles were transformed into agglomerates of hematite 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 modification of hematite zeta potential profile. The biomass adhesion was found more strongly attached for a pH around 3, at a concentration 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. References Araujo, A.C., Viana, P.R.M., Peres, A.E.C., 2004. Reagents in iron ores flotation. Miner. Eng. 18, 219–224. Borges, E., 2011. Biosorption of Co (II) and Ni (II) from aqueous solutions using Rhodococcus ruber strain. MSc. Dissertation – Department of Materials Engineering, Pontifical Catholic University of Rio de Janeiro, Rio de Janeiro, 136p. Botero, A.E.C., Torem, M.L., Mesquita, L.M.S., 2007. Fundamental studies of Rhodococcus opacus as a biocollector of calcite and magnesite. Miner. Eng. 20 (10), 1026–1032.
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Please cite this article in press as: Lopez, L.Y., et al. Fundamental aspects of hematite flotation using the bacterial strain Rhodococcus ruber as bioreagent. Miner. Eng. (2015), http://dx.doi.org/10.1016/j.mineng.2014.12.022