WO2014165955A1 - Heavy metal biosorption method - Google Patents

Abstract

The present invention relates to a heavy metal biosorption method using Rhodococcus ruber bacteria. More specifically, the present invention relates to a method that includes treating R. ruber bacteria with NaOH, contacting with a solution containing Co(II) and (Ni(II) in a medium with a pH of 6.0 and a R. ruber concentration in the order of 3 g.L^-1, for a period of about 30 minutes, at temperatures in the order of 25 °C.

Description

 "HEAVY METAL BIOSSORTION PROCESS"

 Field of the Invention

 The present invention is directed to a heavy metal biosorption process using Rhodococcus ruber bacteria.

 Background of the Invention

 The importance of water as a natural resource is becoming increasingly evident and with this there is an increasing rigor in its legislation. With this, many works and researches have been developed regarding water treatment. One of the worst sources of water pollution is the dumping of heavy metals that can cause serious damage to human health from exposure to them. Conventional methods of heavy metal removal are: Chemical precipitation, coagulation, activated carbon adsorption and ion exchange. There are also alternative treatment methods that use biological material to remove these metals such as biosorption and bioaccumulation.

 Biosorption is the removal of materials, whether compounds, metal ions, among others by sorbent of inactive biological origin due to the "high forces" of attraction. (Volesky and Holan (1995)). This process is based on the ability of some types of biomass to concentrate and bind metals in aqueous solutions through their cellular envelope. This method may be related to cell metabolism or to physical or chemical adsorption processes in the biomass cell wall. The biomass destined for this process can come from various sources such as algae, fungi, bacteria and higher plants. Advantages of this method over conventional ones are low cost, mud minimization, high efficiency and biosorbent reuse. The steps of the general prior art biosorption process are shown in Figure 1.

Among the possible beings of biomass we give special emphasis to bacteria as they are found in basically all environments in the earth's crust including the most inhospitable, also found in harmonious symbiosis with other organisms. Bacteria are beings unicellular cells of simplified structure with no defined cell organization and their DNA and other organelles are free wrapped only by the cell wall. What sets them apart from other living things is the cell wall. Not all bacteria have the same cell wall constitution. Gram-positive bacteria have a thick peptidoglycan layer about 90% of the cell wall constitution (Dijktra and Keck, 1996). Gram-negative bacteria, on the other hand, have a thin layer of peptidoglycan representing 10-20% (Beveridge, 1999). In the present invention one species of bacteria is prominent, Rhodococcus ruber.

 The genus Rhodococcus was established by Goodfellow and

Anderson (1997), where R. rhodochrous are recognized as type species and the others R. bronchialis, R. coprophilus, R. corallinus, R. equi, R. erythropolis, R. ruber, R. rubropertinctus, R. opacus and R .. terrae. These bacteria have a wide suitability in the environment being found mainly in soil but also in lakes, rivers or other aquatic environments. They are generally non-pathogenic, being pathogenic to men and animals R. equi acting mainly as opportunistic microorganism Sánchez et al. (2004). They are usually shaped like bacilli or cocci and are Gram positive, aerobic and resistant to alcohol and acid. These bacteria are widely used in soil and water bioremediation and the opacus strain is already widely used in biosorption.

 The present invention describes the use of Rhodococcus ruber bacteria in heavy metal biosorption processes as an alternative to conventional treatments and an extension to the possibility of biomass to be used.

 The scientific and patent literature points to some prior art documents relevant to the present invention, which will be described below.

US 2008/0009054 A1 discloses the use of a new bacterium, a strain of Bacillus sp., To be used in metal biosorption processes. It also reveals a sporulated industrial inoculant or no, with said bacterium, its method of production and the process for removing metals using said bacterium. The present invention differs from this document in that it is heavy metal biosorption using bacteria of the genus Rhodococcus ruber.

 US 5,789,204 discloses a biosorbent for heavy metal biosorption containing sodiophosphate polyaminosaccharides as the main ingredient for maintaining biomass which may be comprised of Apergillus, Penicillium, Trichoderma or Micrococcus, originating from industrial fermentation or biological treatments. The present invention differs from this document in that it is heavy metal biosorption using bacteria of the genus Rhodococcus ruber.

 US 2007/0202588 A1 discloses a biosorbent used for cleaning petroleum contaminated fluids and petroleum derivatives. The invention absorbs and degrades oil using bacterial strains. Desired strains are immobilized in a hydrophobic sorbent, which is placed on the surface of the fluid to be decontaminated. The present invention differs from this document in that it is heavy metal biosorption using directly the bacteria of the genus Rhodococcus ruber.

 From what can be inferred from the researched literature, no documents were found anticipating or suggesting the teachings of the present invention, so that the solution proposed here has novelty and inventive activity in relation to the state of the art.

 Brief Description of the Invention

 The present invention provides a new and inventive biosorption process using a Rhodococcus bacterium species, Rhodococcus ruber which has superior results in metal biosorption.

It is therefore an object of the present invention a heavy metal biosorption process comprising the step of contacting Rhodococcus ruber bacteria with a medium comprising heavy metals, which is desired to biosorb from the environment. In a preferred embodiment, the above process comprises the decontamination of the environment from biomass and solution containing metal ions, which is subjected to biosorption for solid-liquid separation, where at the end of the biosorption process there is regenerated biomass and metal. In a preferred embodiment the heavy metals are Co (ll) and Ni (ll).

 These and other objects of the invention will be immediately appreciated by those skilled in the art and those of interest in the art, and will be described in sufficient detail for reproduction in the following description.

 Brief Description of the Figures

 The present invention will be described taking into consideration the accompanying schematic figures which illustrate:

 - Figure 1 - Schematic flowchart of a biosorption process where (1) comprises biomass, (2) comprises metal ion containing solution, (3) comprises biosorption, (4) comprises solid-liquid separation, (5) comprises charged biomass, (6) comprises decontaminated effluent, (7) comprises non-destructive regeneration, (8) comprises biomass destruction, (9) comprises regenerated biomass, (10) and (11) comprise metal.

 - Figure 2 - Scheme of batch biosorption experiments.

- Figure 3- Zeta potential of R. ruber (1 gL ^"1 ) without treatment, influence of solution pH on the presence of Co (ll) and Ni (ll) metals (30mg.L ^{" 1} )

- Figure 4- Zeta potential of R. ruber (1 g.L-1) treated, influence of solution pH on the presence of Co (ll) and Ni (ll) metals (30 mg.L-1).

 - Figure 5- Effect of pH on Co (ll) biosorption by untreated R. ruber (Initial Metal Concentration - 50 mg.L-1; Biomass Concentration: 1.0 gL-1; Agitation Speed: 125 rpm contact time: 3 h).

- Figure 6- Effect of pH on Ni (ll) biosorption by untreated R. ruber (Initial Metal Concentration - 50 mg.L-1; Biomass Concentration: 1.0 gL-1; Agitation Speed: 125 rpm contact time: 3 h).

- Figure 7- Effect of Biomass Concentration on Co (ll) Biosorption by R. ruber (initial metal concentration: 50 mg.L-1; pH: 6.0; stirring speed: 125 rpm; contact time: 3 h).

 - Figure 8- Effect of Biomass Concentration on Ni (ll) biosorption by R. ruber (initial metal concentration: 50 mg.L-1; pH: 6.0; stirring speed: 125 rpm; contact time: 3 h).

 - Figure 9- Effect of Initial Metal Concentration on Co (ll) biosorption by R. ruber (pH: 6.0; biomass concentration 3 gL-1; stirring speed: 125 rpm and contact time: 3 h) .

 - Figure 10- Effect of Initial Metal Concentration on Ni (ll) biosorption by R. ruber (pH: 6.0; biomass concentration 3 gL-1; stirring rate: 125 rpm and contact time: 3 h) .

 - Figure 11- Effect of contact time on Co (ll) biosorption by R. ruber (pH: 6.0; Biomass concentration 3 gL-1; Initial metal concentration 30 mg.L-1 and stir rate: 125 rpm).

 - Figure 12- Effect of contact time on Ni (ll) biosorption by R. ruber (pH: 6.0; Biomass concentration 3 gL-1; Initial metal concentration 30 mg.L-1 and stirring speed: 125 rpm).

 - Figure 13- Effect of Temperature on Co (ll) biosorption by R. ruber (pH: 6.0; Biomass concentration 3 gL-1; Initial metal concentration 30 mg.L-1, Contact time 30 minutes and stirring speed: 125 rpm).

 - Figure 14- Temperature effect on Ni (ll) biosorption by R. ruber (pH: 6.0; Biomass concentration 3 gL-1; Initial metal concentration 30 mg.L-1, Contact time 30 minutes and stirring speed: 125 rpm).

 - Figure 15- Application of kinetic results to the pseudo-first linear order model for Co (ll) uptake using R. ruber.

 - Figure 16- Application of kinetic results to the pseudo-second order model for Co (ll) uptake using R. ruber.

- Figure 17- Application of kinetic results to the pseudo-first order linear model for Ni (ll) uptake using R. ruber. - Figure 18- Application of kinetic results to the pseudo-second order linear model for Ni (ll) uptake using R. ruber.

 - Figure 19- Effect of metal concentration on Co (ll) biosorption by R. ruber nonlinear model (Biomass concentration: 3 gL-1; pH: 6.0; Agitation speed: 125 rpm; contact time) : 3 h).

 - Figure 20- Effect of metal concentration on Co (ll) biosorption by R. ruber linearized model (Biomass concentration: 3 gL-1; pH: 6.0; Agitation speed: 125 rpm; contact time: 3 h).

 - Figure 21- Effect of metal concentration on Ni (ll) biosorption by R. ruber nonlinear model (Biomass concentration: 3 gL-1; pH: 6.0; Agitation speed: 25 rpm; contact time) : 3 h).

 - Figure 22- Effect of metal concentration on Ni (ll) biosorption by R. ruber linearized model (Biomass concentration: 3 gL-1; pH: 6.0; Agitation speed: 125 rpm; contact time: 3 h).

 - Figure 23- Application of Langmuir equation for Co (ll) biosorption by R. ruber.

 Figure 24- Application of the Freundlich equation for Co (ll) biosorption by R. ruber.

 - Figure 25- Application of Tenkim equation for Co (ll) biosorption by R. ruber.

 Figure 26- Application of the Dubinin-Radushkevich equation for Co (ll) biosorption by R. ruber.

 - Figure 27- Application of Langmuir equation for Ni (ll) biosorption by R. ruber.

 - Figure 28- Application of the Freundlich equation for Ni (ll) biosorption by R. ruber.

 - Figure 29- Application of Tenkim equation for Ni (ll) biosorption by R. ruber.

- Figure 30- Application of Dubinin-Radushkevich equation for Ni (ll) biosorption by R. ruber. Figure 31- Effect of serial biosorption and biosorbent treatment on the removal of Co (ll) by R. ruber (pH: 6.0; Biomass concentration 3 gL-1; Initial metal concentration 30 mg.L-1 , Contact time 30 minutes and stir speed: 125 rpm).

 - Figure 32- Effect of serial biosorption and biosorbent treatment on Ni (ll) removal by R. ruber (pH: 6.0; Biomass concentration 3 gL-1; Initial metal concentration 30 mg.L-1 , Contact time 30 minutes and stir speed: 125 rpm).

 - Figure 33- Effect of serial biosorption and biosorbent treatment on removal of Co (II) and Ni (ll) in a binary competition system by R. ruber (pH: 6.0; Biomass concentration 3 gL-1; Initial metal concentration 30 mg.L-1, Contact time 30 minutes and stirring speed: 125 rpm).

 - Figure 34- Growth of Rhodococcus ruber in different culture media - Dry weight (g.L-1) versus Culture media.

 - Figure 35- Growth of Rhodococcus ruber bacteria in different culture media - Absorbance versus Time (Hours).

 - Figure 36- Growth curve of Rhodococcus ruber bacteria - Dry weight (g.L-1 x Hours).

 - Figure 37- Growth curve of Rhodococcus ruber bacteria.

 - Figure 38- Correlation between absorbance and number of cells per mL of R. ruber.

 Figure 39- Rhodococcus ruber Infrared Spectrometry without treatment.

 - Figure 40- Infrared spectrometry of the interaction between R. ruber and Co (ll) without treatment.

 Figure 41- Infrared spectrometry of the interaction between R. ruber and Ni (ll) without treatment.

Figure 42- Infrared spectrometry of R. ruber and its interaction with Co (ll) and Ni (ll) without treatment. Figure 43- Infrared spectrometry of R. ruber treated with NaOH.

 Figure 44- Infrared spectrometry of the interaction between R. ruber treated with NaOH and Co (ll).

 - Figure 45 - Infrared spectrometry of the interaction between R. ruber treated with NaOH and Ni (ll).

 Detailed Description of the Invention

 The examples described herein are intended merely to exemplify one of the numerous embodiments of the present invention, however, without limiting the scope thereof.

 The present invention comprises the use of Rhodococcus ruber bacteria in heavy metal biosorption processes.

Rhodococcus ruber

 Rhodococcus ruber comprises the bacteria of the genus Rhodococcus ruber, a non-pathogenic Gram-positive bacterium that is partially resistant to alcohol, acid and also aerobics. They grow well at temperatures ranging from 30 to 37 ° C, and grow in a variety of laboratory cultivation media. Mucoid colony type, smooth or rough and cream colored, red or orange, colorless and it can be seen that, generally, the forms are variants of parental cells. Its cell wall contains mycolic acids with 34-52 carbon atoms and up to 3 double bonds, with most of the saturated and unsaturated carbon chain type and the presence of branched 10-methyl- (stearic tuber) fatty acids, Sánchez et al. (2004).

Biosorption

 The biosorption process according to the present invention comprises a process of concentrating sorbate in a biological sorbent. This process is based on the ability of some biomass to concentrate and bind metals in aqueous solutions through their cellular envelope.

Heavy metals By heavy metals about 40 elements, which are most harmful to human health, have been scaled down in terms of toxicity: As>Cd>Pb>Hg>Co>Cu>Cr>Ni>Se>Zn> AI , the last four metals cause greater damage when found in plants, which we call phytotoxicity but also cause problems in humans and other living beings when administered at high dosages (Volesky, 2001).

 Example 1. Preferred Realization

 The materials, reagents and equipment used in the biosorption study for the removal of Co (ll) and Ni (ll) using R. ruber bacterial biomass, as well as the experimental methodology used are described below.

Initially, we discuss the establishment of the best experimental conditions for biomass growth. Then, we describe the methodologies employed in the study of the interaction between biomass and metals, and then present the analytical procedures for quantitative determination, as well as the techniques employed in the characterization of biomass before and after biosorption.

 Stock Solutions:

From 98% pure CoCl ₂ .6.H ₂ O and NiCl ₂ .6.H ₂ O supplied by VETEC, stock solutions of 500 mg.L ^"1 further dilutions were prepared to obtain the desired concentrations for use in the biosorption assays.

 Metal Ion Solutions for Biosorption:

The biosorption experiments used 50 mg.L ^"1 metallic solutions. During the development of the work, 0.1 M HCl and 0.1 M NaOH solutions were also used to adjust the pH of the solutions.

 Microorganism Used:

The strain of the microorganism R. ruber was obtained from the Culture Collection of the André Tosello Tropical Foundation for Research and Technology - Campinas, São Paulo, registered by the center under number 1879. The growth of the strain was obtained at 28 ° C and kept at lyophilized environment -80 ° C. Influence / Effect of Culture Medium on R. ruber Growth:

Three different types of culture media commonly used for bacterial cell growth were tested. The culture media used were the compound YM: Glucose 10 gL ^"1 peptone, 5 g ^L", 5 malt extract 3 gl ^"1 yeast extract and 3 g ^{L" 1.} GYM medium is composed of : Glucose 10 gL ^"1, malt extract 4 g ^{L" 1} yeast extract and 4 g L ^". The means ¹ TSB culture Himedia ^® brand commercially available, which was used 30 ^{gL" 1.}

 All culture media were adjusted to pH 7.0 and then autoclaved at 1 atm at 121 ° C for a period of 20 minutes.

 It is noteworthy that all culture media used during the experimental phase of this work were liquid in nature, since the bacteria is aerobic.

 Initially the pre-inoculum was prepared in the three culture media.

15 tested. From the strain of the R. ruber microorganism, with the aid of a platinum loop a small amount of cells were removed and transferred to each type of liquid culture medium contained in 250 mL erlemmeyers containing 100 mL of medium from each culture medium. tested. The entire procedure was performed in a microbiological aseptic chamber.

 0 Later the erlemmeyers were wrapped in shaker

(Cientec ^® Model CT-712) at 28 ± 2 ° C with agitation of 125 rpm for a minimum of 12 hours. After growth, the liquid medium was centrifuged using a 5000 rpm centrifuge for 6 minutes. Subsequently, the bacterial cells were resuspended in small amounts of distilled water and centrifuged again under the same conditions. In the second resuspension with sterile distilled water the bacterial cells were placed in previously weighed porcelain crucibles.

 For determination of cell concentration the Standard Methods methodology for Dry Weight of Soluble Solids was used. The O0 were brought to the oven at 103-105 ° C for a period of 24 h.

Maintenance, Cultivation and Quantification of R. ruber: All procedure described above was adopted for maintenance and cultivation of R. ruber. And after obtaining the kiln dry weight, it was estimated the value of the concentrate in gL ^"1. The bacterial concentrate obtained goes to autoclave (PRISMATEC ^® Vertical CS), again for inactivation of bacteria, which occurs in the same way mentioned for autoclaving of culture medium at 121 ° C for a period of 20 minutes.This step aims to kill the cells, since in the biosorption process the organism's life state does not influence the process, making it ecologically correct for the process. later discard.

 In the absorbance biomass quantification, 1 ml_ aliquots of pre-inoculum were deposited in 2L capacity erlenmeyers containing 1 L sterile TSB medium. bench at a temperature of 28 ± 2 ° C at 125 rpm. 10 ml aliquots with three replicates were taken for each reading time in an aseptic laminar flow microbiological chamber. Previous experiments have shown that R. ruber bacteria reach their stationary phase within 12 hours of incubation (28 ± 2 ° C and 125 rpm).

 The TSB culture medium, considered the best for R. ruber growth, an experiment described in item 4.3, has a yellow color and absorbs better light at a different wavelength than deionized water. To establish the correlation between cell number x absorbance measured directly from TSB medium, 5mL aliquots were taken from the culture after 12 hours of incubation in three replicates and successive dilutions (1/5, 1/10, 1/20, 1/50, 100 and 1/200) and each dilution was read on a spectrophotometer at 600 nm (Tufenkji et al., 2009).

In the quantification by the number of bacterial cells by optical density of the suspension, the commonly used method is simple and easy to read. But there is no direct correlation between absorbance and number of cells inhabiting the culture medium. When the number of cells in a sample is to be determined, the technique of spectrophotometry and thereafter to establish a correlation between the value obtained by absorbance optical density versus the number of cells of a suspension determined by a methodology in which we can perform cell counting, where the most appropriate and used technique is the Neubauer.

 The 1/100 dilution, in general terms, is considered ideal for reading bacterial cells comfortably to human vision in the Neubauer Chamber.

 Through the data obtained in the readings, curves were made, where the linear correlation between the two parameters was performed, so that the sample readings would enter the confidence limit, in which the Beer-Lambert Law is considered valid.

 Cell Number x Absorbance Correlation Curve:

 Linear correlation curves were plotted between the number of R. ruber bacterial cells versus the absorbance obtained in the read dilutions described in the item above. This correlation helps us to measure and have a quantitative equivalence of the microorganism used in the biosorption process.

 R. ruber Growth Curve:

The growth curve of R. ruber bacteria was determined with the intention of knowing the kinetic decrease of biomass. Experiments were performed to determine bacterial cell concentration as a function of incubation time. For this, a 1 mL aliquot of pre-inoculum was transferred to an erlenmeyer containing 1 L of TSB medium. This erlemeyer was shaker incubated at 28 ± 2 ° C and 125 rpm. 10 mL aliquots with three replicates were taken for each reading time in an aseptic laminar flow microbiological chamber for SPEC ^® spectrophotometer analysis.

The reading times were 0, 1, 2, 3, 4, 6, 8, 12, 24, 36, 48 and 60 h, where in the TO aliquots were removed as soon as the pre-inoculum was transferred to the culture medium. . The white sample was read with TSB culture medium. without the presence of the bacteria. All readings were taken at 600nm wavelength.

Characterization of R. ruber Biomass

 Calculation of External Surface Area Estimation and Biomass Volume:

 To estimate the surface area of the untreated and treated bacteria, we measured 100 cells in 2 μιη scale scanning electron microscopy image fields where the ellipsoid dimensions were obtained. The difference between untreated and untreated bacteria was estimated taking into account the standard deviation and from there a correlation between the results was performed.

 Infrared Spectrometry:

The infrared spectra of the untreated R. ruber bacteria treated with NaOH, as well as the untreated and treated biomass charged with the metal ions of the study Co (II) and Ni (ll) were analyzed for comparison of functional groups. present on the surface of R. ruber before and after interaction with the heavy metals. The analyzes were performed using FTIR spectrophotometer equipment, kindly provided by Professor Júlio Carlos Afonso of the UFRJ Institute of Chemistry. Spectra were collected at a resolution of 4cm ^"1 using 120 scans.

Biosorption solutions with initial metal concentration of 30 ppm or 30 mg.L ^"1 and biomass concentration of 3 gL ^{" 1} were prepared for both untreated and NaOH treated bacteria. All volumes of infrared prepared samples were 100 ml, to ensure sufficient sample mass for analysis, including samples containing only untreated and NaOH-treated bacteria where the volume was supplemented with deionized water. All samples were centrifuged after the contact time set at 30 minutes at a speed of 5000 rpm for a period of 6 minutes, the volumes were resuspended in aliquots of deionized water and set for oven drying. Suspended Soluble Solids described in item 4.2. After 24 hours the dried material was massaged in a mortar and sent for analysis in powder form. The samples were homogenized with 500mg of potassium bromide (KBr) and then analyzed.

Zeta Potential Measurements:

The device used to perform the zeta potential readings was the Zeta Meter System 4.0 ⁺ . The samples were prepared with R. ruber bacterial cells before and after contact with Co (II) and Ni (ll) metals, taking into account that the samples were made with untreated bacteria and treated with NaOH. Potential zeta readings aim to detect any possible biosorption mechanism and the interference of metals under study with the bacterial membrane surface.

The solutions are prepared with the so-called indifferent electrolyte from the 0.01M NaCl salt. The NaCI has a function of physical loading of the particles in the electrode channel, not interfering with the biosorption process or the charge of the particles to be visualized. . The concentration of biomass in solution was 1 gL ^" and the metallic solutions were 30 ppm or 30mg.L ^{" 1} . The samples were adjusted to pH ranging from 2 to 8, in the case of bacterial solutions only after contact for five minutes and the samples were analyzed after two minutes of deposition in the apparatus for analysis of zeta potential, being the supernatant the object of reading. .

 Already the solutions containing biomass and the metals Co (ll) or Ni (ll) were left in contact for 30 minutes, the temperature of 28 ± 2 ° C and stirring of 125 rpm, after this period the samples were adjusted to pH that varied. from 2 to 8 being packed in apparatus for analysis of zeta potential and analyzed after two minutes, where the supernatant was read.

 Potential zeta results were obtained with an average of twenty readings for each sample analyzed.

 Sample Preparation and Analysis in Scanning Electron Microscopy:

The samples analyzed from both untreated and treated bacteria as well as from metal interactions with untreated and treated biomass were submitted to the fixation protocol for biological samples, where it was used the modified Karnovsky fixative which is composed of 2.5% gluteraldehyde, 2.5% formaldehyde in 0.05M sodium cacodylate buffer and pH 7.2 CaCl ₂ Ο, ΟΟ Μ. The fixative volume was about 10 times that of the sample.

Then the samples were washed in aldehyde with three 10 minute passes each in 0.05M cacodylate buffer and immersion in 1% osmium tetraoxide solution (Os0 ₄ ) in 0.05M cacodylate buffer pH 7.2 for one hour, procedure performed in a chapel at room temperature 25 ± 2 ° C. The samples fixed in Os0 were washed with distilled water and subjected to increasing concentrated ethanol solutions (30, 50, 70, 90 and 100%) remaining for 10 minutes each. In the 100% solution the samples were passed three times. . The samples were also placed in individual cages, properly identified for drying to the critical point, procedure described by Kitajima & Leite (1999).

 All samples for SEM analysis were sent to the Histopathology and Structural Plant Biology Laboratory by Biologist Mônica Lanzoni Rossi, under the coordination of Dr. Neusa de Lima Nogueira, from the Center for Nuclear Energy in Agriculture, University of São Paulo, where the above procedures were performed. The equipment used for scanning microscopy analysis was LEO 435 VP, and the samples were analyzed at the Research Support Center / Electron Microscopy Applied to Agricultural Research at the Luiz de Queiroz College of Agriculture, University of São Paulo, under the coordination from Professor Dr. Elliott Watanabe Kitajima.

The scanning electron microscope (SEM) (LEO 435 VP) was used to evaluate biomass morphology before and after sorption of different metallic species and thus to qualitatively analyze the biosorption process. SEM has a unique ability to analyze surfaces. The incidence of the electron beam on the sample surface promotes the emission of backscattered and absorbed secondary electrons as well as characteristic x-rays. These electrons are used for image formation. Electrons have much shorter wavelengths than light photons, and shorter wavelengths are capable of generating high resolution information. Enhanced resolution allows for higher magnifications without losing detail.

5 SEM micrographs can maintain the three-dimensional appearance of surface texture (Gabriel, 1985). The combination of high resolution above 4nm, extensive magnification range (10 to 2x10 ⁵ times), and high depth of field (voltage 0.2 to 40 Kv) makes the SEM suitable for the study of microbial biomass surface.

e Sample Preparation and Analysis in Transmission Electron Microscopy:

 R. ruber suspension as well as R. ruber plus metal interaction (Co and Ni) were fixed in modified Karnovsky's solution: 2% glutaraldehyde, 2% paraformaldehyde, 5 mM calcium chloride in buffer

15 0.05M sodium cacodylate pH 7.2 for 24 hours, washed in 0.1 M cacodylate buffer, powders fixed for 1 hour with 1% osmium tethoxide in 0.1 M sodium cacodylate buffer. 0.9% saline. Each change of solution required a centrifugation at 500rpm for 10 minutes to allow the bacteria to settle. They were then stained "block" with 2.5% uranyl acetate in overnight water. Dehydration was performed with increasing series of acetone in water (25%, 50%, 75%) for 5 minutes, followed by 2 10-minute treatments with 90% acetone and 3 20-minute treatments with 100% acetone, always centrifuging. .

5 After infiltration the samples were included in Spurr resin within the ependorff itself. Ultrathin sections (60-90nm) were deposited on colodion-coated copper screens, contrasted with 2.5% uranyl acetate and lead citrate (Reynolds 1963). The sections were examined and photographed with the Zeiss EM-900 electron microscope and the digitized images.

Preparation of Biosorption Test Solutions: The metal solutions were thus prepared from 500 ppm or 500 mg.L ^{"1 stock} solutions to final solutions containing 50 ppm or 50 mg.L ^{" 1} of Co (ll) and Ni (ll) in a final volume of 50 mL solution. For the pH parameter, the biomass concentrate obtained from R. ruber with a concentration of 1 gL ^{"1 was added} and, in the case of the parameter Biomass Concentration, the same final concentrations of 50 ppm or 50 mg.L ^{" 1} of the solutions. Co (ll) and Ni (ll) were prepared by varying the biomass concentrate obtained from R. ruber cultures. For the pH parameter, solutions from 2 to 9 of Co (ll) were prepared. In the case of Ni (ll), only solutions from pH 4 to 7 were prepared, considering that at pH below 4 the bacterial cells collapse by osmosis and at pH above 7 Ni precipitates and we cannot say for sure if the metal was removed by the bacteria or simply precipitated into solution. For the biomass concentration parameter, all metallic solutions were prepared with concentrations of 1; 2; 3,4; 4.5 and 5 g / L R. ruber, with the respective pH obtained in the respective item. The samples were shakered after mixing the solution and biomass concentrate for a contact time of 3 hours, at 25 ± 2 ° C and shaking at 125 rpm. It is noteworthy that solutions called white with 50 ppm of the metal in question were prepared to be used as a reference in the analysis.

 Factors affecting the adsorption rate and biosorbent uptake capacity were examined on a bench scale. All assays were performed at 28 ° C with the cell suspension in erlenmeyer flasks under constant agitation of 125 rpm to elucidate the appropriate conditions (pH, biomass concentration, metal concentration, contact time and temperature).

PH effect:

For the pH parameter, solutions from 2 to 9 of Co (ll) were prepared. In the case of Ni (ll), only solutions from pH 4 to 7 were prepared, considering that at pH below 4 the bacterial cells collapse by osmosis and at pH above 7 Ni precipitates and we cannot state with certainty whether the metal was removed by the bacteria or simply precipitated into solution.

 The removal of Co (II) and Ni (II) and R. ruber metal ions were prepared in a volume of 50 ml of metallic solution to pH values in the desired range.

 The pH adjustment was made using 1 M NaOH and / or 0.1 M HCl solutions. Afterwards the bacterial suspension was added and the samples were kept in contact for three hours at a rotation speed of 125 rpm in incubator shaker and at 28 ° C. After equilibration the samples were taken and centrifuged for 6 minutes at 5000 rpm. The scheme of batch biosorption experiments is presented in Figure 3.

 The samples were frozen in a freezer for preservation and subsequent analysis of the residual metal concentration by the atomic absorption spectrophotometry method in less than seven days.

 Table 1 shows the experimental conditions employed in the tests.

Table 1 - Conditions employed to determine the influence of pH on the biosorption process.

 Parameter Co (ll) Ni (IU)

 Vol. Metal solution (mL) 50 50

Cone. Initial Metal (mg.L ^"1 ) 50 50

Cone. biomass (gL ^" ') 1 1

 Speed Agitation (RPM) 125 125

 Temperature (° C) 28 ± 2 28 ± 2

 pH 2.3; 4; 5; 6; 7; 8 and 94; 5; 6 and 7

 Time (h) 3 3

We used as control of the tests, whites containing only deionized water and biomass, in order to reduce the effect of possible reading deviations caused by the presence of organic material released by the biomass.

Biomass Concentration Effect: The effect of biomass concentration was studied to know the appropriate concentration of R. ruber to obtain the maximum biosorption of metal ions. For this, assays were performed with different biosorbent concentrations, using the differentiated pH values.

 Table 2 shows the experimental conditions employed.

Table 2 - Conditions employed to determine the influence of the concentration of

 Biomass in the biosorption process.

Balance Time Determination:

 The contact time between the biomass and the solution containing the metallic species was studied in order to determine the residence time required for the maximum biosorption of metals by R. ruber. Therefore samples were collected at different time intervals to verify the equilibrium reached. The pH value used to determine the equilibrium time was obtained in a specific assay.

 Table 3 shows the experimental conditions employed.

Table 3 - Conditions employed in determining equilibrium time.

Initial Metal Concentration Effect:

The effect of metal concentration on the uptake capacity of Co (ll) and Ni (ll) ions by R. ruber io \ evaluated in the range of 5 to 75 mg.L ^"1. The experiments were performed at pH 6.0 ± 0.2 for Co (ll) and Ni (ll) adjusted with HCl or NaOH, and with the predetermined biomass concentration The results obtained in these tests were used to construct the isotherms.

 Table 4 presents the experimental values adopted for each of the parameters.

Table 4- Conditions employed for determining the influence of

 Initial Metal Concentration in Biosorption Process

Biomass Treatment:

 Biomass treatment has been used by several authors in biosorption work (Huang et al, 1988; Kuyucak and Volesky., 1988;

Paknikar et al., 1993; Kapoor and Viraraghavan., 1998). Physical methods include autoclaving, lyophilization and distillation; while in the chemical treatment organic acids, bases and chemicals are used. In general, these treatments increase or reduce the adsorption capacity of metals through biosorbents.

 R. ruber bacteria grown as described above and chemically treated using 20 mL of NaOH (approximately 20% of the total volume of the bacterial concentrate obtained at a concentration of 0.01 M in

200ml of concentrated biomass). This mixture was stirred for 2h at 125 rpm. Then the mixture was centrifuged and washed twice with deionized water. The biomass pH was adjusted with 0.1M HCl to pH 7.0 to perform the biosorption tests. Biomass treatment was used when all parameters of untreated bacteria were established to compare the results.

 Effect of Ni (ll) Presence on Co (ll) and Vice Versa Biosorption:

Competitive biosorption between Co (ll) and Ni (ll) was investigated. To determine the competitive biosorption characteristics between Co (ll) and Ni (ll) the initial concentration of metals was 50 mg.L ^"1. The assays were performed following the same procedure described in item 4.8.4, by example, passing only the total volume of the solution to be 100mL.

Quantitative Analysis of Metal Concentration:

The residual concentrations of the metallic species Co (ll) and Ni (ll) obtained at the end of the experiments were determined by atomic absorption spectrophotometer (ContraAA 300) carried out at the Chemical Analysis Laboratory of the PUC-Rio Department of Chemistry. The removal percentage was calculated from the equation below:

Where:

 Re (Me%) = Percentage of metal removed;

Ci = initial metal concentration (mg.L ^"1 );

Cf = final metal concentration (mg.L ^" ) after contact with the biosorbent.

The uptake capacity of Ni (ll) and Co (ll) by R. ruberi \ biomass determined using equation 2:

(Co - C.W (2)

M Where:

q _e = amount of metal ion captured by biomass (mg.g) at equilibrium; C ₀ = initial metal ion concentration (mg.L ¹ );

C _e : = final or equilibrium metal ion concentration (mg.L ^'1 );

V = volume of metal ion solution (L);

M = biosorbent mass (g).

All biosorption experiments were performed at room temperature (28 ± 2 ° C).

Results and discussion:

 Potential Determination - Electrokinetic Studies:

 The purpose of these analyzes is to determine the electrical charge of the microorganism R, ruber (untreated and after NaOH treatment) and also the interaction between the bacteria (untreated and untreated) and the metals in solution of the present study Co (ll) and Ni (11). The pH variation was the parameter that evaluated the change of R. ruber surface charge (mV) and its interaction with Co (ll) and Ni (II) metal ions. This tool indicates an interaction between metal species and bacterial active sites.

 Figure 3 shows the zeta potential curves as a function of pH for untreated R. ruber before and after biosorption of Co (ll) and Ni (lll) in the pH range ranging from 2.0 to 8.0. . The results demonstrate that R. ruber isoelectric point (PIE) or zero charge point (PCZ) was close to pH 2.5. After PIE or PCZ as the pH values approach neutrality the zeta potential becomes more negative, reaching a stabilization in the load value in the pH range 5.0 (-15.6 mV) at 8 .0 (-15.56 mV).

When we started for the interaction between untreated R. ruber and the metals, we observed a slight variation in the potential zeta values compared to the bacteria values without treatment. Less negative charge values were visualized between R. ruber + Co (II) and R. ruber + Ni (ll) over the tested pH range. Although it was not a difference Significantly, this is explained as the occupation of the active sites of the bacteria by Co (II) and Ni (ll) ions.

PIE ^' s or PCZ ^' s for cobalt and nickel were respectively found at pH around 2.8 and 2.7. The curves plotted on the potentials of R. ruber plus metals showed a similar tendency to untreated bacteria. The highest electronegativity values obtained for the interaction bacteria plus Co (ll) were obtained between pH 7.0 and 8.0 reaching -14.44 mV.

In the case of the interaction between the bacteria plus Ni (ll) the most electronegative value was also obtained between pH 7.0 and 8.0 with a value of -13.38 mV.

 The results obtained are consistent when compared to the article published by Cayllahua et al. (2009) when they used R. opacus bacteria in their Ni biosorption studies (ll). The authors obtained the bacterial EIP or PCZ at approximately pH 3.4 and the highest electronegativity was obtained at about -25 mV. After R. opacus contact with Ni (ll) the PIE or PCZ was established at pH 3.7 approximately and the highest electronegativity value was close to -2mV, being technically "stable" in the pH range between 5.0 and 6.5.

 These results confirm that the active sites of the bacteria are occupied by metal ions, a fact that occurred in the works of other authors (Bueno, 2007; Silva, 2010). Although Silva (2010) failed to establish PIE or PCZ in his electrokinetic testing with metals

Co (ll) and Cu (ll) and R. opacus bacteria, results that disagree with those obtained in our study and the results obtained by Bueno (2008) who found the IPP or PCZ in R. opacus biosorption with Pb ions, Cr and Cu.

 Electrokinetic studies were also performed with R. ruber bacteria treated by NaOH for a contact period of 2 hours. The results were plotted and compiled in Figure 4.

The results show that when compared to the untreated and treated R. ruber bacteria, the EIP or PCZ shifted from pH close to 2.5 to pH 5.4 and the maximum electronegative load increased from -15.6 mV to -36.48 mV at pH 8. In percentage terms it represents an increase of approximately 134% in electronegativity. Suggesting that the adsorption is physical in nature, since the bacteria have negative charges and the treatment only favors the active sites that make up the cell wall, the removal values would be proportionally higher in terms of removal in the biosorption of metals.

 The zeta potential of R. ruber plus the metals involved in the biosorption process Co (ll) and Ni (ll) presented three PIE or PCZ, respectively for Co (ll) at pHs close to 2.6; 4.4 and 6.5 respectively. For Ni (ll) the PIE or PCZ were pH close to 3.0; 4.3 and 5.6 respectively. And the highest electronegativity peaks were -26.6 mV for Co (ll) and -10.2 mV for Ni (II) at pH 8.0.

In electrokinetic studies with Pb (ll), Cu (ll) and Cr (ll) metals using R. opacus biosorbent, Bueno (2007) observed similar results to those obtained in this work. A second charge reversal was obtained by the author for the three metals of her study, which is attributed a specific adsorption of the first hydroxo-complexes: Pb (OH) ⁺ , Cu (OH) ⁺ and Cr (OH) ²⁺ on the surface. by R. opacus. Fuerstenau and Pradip (2005), carried out the research of zeta potential in the flotation of mineral oxides and silicates and pointed this same type of behavior to the studied minerals. Batch Biosorption Tests:

 Biosorption is known as one of the new alternatives for wastewater treatment, and is interesting from an environmental point of view for working with inactive biomasses that bind to heavy metals in contact with aqueous solution. These metals can be later regenerated, reintroduced in the process or simply disposed of correctly in industrial landfills. In the case of biomass, the process can be recovered and feedback, known as desorption.

The study of essential parameters for the development of the biosorption process was carried out to determine the best conditions to obtain a higher efficiency in the removal of Co (ll) and Ni (ll) from aqueous solutions using R. ruber as a biosorbent. It is noteworthy that All experiments were performed on a batch scale. For the determination of the best parameters of by batch tests only one biomass, R. ruber without treatment was considered.

PH influence:

 The pH is considered among the parameters that influence the biosorption process, one of the most important. For it has a determinant action in speciation of ions involved and contained in aqueous solutions, ion charge and sorption sites of the various types of biomass (Gao and Wang, 2007).

 Initially the precipitation points of the studied metals Co (ll) and Ni (ll) were known, to define in which pH range would be considered in the determination of the parameter. For Co (ll) the pH range was 4.0 to 9.0 with a variation of 1.0 between the points of the established range. In the case of Ni (ll), the established working range of pH was 4.0 to 7.0, since after pH 7.0, in the case of the two metals in solutions, they began to show color change, indicating precipitation. From the metals in question, as they are metals very close in physical and chemical characteristics, it was established that above pH 7 it would be impossible to distinguish the uptake by biomass from chemical precipitation.

 The reaction of heavy metals with biomass in solution can be represented by the equation:

H _n B + Me ^{n +} = MeB + nH ⁺ (3) where:

Me = Metal;

N = metal load;

B = Active sites of biomass.

The above reaction denotes that pH may influence biosorption of metal ions by competition between metal and H ⁺ ions by active. Several functional groups present in the constitution of the bacterial cell wall, being carboxyl, amine, hydroxyl, sulfides are highly dependent on their action in relation to the pH of their charge configuration. Carboxyl grouping, for example, at very low pH gives the cell wall positive charges, as the variation of its ionization constants has been established around pH between 3 and 4 (Eccles and Hunt, 1986; Gardea-Torresdey et al, 1990). ).

 The increase in metal uptake with the increase in pH can be explained using the analogy between the metal hydrolysis reaction and the reaction between the sorbent active sites and the metallic species, where in both reactions the hydrogen bond is disrupted. and hydrogen ions are released and replaced by metal (Tipping, 2002; Pagnanelli et al., 2003).

Two important aspects should also be considered when dealing with pH variation, the first will be the speciation diagram of the metals in question, as as commented at the beginning of the topic, metal precipitation may mask the results of biosorption studies. The isoelectric point (PIE) or zero charge point (PCZ) of the biomass involved in the process (R. ruber) and its interaction with metals is another very important aspect in considering the pH parameter results, since values above the PIE cannot be considered as ideal for setting the parameter because they have positive charges where the activation sites have already been occupied by H ⁺ resulting in low or no uptake (Singh et al, 2009; Wang et al., 2008) .

Influence of pH - Co (ll):

 Figure 5 shows the uptake of Co (ll) metals as a function of pH.

It was observed that the best results occurred for pH 6.0, where 37.8% of the metallic species was obtained by R. ruber biomass. Figure 5 considers the removal (R) and uptake (q) values as a function of pH in Co (ll) biosorption. Again emphasizing that the PIE or PCZ of untreated bacteria was at pH 2.5 and R. ruber bacteria plus Co, the PIE or PCZ was at pH 2.8. Therefore we discard pH values below 3. Began to be established at pH 4 by previous knowledge that also at pHs below this value the bacteria ruptures by osmotic pressure.

It is observed that at pH 4.0 removal and uptake are very low, between pH 5.0 and 6.0 the removal and uptake values were identical 37.8% e18.9mg.g ^{~ 1} respectively. The value considered as the best removal parameter was pH 6.0 as it is closer to neutrality.

 Liu et al. (2010) in their biosorption study with cobalt and cadmium metals from the bacterium Pseudomonas fluorescens, found the optimum pH for Co at 6.0 but how the pH determination was shared with the other cadmium metal that obtained better around 5.5, pH 5.5 was established as the best standard. Esmaeili et al. (2007) in a biosorption study using Sargassum sp. as biomass and cobalt, copper and nickel metals, they find the ideal pH for nickel and cobalt 7.0. Silva (2010) in his dissertation Removal of Cu and Co contained in Aqueous Solutions by Biosorption, used R. opacus biomass as a biosorbent, despite having considered the NaOH-treated bacterium variable the ideal for the establishment of the best parameters in the process. Untreated bacteria was also tested and the result was similar and the ideal pH was 7.0 in agreement with Esmaeili et al. (2007), although they are completely distinct biomasses. Mulaba-Bafubiandi et al. (2009) in the paper entitled Cobalt and copper biosorption of hydrometallurgical solutions mediated by Pseudomona spp. obtained the same result of our work in relation to the metal Co (ll) in the pH parameter.

Influence of pH - Ni (ll)

 Figure 6 shows all biosorption results obtained in the pH parameter, where the blue line refers to the removal values and the red line refers to the uptake values.

EIP or PCZ of untreated bacteria plus Ni was obtained at pH 2.7. The best biosorption result for the pH parameter was obtained at pH 7.0, but as the beginning of the precipitation process was visibly observed. In adjusting the pH of the solution, we considered pH 6.0 as the best result of removal and uptake of 27% and 13.5 mg.g ^"1. Considering that metals of extreme chemical proximity are the same pH value for both metals. becomes consistent with R. ruber biosorption.

According to Cayllahua et al. (2009) in their biosorption studies with the R. opacus bacterium in relation to Ni (ll), 40 mg.L ^"1 of metal were used and obtained as the best parameter pH 5.0 disagreeing with the results obtained in our experiments. By performing biosorption experiments with Bacillus cereus bacteria on Ni (ll) removal, the pH considered as the best parameter after testing was 6.5, another result that differs from the present study (Zang et al., 2010).

 In Pb (ll) and Ni (ll) bioremediation studies with live and inactive bacterial cells with Pseudomonas aeruginosa where the best pH removal result was 7.0 for Ni (ll) (Gabr et al., 2008). Swazo-Madrid et al. (2010) in Ni (ll) metal ion biosorption experiments the ideal pH found was 7.5 where the biosorbent was the yeast Rhodotorula glutinis.

 In the establishment of the next stages of biosorption between R. ruber and the metals Co (ll) and Ni (ll) pH 6.0 was established.

Effect of R. ruber Concentration:

The R. ruber biosorbent concentration parameter at the initial uptake rate of Co (ll) and Ni (II) metals. The bacterial concentration was varied from 1.0 to 5.0 gL ^"1 to determine the effect of the microorganism concentration on the initial sorption rate.

Effect of Concentration of R. ruber-Co (ll):

Figure 7 shows the removal results represented by the blue line and the capture results were plotted on the red line. The effect of biosorbent concentration is considered an important parameter for establishing the best biosorption conditions. From the logical point of view, the larger the amount of biosorbent in solution, theoretically, the greater the removal and uptake of metal ions involved in the process. But this logic is confirmed in some cases and in others is not proven.

According to Figure 7 showing the data of R. ruber concentration in relation to the initial concentration of Co (ll) (50 mg.L ^"1 or ppm), an increase in the removal of Co (ll) at concentrations of 1 , 0 to 3 g / L ^"1 , where the maximum value has been reached 30%. This is due to the increase in surface area of adsorption provided by the amount of biosorbent available in solution and consequently the largest amount of active sites available for the sorption process to occur, this fact was also observed by Bueno (2007).

Higher values of R. ruber concentration (4.0, 4.5 and 5.0 gL ^"1 ) showed decreasing removal and uptake results. One of the factors that explain this event is the formation of aggregates during biosorption, since Biomass concentration is high in solution, decreasing the adsorption area, a fact confirmed by the authors Ekmekyapar et al. (2006), Bueno (2007) and Silva (2010).

 Even establishing the R. opacus biomass concentration parameter with NaOH-treated bacteria in Co (ll) biosorption studies, the results obtained by Silva (2010) are consistent with those presented in this work.

 Effect of R. ruber - Ni (ll) Concentration:

 Figure 8 shows the respective removal data (blue line) and uptake (red line) of the R. ruber biosorbent in relation to Ni (ll) metal ions.

The data show a great similarity in behavior in relation to those obtained by the metal Co (ll) once again making inferences of the proximity of existing chemical properties between the two metals. There is an increase in removal and higher uptake rates in the R. ruber biomass concentration range from 1.0 to 3.0 gL-1 when the removal peak reaches 28%. When an increase in biomass, the larger the area adsorption surface and more activation sites are available, Bueno (2007).

 Biomass concentration values above 4.0; 4.5 and 5.0 g.L-1 of R. ruber were decreasing in relation to the initial concentration of Ni (ll) metal, a fact observed for Co (ll) metal in this study. Other authors also observed this behavior, being attributed to the biomass aggregation when it is in high concentrations in solution, consequently decreasing the adsorption surface area (Ekmekyapar et al. (2006); Bueno (2007) and Silva (2010)). Another observation to be clarified is that the uptake values decrease proportionally in relation to the amount of R. ruber, because even presenting more active sites due to the increase of biosorbent to establish the biosorption, these are unsaturated (Bueno, 2007).

The results disagree with those presented by Cayllahua (2008) for Ni (ll) metal using R. opacus biosorbent. The best biomass concentration biosorption result was 5g.L ^"1 , obtaining 54% removal.

In order to establish the next steps of the biosorption process, the concentration of R. ruber biomass in the removal of Co (II) and Ni (ll) metal ions was fixed at 3.0 gL ^"1 .

Initial Metal Concentration Effect:

In this biosorption step, metallic concentrations of Co (ll) and Ni (ll) ions were tested in the proportions of 5 mg.L ^"1 , 10 mg.L ^{" 1} , 15 mg.L ^", 30 mg.L ^" 1,50 mg.L ^"1 and 75 mg.L ^{" 1} being the pH adjusted to 6.0 for both metals and also the biomass concentration set at 3.0 gL ^"1. Effect of Initial Metal - Co (ll Concentration) ):

 The results obtained in the tests of this parameter were plotted in figure 9 below where the removal data (blue line) and the capture data (red line) are described.

Initial metal concentration is also an important parameter in biosorption studies, as this is how we can approximate But experimental laboratory work in relation to a real situation, in contamination of natural resources can not find a single element and the concentrations can be as varied as possible.

Regarding the variation of the initial concentration of Co (ll) metal by R. ruber, we can observe that the highest removal was obtained at a concentration of 5 mg.L ^"1 around 60%. From that point on, the removal values began to increase. decrease as the metal ion concentration increases.

Considering the value with the highest removal very low in terms of metal concentration, but with a reasonable removal in terms of setting parameters, a value of 5 mg.L ^"1 was not adopted, the same value established for the biosorption Co (ll) metal. by R. opacus (Silva, 2010).

As the best metal concentration parameter, 30 mg.L ^"1 was chosen, since it is a metal concentration closer to what can occur in terms of contamination in the real situation, with removal value close to 45%.

Effect of Initial Metal Concentration - Ni (ll):

 In Figure 10 the removal (blue line) and uptake (red line) data were plotted to determine the initial concentration of Ni (ll) metal by R. ruber mediated biosorption process.

Speaking of the initial concentration parameter against the metallic ion Ni (ll) by R. ruber, a behavior different from that presented by Co (ll) was demonstrated. Two removal peaks appeared in the variation studied, at the concentration 5 mg.L ^"1 the removal was a little over 36% and the second peak was signaled at the concentration of 30 mg.L ^{" 1} with the highest expression removal. percentages, approaching 42%. This also favored the choice of metallic concentration of 30 mg.L ^"1 for the two metals of the present study. From this parameter could be established the studies of reaction kinetics. The adsorption isotherms will be described after the parameters of biosorption. For metals Co (ll) and Ni (ll) treated by biosorption with R. ruber bacteria, the initial concentration values of the metal 30 mg.L ^"1 are fixed.

Contact Time Effect:

 The contact time is one of the most important biosorption parameters, because it is where we define how long the reaction kinetics is processed. In terms of industrial processes or wastewater treatment, the case of our object of study is one of the optimization parameters. Time is undoubtedly one of the biggest villains in considering cost and energy expenditure in the process area.

Contact Time Effect - Co (ll):

 Figure 11 shows the contact time data for R. ruber-mediated Co (ll) metal biosorption, blue line (removal) and red line (uptake).

 The results are very clear and show that the reaction kinetics occur within the first 30 minutes of the contact of Co (ll) metal with the biosorbent. Kinetics can be said to occur in four phases. The first occurs in an interval of 0 to 15 minutes where there is no major representation in terms of removal. From 15 to 20 minutes a very significant increase in removal occurs, therefore it is suggested that most reactions of the biosorption process occurred in this range. From 30 minutes to 150 minutes the increment that occurs in the removal is not considered significant, or can even be attributed to laboratory failures in sample preparation or readout errors due to the sensitivity of the analyzer to atomic absorption. The fourth phase is considered after the contact time of 150 to 180 minutes, which is when a decrease in removal begins.

This fact also occurred in the work done by Yamaura et al. (2008) although they worked with Ni (ll) and sugarcane bagasse as biosorbent, the removal curve after 50 minutes of contact time began to decrease. The authors hypothesized the possibility of competition for the active sites for other ions. contained in solution, such as Na ⁺ NaOH added to adjust the pH of the solution.

In a study by Silva (2010), the results using R. opacus bacteria in the removal and uptake of Co (ll) ions under ambient temperature, 3.0 gL ¹ of biosorbent, pH 7.0 of the solution and agitation of 125 rpm, suggests two kinetics steps one very fast that occurs in the first minute of contact and with great removal increment and the second much less significant in percentage terms reaching the maximum removal at 240 minutes. Bhatnagar et al. (2010) used lemon peel as a biosorbent in the removal of cobalt present in aqueous solution and signaled the same observed by the author above, where they obtained 50% removal results in 2.5 minutes and the equilibrium was reached with seven hours. contact time. Liu et al. (2010) using Pseudomonas fluorences in cobalt and cadmium biosorption, observed two steps in the process kinetics, agreeing with the results obtained by the other authors mentioned, the equilibrium was reached with 60 minutes.

Contact Time Effect - Ni (ll):

 Figure 12 presents the removal (blue line) and uptake (red line) results.

 The reaction kinetics in the reaction of R. ruber biosorption with the metallic ion Ni (ll) occurs in two stages. The step that happens in the first 30 minutes of the process is the most representative because in it the extra removal is configured. In the second step after 30 minutes is the process equilibrium, removal and capture stabilize consistently and the difference between the points is not considered significant.

The results obtained in the present study in terms of curve behavior agree with Cayllahua et al. (2009) when he studied Ni (ll) biosorption by R. opacus. The authors also assigned two steps in the first 30 minutes, as happened in our experiments. Monteiro et al (2008) in their studies of nickel adsorption by green coconut fiber obtained kinetic equilibrium results in the first five minutes of contact. Noting that they used the metal concentration of 0.1 gL ^'1 and biosorbent concentration of 50 mg.L ¹ .

In Ni (ll) removal studies using sugarcane bagasse the maximum removal was 31% in the first twenty minutes of contact. Under the conditions of 20 mg.L ^"1 Ni (ll), 50 mg of crushed sugarcane bagasse per liter, pH 6.4 and 300 rpm stirring (Yamaura, 2008).

 The equilibrium of biosorption process kinetics are totally influenced by aspects such as biosorbent type (numbers and functional groups), biosorbent size and shape, physiological state of biomass and the metal involved in the process (Padmavathy et al, 2003 ).

Temperature Effect:

 Temperature is the biosorption evaluation parameter that analyzes various thermodynamic aspects of the process. Through temperature various aspects of biosorption may be favored or disadvantaged. Thus, the experiments performed in the item aim to optimize the biosorption process in the study of the treatment of aqueous solutions containing Co (ll) and Ni (ll).

Temperature Effect - Co (ll):

 Figure 13 shows the activation energy data over time over time. The blue line refers to the results obtained at 25 ° C or 298 ° K, the red line corresponds to the temperature of 35 ° C or 308 ° K and the green line represents the temperature 45 ° C or 308 ° K when removing Co (11) by R. ruber.

According to Figure 14, the temperature of 25 ° C or 298 ° K shows its maximum activation energy range between 15 and 30 minutes of contact (close to 0.115 KJ.g ^" ), while temperatures of 35 ° C or 308 ° K and 45 ° C or 318 ° K already practically stabilize their activation energy values at the first reading taken at 15 minutes, so the maximum activation energy variation occurred before 15 minutes. The important thing to note is that in the 30-minute time period established as the optimal biosorption parameter it was found that the temperature of 45 ° C or 318 ° K (approximately 0.120 KJ. G ^"1 ). ° C or 298 ° K, does not remain stable over the evaluation time, decreases and reaches values without significant difference at a temperature of 35 ° C or 308 ° K.

The temperature of 35 ° C or 308 ° K was the most stable over time (value ranges from 0.105 to 0.110 KJ.g ^"1 ) of evaluation decreasing from 120 minutes and becoming without significant difference from at 45 ° C or 318 ° K (0.105 KJ.g ^"1 ). The temperature of 25 ° C or 298 ° K presented within 30 minutes was close to 0.115 KJ.g ^"1 and maintained activation energy values among the highest among the temperatures evaluated over time, decreased from 120 minutes but closed the time of 180 minutes higher than the other temperatures.

 In order to finalize the temperature effect parameter, we not only considered the activation energy plus the removal value as an evaluation and, therefore, the temperature of 25 ° C or 298 ° K that showed a removal result, slightly above 30%, was evaluated. considered the best biosorption parameter.

 Temperature Effect - Ni (ll)

 Figure 14 presents the results in graphical form where the green line refers to the temperature of 45 ° C or 318 ° K, the red line represents 35 ° C or 308 ° K and the blue line refers to the temperature of 25 ° C. C or 298 ° K in Ni (ll) removal by R. ruber bacteria.

The temperature of 45 ° C or 318 ° K presented the highest activation energy value among those tested. The maximum value of Ea was presented at 15 minutes, the first point of evaluation of the parameter, thereafter Ea began to decrease steadily reaching the last point of evaluation time equaled the temperature of 35 ° C or 308 ° K. The value of the activation energy reached its apex near 0.131 KJ.g ^"1 . As with Co (ll) for Ni (ll) the temperature of 35 ° C or 308 ° K showed the highest activation energy regularity over time (range 0.103 to 0.106 KJ.g ^"1 approximately). And at the temperature of 25 ° C or 298 ° K also similarity occurred when comparing with the metal Co (ll) the largest variation of the activation energy occurred between 15 and 30 minutes, with the peak of the energy of activation at 0.108 KJ.g ^"1 . After this peak, Ea began to decrease and obtained the lowest value of the energies tested in the time of 180 minutes (approximately 0.098 KJ.g ^"1 ).

 So also with Co (ll) was not only considered the value of Ea to establish the best biosorption temperature value, the removal value was also considered and it was concluded that the best temperature was 25 ° C. ° C or 298 ° K having removal value close to 26%, thus constituting an endothermic reaction. The results presented in this topic are consistent with those presented by Cayllahua et al (2009).

 It has been established that for Co (ll) and Ni (ll) metals in the removal of aqueous solutions using biosorbent R. ruber bacteria was 25 ° C or 298 ° K.

Biosorption Kinetics:

 Biosorption Kinetics - Co (ll):

 Figures 15 and 16 demonstrate the proposed kinetic models to evaluate R. ruber biosorption for the treatment of aqueous solutions containing heavy metal Co (ll).

In the kinetics studies of the reactions involving the Co (ll) biosorption process, two models of pseudo-first and pseudo-second orders were used. In the case of the studies of the present invention, the best-fit model was the pseudo-second order where R ² was 0.9687, the pseudo-first order model also showed a good fit with R ² of 0.9296. . In general work with biosorbent materials fits better with pseudo-second order kinetic models. Bhatnagar et al (2010) in their work on removing cobalt contained in aqueous solutions by the lemon peel biosorbent fit the pseudo second-order model with R ² of 0.9903.

In the study proposed by Lui et al. (2010) in the use of the bacterium Pseudomonas fluorescens treatment of solutions containing Co (ll) and Cd (ll) also the pseudo-second order model adjusted more satisfactorily with R ² to 0.997 for Co (ll) metal ions. . This result was also obtained by Silva (2010) in his biosorption studies with aqueous solutions containing Co (ll) and Cu (II) with R ² of 0.9999 for the pseudo-second order model.

Biosorption Kinetics - Ni (ll):

 Figures 17 and 18 show the two models of kinetic equation plotted for Ni (ll) biosorption in aqueous solution by R. ruber.

In kinetic studies of biosorption reactions kinetic models are applied and of fundamental importance for the understanding of the process. In most works to which biological agents are applied as adsorbents the pseudo-second order model fits better. This statement applies to the item in question, since the pseudo-second order model presented an R ² of 0.9944 for the sorption of Ni (ll) by R. ruber bacterial biomass. The pseudo-first order model showed a not very adequate fit, with R ² at 0.7513.

 When compared with the available literature, we find that many authors present similar results regarding Ni (ll) removal even when other types of biosorbents are available.

Mimura et al. (2010) used rice husk as adsorbent in solution containing Cu (ll), Al (lll), Ni (ll) and Zn (ll), tested the kinetic parameters in the individual metals in a competitive way. and competitive without Al (III). They concluded that Ni (ll) individually and competitively without Al (lll) fits the pseudo-second model better. order with R ² respectively of 0.989 and 1.000. For the competitive form, the pseudo-first order model was ideal and the authors indicate this fact to the formation of oxide-hydroxide films on the biosorbent surface by the presence of Al (lll). According to Cayllahua et al. (2009) in their studies of kinetic, thermodynamic and Ni (ll) biosorption equilibrium by R. opacus, the pseudo-second order model also showed the best fit, the parameters used by the author were 5 mg.L ^"1 of Ni (ll) solution, 5 gL ^{" 1} of R. opacus and pH 5.0 at 60 minutes contact time.

Biosorption Isotherms:

To describe the distribution of the solute in the solid phase and the liquid phase for the equilibrium condition, it is necessary to express the amount of adsorbed solute per unit weight of sorbent, q _e , as a function of the equilibrium residual concentration, C _e , of the solute. remaining in the solution. The expression of this relationship is called adsorption isotherm.

Sorption isotherms were experimentally determined for R. ruber biomass. These isotherms were derived to a predetermined pH value for each metal. A metal solution of 1000 ml of 100 mg.L- ¹ of Co (ll) and Ni (ll) was prepared from the salts of Cl ₂ Co 6 (H ₂ 0) and Cl ₂ Ni 6 (H ₂ 0), Samples of the solutions were diluted to prepare the working metal concentrations. The experiments were designed for an initial metal concentration range of 5 to 75 mg.L ^"1 to achieve the maximum uptake capacity.

Figures 20 and 22 can be seen that for Co (ll) and Ni (ll) sorption initially the uptake capacity increases linearly with increasing equilibrium concentration. This is the result of the increment of the concentration gradient due to the increment of the initial concentration of the metal ions. The captured capacity is eventually limited by the fixed number of active sites in the biomass and a resulting plateau can be observed. This plateau can represent the maximum capacity captured by biomass for each metallic species. In figures 20 and 22 were found aq _and for Co (ll) and Ni (ll), it is 19.05 and 5.87 mg.g ^" , respectively, for an initial metal concentration of 75 mg.L ^{" 1} .

Adsorption Isotherm Models:

 Several mathematical models have been developed to quantitatively represent the relationship between sorption extent and residual solute concentration. The most widely used models are the Langmuir and Freundlich isotherm models, we also tested the Temkin and Dubinin-Radushkevich models.

Freundlich's isotherm is capable of describing adsorption of organic and inorganic compounds in a wide variety of adsorbents, including biosorbents. As it is characterized by a robust equation, it fits in sorption and desorption cycles and is excellent for heterogeneous systems, satisfactorily in biosorption processes (Febrianto et al., 2009). The adsorption data with respect to the two metallic species presented better fit to the Freundlich Isoterma model (Figures 24 and 28). Freundlich isotherms fit best under the conditions of the two metals Co (ll) and Ni (ll) with R ² respectively of 0.9608 and 0.9616. Table 4 below shows some comparative data found in the literature on references to adsorption isotherms, metal and biosorbent material.

Table 4 - Relationship between biosorbent, adsorbate, conditions and R ² in Frendluich isotherms.

Biosorbent Adsorbate Operating Conditions R ² Reference

 pH Temperature (°

 Ç)

 Cassia fistula Ni (II) 6.0 30 0.789 Hanif e / al. (2007)

Co (II) shell 6.0 0.979 Vijayaraghavan et crab al. (2006)

 Dry sludge Ni (II) 4.5 0.994 Aksu et al. (2002). activated

 Co (II) Residues 6.0 0.923 Javed et al. (2007) biomass of

 roses

 Ni (II) Waste 4.0 45 0.968 Malkoc and Nuhogiu Tea Industry (2005)

 Ni (II) Alginate 5.0 0.986 Vijaya et al.

 calcium (2008)

 Myriophyllum Co (II) 20 0.760 Lesage et al.

spicatum L. (2008) Ark Shell (II) 0.921 Dahiya and Tripathi Pretreated Ni (II) 0.725 (2008)

 Source: Febrianto et al. (2009).

Best Biosorption Parameters:

 After establishing the best biosorption parameters for Co (ll) and Ni (ll) metal ions contained in aqueous solutions using R. ruber bacterial biomass.

Best Biosorption Parameters - Co (ll):

 Figure 31 shows the blue stain removal data and the red line shows the data on Co (ll) uptake by the R. ruber biosorbent. We consider as the best parameters for Co (ll):

 • pH: 6.0;

• Biomass Concentration: 3g.L ^"1 ;

• Initial Metal Concentration: 30 mg.L ^"1 ;

 • Contact Time: 30 minutes;

 • Temperature: 25 ° C or 298 ° K.

 At this stage, the following variables, serial biosorption and treatment were evaluated:

 • Better biosorption parameters with the untreated biosorbent (1 biosorbent cycle);

 • Better biosorption parameters with a new biosorbent being fed and again undergoing biosorption (2 cycles);

 • Better biosorption parameters with a second new biosorbent being fed and again undergoing biosorption (3 cycles);

 • Better biosorption coatings (obtained with untreated biosorbent) treated with NaOH.

They were compared through the best biosorption parameters obtained from the batch experiments of solutions. (II) -containing water by R. ruber. What we call a series biosorption cycle is the gathering of the best parameters in the evaluation of the biosorbent in question. In the case of two biosorbent cycles, it is understood when we perform a new biosorption process with reintroduction of a quantity of biosorbent and therefore three cycles, plus a new biosorbent reintroduction and a new biosorption totaling three biomass passages. In the case of the treated biosorbent, the bacterial concentrate was left in NaOH solution for a period of 2 hours of contact and then washed for two hours and resuspended in deionized water and only then used in the biosorption.

In the results presented in Figure 32, the best parameters with untreated R. ruber biosorbent or 1 cycle showed removal in the order of 44% and uptake of 22 mg.g ^"1. When a new reintroduction of R. ruber biosorbent or 2 cycles, it was increased we had results of 50.6% and 25.3 mg.g ^"1 and in the last case of serial biosorption we call 3 cycles we obtained 52.5% and 26.2 mg removal results. g ¹ .

 We can see that there was little increase in removal when we consider that when reintroducing a new R. ruber biosorbent volume the active sites would be available and the volume of metal ions would be smaller, thus providing a larger increase in removal in the overall appearance. But this fact has not been confirmed, and there is no chemically plausible explanation for a given event.

In the case of the treatment of R. ruber bacteria with NaOH in a single biosorption cycle, it presented excellent removal results reaching a value close to 97% and uptake of 48.46mg.g ^_1 . As the most interesting and promising result in terms of the use of biosorbent in the removal of aqueous solutions containing Co (ll). The results are encouraging from the standpoint that compared to a serial biosorption would not have several more steps in the process and with higher actual removal values. The only further step in the process It would be the biosorbent treatment, which can be considered as an extra cost and would also spend more time when considering effluent treatment scales. In return the cost and time would be offset by a significant removal increment.

Silva (2010), when performing his work with aqueous solutions containing Co (ll) and using R. opacus biosorbent treated with NaOH, obtained similar results, although he worked with low metal concentration (5mg.L ^{~ 1} ) and at pH 7.0 their results were similar to our study, where it reached the removal of 97%.

The fact that the increase in the removal of Co (ll) by R. ruber was due simply to the increase in the electronegativity of the bacteria wall conferred by the action of the chemical agent. According to Pai et al (1998) in their work with Fe (III), they explain that treatment with 0.1 N NaOH is interesting because it easily promotes ionic exchange of Na ⁺ -filled sites with Fe ⁺³ when there is protonation of active sites. .

In their work on Ascophyllum nodosum uptake of Co (ll) the authors observed a higher uptake when K ⁺ was used as competition metal. In some cases, competition between metals of the same charge favors biosorption or ion exchange (Kuyucak and Volesky, 1989).

Best Biosorption Parameters - Ni (ll):

 Figure 32 shows the blue line removal data and the red line refers to Ni (ll) uptake data by the R. ruber biosorbent. We consider the best parameters for Ni (ll):

 . pH: 6.0;

· Biomass Concentration: 3g.L ^"1 ;

• Initial Metal Concentration: 30 mg.L ^"1 ;

 • Contact Time: 30 minutes;

 • Temperature: 25 ° C or 298 ° K.

In this stage, the following variables, serial biosorption and treatment were evaluated: • Better biosorption parameters with untreated biosorbent (1 cycle of biosorbent);

 • Better biosorption parameters with a new biosorbent being fed and again undergoing biosorption (2 cycles);

 • Better biosorption parameters with a second new biosorbent being fed and again undergoing biosorption (3 cycles);

 • Better biosorption coatings (obtained with untreated biosorbent) treated with NaOH.

As already described above, the interpretation of the variables of the item in question goes straight to the results presented regarding the removal and uptake of Ni (ll) contained in aqueous solutions mediated by R. ruber. When dealing with a single biosorption cycle or the combination of the best parameters obtained throughout the experiments, we obtained a 36.6% removal and 18.3 mg.g ^"1 uptake, we can suggest that all results in relation to Co (ll) in this study presented values slightly below Ni removal (11), which is explained by the fact that the cobalt ionic radius is smaller than nickel, thus favoring the occupation of active sites in the biosorbent wall.

In the case of a biosorbent reintroduction in the process, or what we call 2 cycles of biomass, the arranged results were 40.3% removal and 24.2 mg.g ^"1 uptake. And for when a second new biomass reintroduction or 3 serial biosorption cycles, we had 56.2 removal and 28.1 mg.g ^"1 uptake.

For the biosorption where R. ruber bacteria was treated with NaOH prior to contact with the aqueous solution containing the Ni (ll) metal ions, the removal also as previously with Co (ll) was exceptionally presented in relation to the biosorption. serial biosorption. Removal was 89.2% and uptake 44.6 mg.g ^'1 . This result with R. ruber NaOH treatment for a period of two hours prior to biosorption was similar to that obtained by Cayllahua et al. (2009) (92%) with some caveats, the authors in this study did not perform the treatment of R. opacus biosorbent, but used a dosage of Ni (ll) metal ions far below the proposed by our study, 5 mg.L- compared to our 30 mg.L ^{"1. In addition} , they used a higher amount of biosorbent, which compares to 5 gL ^{" 1} compared to our experiments where 3 gL ^{"1 was used} . The amount of biosorbent, the amount of metal involved in the process among other factors directly influence biosorption (Faroq et al., 2010).

 Best Biosorption Parameters in Binary Systems - Co (ll) and Ni (ll):

 Figure 33 shows the results obtained in binary competition system between Co (ll) and Ni (ll) metal ions contained in aqueous solutions mediated by R. ruber.

 In biosorption studies where the competition between the two metals involved in the process were evaluated in relation to the best parameters obtained during the experimental studies, the biosorption in series with 2 and biosorbent cycles, besides the treatment of R. ruber bacteria was also evaluated. NaOH for a period of 2 hours prior to contact with the solution containing Co (ll) and Ni (ll).

 The results of a biosorption cycle with the best parameters obtained the removal of Co (ll) in the binary competition system was 42.1% and for Ni (ll) it was 24%. For the serial biosorption being reintroduced a new cycle of R. ruber biosorbent the results for Co (ll) was 53.2% and in the case of Ni (ll) 38%. Already for the reintroduction of the second cycle of biosorbent, where 3 cycles are completed in series, we present them as removal values for Co (ll) 67.6% and Ni (ll) 51.2%.

When we talk about the treated bacteria being used as a variable in the binary system containing Co (ll) and Ni (ll) the respective removal results were obtained 60.1% and 62%. In situations where we treat with real effluents we commonly find two or more metallic species and even find pollutants of an organic nature. This scenario can affect and modify the uptake of metal ions through the biosorption process. Compared to the sorption system where there is only one metallic species, the binary system will present on the biosorbent a preference for the occupation of the active sites, in the case of bacteria, as in our study, the cell wall will offer a preference for adsorption. of a kind.

 Analysis of this competition in environments containing more than one metal requires the analysis of metal speciation, metal concentrations and the nature and concentration of biosorbent. These analyzes have already been proven by several studies on biosorption metal competition in binary and tertiary systems (Bueno, 2007) and even in quaternary systems (Mimura et al., 2010).

 Figure 34 clearly shows us that in a binary system containing Co (ll) and Ni (ll) in R. ruber biosorption without biomass treatment and using the serial process strategy with up to 3 biosorbent cycles, The metallic species Co (ll) is noticeable in relation to Ni (ll) because the removal results showed a preference for the R. ruber cell wall over the metallic Co (ll) ions, due to the fact that the ionic radius of Co (0) 63 A) is smaller than Ni (0.83 A) and therefore has a higher density. This was also evidenced by Bueno (2007) and Mimura et al (2010).

In the biosorption with treated R. ruber biomass there was no significant difference between the removal between Co (ll) and Ni (ll). No literature was found in the literature dealing with binary or tertiary systems that worked with treated bacterial biomass, thus assuming that as more active sites are made available due to the increased negativity of the bacterial cell wall, nickel ions ( ll) who has no preference for initially occupying the active sites already filled by cobalt ions (11) more negative charges would be available to adsorb.

 Microbiological Assays:

 R. ruber Growth in Different Cultivation Media:

 Figure 34 shows the growth results of three culture media of Rhodococcus ruber bacteria.

 Growth tests in different culture media are important because they define which nutritional conditions the microorganism develops satisfactorily. Three culture media commonly used in the laboratory for bacterial growth were tested. For example, the bacterium R. opacus was grown in YMA medium in the works by the authors (Bueno, 2007; Cayllahua, 2009 and Silva, 2010).

From the results visualized in Figure 31, the YMA culture medium did not present as satisfactory a dry weight growth result as the TSB. And the GYM culture medium presented the worst results among those tested. The temperature at which the tests were done at 30 ± 2 ° C, was reported by Sanchez et al. (2004).

 Therefore it was defined that the best liquid culture medium under the conditions established for R. ruber was TSB (Tryptone Soy Brotch).

 Figure 35 shows in absorbance data the growth of R. ruber in different culture media and over time, shown at intervals of 12, 16, 24, 36 hours.

R. ruber Growth Curve:

In Figure 36 the data of R. ruber growth curve are described having as variables the dry weight (gL ^"1 ) and time in (hours) in TSB medium.

The R. ruber growth curve shows very fast behavior. In relation to the three phases presented in Figure 38, the adaptation phase or also known as lag phase, is where the microorganism is literally adapting to the environment, often the bacteria comes out of an environment with poor nutritional condition and when packed in a kinda culture, it takes time to adapt to local conditions. This phase is suggested between 0 and 1 hour. Then comes the logarithmic phase that is when the already adapted microorganism begins its full physiological and reproductive condition, at this stage the bacteria grow exponentially, because they are in a culture medium that favors their multiplication. The log phase was established in Figure 33 from the period after the first hour to 12 hours. The end of the logarithmic phase is interesting because in it the number of cells that divide or multiply is theoretically equal to the number of bacteria that die, so from that moment we can consider the maximum bacterial growth proposed for R. ruber in culture medium. TSB. As in our biosorption work the microorganism was inactive, the incubation period was considered from 12 hours. This collaborates as an optimizer in establishing a pilot plant for a biosorption process in the treatment of industrial effluents.

Another curious fact is that the number of cells from this moment becomes constant the number of cells in the order 10 ⁹ cells. mL ^"1 , the same result found by Sierra (2010) when he carried out Escherichia coli growth in his biocolloid transport experiments in a saturated sand. Thus we can estimate" less inaccurately "the amount of inoculum and infer more consistent with the microbial population present in the experiments.

 The stationary phase began at 12 o'clock and extended until the end of the evaluations performed at 48 hours, variations were noted in the readings that may have been attributed to human manipulation or the sensitivity of the devices used in reading the data.

 Correlation Curve Between Absorbance and Number of Cells:

The correlation curve between absorbance at a wavelength of 600 nm and the number of cells per mL of R. ruber growth in TSB medium was plotted. Figure 38 shows the linear adjustment of the correlation where we obtained, R = 0.9918. Table 5 correlates dilutions, cell number and absorbance at wavelength 600 nm.

Table 5 - Relationship between dilutions, cell number and absorbance of R. ruber.

 Dilutions Cell No. / mL Absorbance (600 nm)

 1 4.00E-09 1, 203

 ½ 2.00E-09 0.69

 1/5 8E-10 0.32

 1/10 4E-10 0, 16

 1/20 2.00E- 10 0.07

 1/50 8.00E-1 1 0.0222

 1/100 4.00E- 1 1 0.002

 1/200 2.00E-1 1 0

We actually make this correlation to understand quantitatively from a microbiological point of view, as a concentration of bacteria in grams per liter can vary greatly depending on the conditions under which the inoculum was made available. In the biosorption works developed at PUC-Rio's Laboratory of Mineral and Environmental Technology, microbiological biosorbents are measured following a trend of other works in the segment. Where the microorganism is concentrated in low amounts of distilled water and after weighing for 24 hours in an oven at 103-105 ° C, the concentration in gL ^{"1 is determined} . For this purpose, the calculations performed in this item could be used in future work. with the amount in gL ^"1 of the culture medium that has been established. If we have 4 x ^{10 "9} bacterial cells per mL of TSB medium we get the ratio of 4 x 0 ^{" 12} R. ruber cells in 1 liter of culture medium, knowing the biosorbent concentration in gL ^"1 to be used in the biosorption process. We will more accurately estimate the amount of cells that will act in the system.

External Surface Area of R. ruber.

In order to differentiate in terms of external surface area R. ruber bacteria, 100 R. ruber cells were measured in SEM images obtained on a 2pm scale and then the external surface area of the bacterium was calculated. ellipsoid, since cells have no true spherical shape, although they visibly have this shape or as they are commonly called "cocus".

 The results show that there was a 5% difference in standard deviation when considering the area of untreated and treated bacteria, that is, the treated bacteria are 5% smaller in surface area when compared to bacteria not treated with NaOH. This decrease gives us the idea that the smaller the surface area of a body, the larger the total contact area in a given area.

 Not explaining the increased removal between untreated and untreated bacteria, but collaborates with other factors explained throughout this dissertation, which would be a set of events to clarify this difference.

Biosorbent Characterization - R. ruber:

 R. ruber bacteria were characterized by light microscopy, scanning electron microscopy in contact with Co (ll) and Ni (ll) metals, as well as bacteria also treated with NaOH.

Light Optical Microscopy - R. ruber:

 The characteristics of the R. ruber bacteria wall can be demonstrated. It can be confirmed that the bacterium R. ruber is Gram positive, as the result of the bluish staining indicates that the dye could not penetrate inside the cells due to the thick layer of peptidoglycan that constitutes this type of wall. Otherwise Gram-negative bacteria are red in the presence of violet blue which is the recommended dye for this type of test. Sanchéz et al. (2004) in their didactic review regarding the genus Rhodococcus designates bacteria belonging as Gram-positive.

 Scanning Electron Microscopy - R. ruber:

Images were obtained showing morphological aspects of R. ruber bacteria before and after contact with Co (ll) and Ni (ll) metal ions separately and in mixture. In addition to demonstrating aspects of bacteria treated with NaOH before and after contact with the metals Co (ll) and Ni (ll) individually.

 Microphotography A presents aspects of the R. ruber bacterium as a control, that is, it is not interacting with any other type of compound. It is noticed that the bacterium has dimensions of 2μηι in length and 1pm in thickness, data consistent with the literature found and similar works using the species of the same genus, R. opacus (Mesquita, 2000; Alvarez- Vazquez et al., 2004; Sánchez et al, 2004; Bueno, 2007; Cayllahua, 2008; Silva, 2010).

 The morphology of R. ruber bacteria observed in the micrograph A, as many in the other micrographs, are mostly presented when we consider the focused region of the samples and reproduced in the images, in the form of "coccus". But some cells also appear in the slightly longer "bacillary" form, a characteristic feature of the genus (Sánchez er al, 2004). A curious aspect observed in the images is that the bacterium R. ruber has as one of its characteristics to line up in 3 or 4 "cocus" cells, very similar to a septate fungus hyphae.

 In microphotography B we visualize the interaction between the bacterium R. ruber and the metallic ions of Co (ll), we can suggest a more crowded aspect of the bacteria as a tendency to capture the metal present in aqueous solution. Again emphasizing that the photographs taken are of specific points of the samples prepared for scanning electron microscope purposes and on many occasions do not reflect the overall appearance of the sample.

Microphotography C demonstrates the interaction between R. ruber and Ni (ll) metal ions and D. ruber after metallic contact with the mixture of Co (ll) and Ni (ll) ions in competition system. Apparently two very similar microphotographs, where in C presents a higher concentration in the number of cells in the region photographed in first and In the background and image D the bacterial cells of R. ruber are more spaced in the foreground and background.

 The photographs indicated with the letters E and F refer to R. ruber bacteria treated with NaOH and controls. In microphotography E the increase was 15,000 times, demonstrating a clustering aspect similar to that reported in image B, but with an aspect of more cell concentration. Image F once again highlights the previously commented detail with a zoom of 25,000 times closer.

 In the interaction of R. ruber treated with Co (ll) metal ions seen with approximation of 15,000 times by the SEM technique in the G image, the agglutination of the bacterial cells is again observed and suggested. But in contrast to image H where the interaction between treated R. ruber and Ni (ll) metal ions is not visualized this clustering aspect, on the contrary, the bacterial cells are more dispersed and yet , lining up at times in sets of 4, 5 and even 6 cells.

Transmission Electron Microscopy - R. ruber:

 Images showing superficial aspects of R. ruber bacteria before and after contact with Co (ll) and Ni (ll) metal ions separately and in mixture were obtained. It also demonstrates aspects of NaOH-treated bacteria before and after contact with Co (ll) and Ni (ll) metals individually.

 Transmission electron microscopy or MET give us two positive aspects regarding SEM, as this type of technique gives us larger magnifications and visualization of the external surface of the bodies to be scanned. As an apparent advantage, SEM photos give us third-dimensional perspectives, which in superficial terms also have their function.

In microphotography A we see R. ruber cells as a control and without treatment. Although image B reports the interaction between the bacterium and the metal Co (ll) are similar in image, in the magnifying aspect (20,000 times) and 2pm scale. Enabling us to suggest some features. In photograph A, even though the contrast is not the same for both images, this makes comparison somewhat difficult. We can more attenuate the organelles and constituents of R. ruber cells.

 In image B we suggest a concentration of Co (ll) ions at the edge of R. ruber cells. Since the regions closest to the bacterial cell wall show a more intense dark color. And since the principle of biosorption is adsorption and nonabsorption, this result was inferred, once again remembering that they are conjectures constructed from the analysis of the images, since we had no semi-quantification or qualification aspects as in the case of SEM. couple an Energy Dispersive System (EDS) that can assure us of this statement.

 Microphotography C shows the interaction between R. ruber and Ni (ll) metal ions and microphotography D shows the interaction of R. ruber in a binary system containing Co (ll) and Ni (ll). Image E represents the R. ruber bacterium treated with NaOH and as a control. Microphotographs show the interaction between the treated R. ruber bacteria and the metal ions Co (ll) (F); R. ruber treated plus Ni (ll) metal ions (G and H). In all the images of the interaction between the bacteria and the metals, a darker hue is observed near the cell wall - the R. ruber cells and the metals involved in the process may be adsorbed.

 Infrared Spectroscopy Analysis:

Infrared spectroscopy analyzes aim to identify functional clusters, organic constituents present mainly in the cell wall of R. ruber bacterial cells. Analyzes were performed from samples prepared with untreated bacteria and their interaction with metals Co (ll) and Ni (ll). Also samples with the bacteria treated with NaOH were prepared for analysis, as well as its interaction with the metallic ions of Co (ll) and Ni (ll). The infrared wavelength range in which we performed the analyzes was from 500 to 4000 cm ^"1 , thus identifying the constant clusters in the bacterium R. ruber, we can characterize them as responsible for the biosorption process and what are the mechanisms present interaction between metals and biosorbent Table 10 correlates the wavelengths and the corresponding functional groups responsible for the existing adsorption of metals and the charge presented by the bacteria in question.

It should be remembered that all samples containing both untreated and treated bacteria were prepared from solutions containing 30 mg.L ^"1 of Co (ll) and Ni (ll) metal ions and the R. ruber biosorbent concentration of 3 gL ^"1 , all adjusted to pH 6.0 before contact with the bacteria.

Table 6 - Infrared wavelength (cm ^"1 ) and relationship between their corresponding functional groups.

Wavelength (cm ¹ ) Functional Groupings

 Values close to 3350 -OH and N-H group stretch

2920 -CH Asymmetrical CH ₂ Stretch

Range close to 2850 -CH symmetrical CH ₂ stretch

 Range near 1650 Amide Group I

 Range near 1540 Amide Group II

Range near 1400 C = 0 symmetrical COO stretch ^"

1250-1220 P = 0 symmetrical P0 ₂ ^" stretch

 Near Range 1070 -CN

 1200-900 C-O-C and OH of polysaccharides

 Source: Bueno (2007).

 Infrared Spectroscopy Analysis - R. ruber without treatment:

 The bands representing the infrared R. ruber constitution are shown in Fig. 39 where we visualize the 3294 band corresponding to the region of the -OH and -NH groupings of glycoses and proteins (Ashkenazy et al., 1997; Silverstein et al., 2007). Cayllahua et al. (2009) and Bueno (2007) obtained similar bandwidth results when they used R. opacus bacteria in their biosorption work.

Already when we denote bands of intensities corresponding to 2960 and 2928 are attributed respectively to the asymmetric vibrations and CH ₂ radicals as described in table YY. Fact also observed by the authors Bueno (2007) and Cayllahua et al. (2009). Amide groups I and II are observed at peaks 1652 and 1537 (Silverstein et al, 2007). In bands peaks 1457 and 1398 are responsible for grouping C = O and COO ^"The peak 1234 corresponds to the grouping P = 1066 is the indication that the grouping protein -CN;. , And finally the peaks 782 and 541 are torsional oscillations of CH and NH (NH ³⁺ - amino acid) respectively (Silverstein et al, 2007).

When we analyze the interaction spectra between untreated R. ruber and Co (ll) metal ions, we can observe a decrease in bands in the peak bands corresponding to 1536, 1233, 1065, 781 and 534 cm ^"1 . amide II, P = O, -CN, CH and NH with higher intensity in terms of band variation (541 to 534) the other peaks varied from 1 point in relation to the bacteria without contact with the metal. by R. ruber biosorption by the metallic ions of Co (ll), it is interesting to highlight the role of the NH (NH ³⁺ ) grouping due to its high variation, which disagree with those found by Bueno (2007) and Cayllahua et al (2009). When they worked with R. opacus, they observed considerable participation of the group - OH, which was not verified in our work.

Analyzing the untreated R. ruber bands in interaction with Ni (ll) metal ions, we found a decrease in the following peaks 1536, 1452, 1233, 1065 and 537. These correspond to the respective functional groups, amide II, C = O, P = What are symmetrical stretches of COO ^" and PO ₂ ^" respectively; -CN is the NH (NH ³⁺ ) group. Once again highlighting peak 537 where the largest difference (541 cm ^"1 of the bacteria without contact with the metal was obtained.) This group is considered important in the biosorption process for both Ni (ll) and ions metal ions. of Co (11).

 The results disagree with those obtained by Cayllahua et al.

(2009) in their work on interaction of R. opacus bacteria with metal ions Ni (ll) where the largest differences were presented in the wavelength ranges 3414 and 2937 which are responsible for the - OH and -CH clusters.

When we superimpose the infrared spectrometry curves in figure 42, we observe that the behavior of the metallic ions of Co (ll) and Ni (ll) are extremely similar and we also realize that the difference between the untreated bacterium R. ruber and the its interaction between the metallic ions Co (ll) and Ni (ll) is not significant results that agree with the presented by Cayílahua et al (2009) when we consider the interaction of R. opacus with Ni (ll), except in the range of greater differentiation than for the authors was in the range between 3414 and 2937 and in our study the highest interaction range was marked in the range of 541 cm ^"1. These results can confirm the low removal obtained by the bacterium R. ruber not treated with the ions. (ll) and Ni (ll) metals in biosorption assays Infrared Spectroscopy Analysis - R. ruber Treated with NaOH:

 The bacteria, after being treated with NaOH for a period of 2 hours, were placed in contact with metallic solutions containing Co (ll) and Ni (ll). From there the samples were prepared for analysis in Infrared Spectrometry. Following are the results of the analyzes.

Figure 43 presents the results regarding the vibration peaks in the bands that relate the existing clusters in the organic constitution of R. ruber bacterial cells after NaOH treatment. The 3417cm ^"1 band peak that corresponds to the functional group - OH was not evidenced in this same study when we treated R. ruber bacteria without chemical treatment with NaOH. The 3294 band evidenced in the untreated bacterium did not mention the analysis. even though its presence is perceived as a less evident peak in figure 40, considering it close to the 3417 wavelength value from left to right. The peaks 2960 and 2928 cm ^"1 were similar to those described in the item R. ruber without treatment, and the functional groups referring to -CH with asymmetric CH ₂ stretching were responsible for the metal ion uptake process in this study. The 1648 band responsible for the amide II functional grouping was lower in absolute value than the untreated bacteria (1652 cm ^"1 ).

Of particular note is the wavelength in the 1462 range, which has been shown to be higher in terms of absolute value than the untreated bacteria (1453 cm- ¹ ). This may configure new active sites for the removal of Co (ll) and ions. Ni (ll) by R. ruber bacteria These results agree with Bueno (2007) and Cayllahua et al. (2009) when they worked with R. opacus bacteria even without chemical treatment.

Figure 44 plotted the curve of the bands corresponding to the interaction of R. ruber with the metallic ions of Co (ll). Where we perceive the timid but qualitative participation from the qualitative point of view of the -OH group in the process. This did not occur in interaction with untreated bacteria. The peak 1451 cm ^"1 was expressive for the interaction of the treated bacteria with Co (ll) since its peak related only to R. ruber treated was 1462 cm ^{" 1} , and the group C = O was responsible for metal adsorption by biosorbent. The -CN group also showed a slight decrease (from 1064 to 1063 cm ^-1 in comparison).

When we describe the interaction between the bacterium R. ruber treated with Ni (ll) metal ions we observed a large participation of the -OH group in the process, because the decrease in band size in wavelength was expressive, went from 3417 in the bacterium. 3404 cm ^"1 in the interaction with Ni (ll) ions. Finally, the 1452 band that corresponds to the C = O group with symmetrical COO stretching ^" were considered responsible for the R. ruber biosorption process of the ions. Ni (11).

In general, the functional groups deserved more prominence when we consider R. ruber bacteria chemically treated by NaOH This fact culminates in the statement that functional clusters together with a number of other environmental factors favored the greater removal by R. ruber. Even more from the point of view of the higher qualitative action of several functional groups in the interaction with the ions of Co (ll) showed a removal that reached the level of 97% and the two groups -OH and C = O expressed a result of 89% removal for Ni ions (ll).

 The removal of two metallic species of Co (ll) and Ni (ll) was analyzed using Rhodococcus ruber bacteria as biosorbent. The metals were chosen according to their industrial use, due to their degree of dangerousness in relation to living beings and the environment.

 Thus, by all the results obtained and presented above, the heavy metal biosorption process according to the present invention comprises the use of Rhodococcus ruber bacteria acting in a pH-adjusted biosorption medium with 1 M NaOH and 0.1 M HCl at temperatures of the order of 28 ° C ± 2 ° C for a time of about 3 hours, said biosorption medium being subsequently centrifuged for about 6 minutes for heavy metal separation and bacterial regeneration.

Preferably, when it comes to the metallic species Co (ll) and Ni (ll), the process according to the present invention comprises the use of NaOH-treated R. ruber bacteria in a biosorption medium with a pH of 6.0 and concentration R. ruber in the order of 3 gL ^" for a time of about 30 minutes at temperatures in the order of 25 ° C.

 Under these conditions, the process according to the present invention provides removal results of about 97% for Co (11) and about 89% for Ni (11).

 However, those skilled in the art will appreciate the knowledge presented herein and may reproduce the invention in the embodiments presented and in other embodiments, without thereby departing from what is described and claimed herein.

Claims

Claims

 1. HEAVY METALS BIOSORATION PROCESS, characterized in that it comprises a step of contacting Rhodococcus ruber bacteria with a medium comprising heavy metals, which it is desired to biosorb, then regenerating the biomass and recovering the heavy metal.

 Heavy metal biosorption process according to Claim 1, characterized in that it comprises the use of Rhodococcus ruber bacteria acting on a biosorption medium at pH 4-7, adjusted with 1 M NaOH and 0.1 M HCl. at temperatures of the order of 28 ° C ± 2 ° C for a time of about 3 hours, said biosorption medium being subsequently centrifuged for about 6 minutes for heavy metal separation and bacterial regeneration.

 Heavy metal biosorption process according to claim 1, characterized in that the heavy metals to be biosorbed are Co (ll) and Ni (ll).

Heavy metal biosorption process according to claims 1 and 3, characterized in that it comprises treating R. ruber bacteria with NaOH for a period of 2 hours prior to contact with the solution containing Co (ll) and Ni. (ll), a biosorption medium with a pH of 6.0 and R. ruber concentration in the order of 3 gL ^"1 , the contact being made for a time of about 30 minutes, at temperatures of the order of 25 ° C.

 Heavy metal biosorption process according to claim 1 or 3, characterized in that the removal of Co (ll) in the binary competition system is obtained by about 42.1% and for Ni (ll) by about of 24%.

 6. Heavy metal biosorption process according to claim 1 or 3, characterized in that in the series biosorption a new cycle of the R. biosorbent is reintroduced. The removal results for Co (ll) are about 53%. , 2% and for Ni (11) to be about 38%.

7. Heavy metal biosorption process according to claim 1 or 3, characterized in that in biosorption with If the second biosorbent cycle is reintroduced, where 3 series cycles are completed, the removal values of Co (ll) will be about 67.6% and Ni (ll) will be about 51.2%.

 Heavy metal biosorption process according to claim 4, characterized in that removals of about 97% for Co (ll) and about 89% for Ni (ll) are obtained.