Hui Niu , Bohumil Volesky * and Newton C. M. Gomes^a

http://www.mcgill.ca/biosorption/biosorption.htm

Department of Chemical Engineering , McGill University                 *e-mail: boya@chemeng.Lan.mcgill.ca
3610 University Street, MONTREAL, Canada H3A 2B2              *corresponding author

^aInstituto de Microbiologia Prof. Paulo de Goes, CCS
Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil

 
    The presence of L-cysteine increased gold-cyanide biosorption by protonated Bacillus subtilis, Penicillium chrysogenum and Sargassum fluitans biomass at pH 2 by (148 ~ 250)%. The respective Au uptake by these biomass types was 20.5 mmol/g ,14.2 mmol/g and 4.7 mmol/g of Au. Au-loaded biomass can be eluted with 0.1M NaOH. The elution efficiencies exceeded 90% at pH 5.0 with the Solid-to-Liquid ratio S/L = 4. Biosorption of anionic AuCN₂^- complex involved ionizable protonated cysteine-loaded biomass functional groups. An adverse effect of increasing solution ionic strength (NaNO₃) was explained by a triple layer surface complexation mechanism. The NO₃^- anion competed with AuCN₂^-. Results confirmed that certain waste microbial biomaterials are capable of effectively removing and concentrating gold from solutions containing residual cyanide.

1. INTRODUCTION
 
    Recent experimental results demonstrated that Bacillus subtilis, Penicillium chrysogenum and Sargassum fluitans biomass could extract Au from cyanide solution [1]. The main mechanism of Au biosorption involved anionic AuCN₂^- species adsorption onto N-, P-, or O- containing functional groups on biomass through ion-pairing (H^+.- AuCN₂^-). However, the capacities for Au biosorption by Bacillus subtilis, Penicillium chrysogenum and Sargassum fluitans biomass were not encouraging .
    Proteins are known to be capable of complexing with metal ions. Cysteine, which figures prominently in discussions of metal ion binding to proteins, has three possible coordination sites, namely sulfhydryl, amino and carboxylate groups [2]. Hussain attributed the protection of isolated human lymphocytes from silver toxicity to cysteine through the formation of Ag-thiol complexes [3]. The complexation of Cu-cysteine was ascribed to the complexing of Cu to thiol as well as amino groups [4]. These results showed that cysteine had a tendency to combine well with metals. However, the behavior of L-cysteine in Au-cyanide complex biosorption have never been examined.
    The objectives of this work are to investigate the effect of L-cysteine on Au biosorption from cyanide solution by dead Bacillus subtilis, Penicillium chrysogenum and Sargassum fluitans biomass. The mechanism of Au-cyanide biosorption under these unconventional conditions was also examined.
 

2. MATERIALS AND METHODS

2.1 Biosorbent preparation
    Waste industrial biomass samples of Bacillus subtilis and Penicillium chrysogenum were collected from Sichuan Pharmaceutical Company, Chengdu, P. R. China. Sargassum fluitans seaweed biomass was collected beach-dried on the Gulf Coast of Florida. Biomass was ground into particles around (0.5-0.85) mm in diameter, then washed with 0.2N HNO₃ for 4 hrs and rinsed with distilled water to pH~4.5. Finally, the biomass was dried in the oven at 50⁰C for 24 hrs to a constant weight.

2.2. Acidification of gold cyanide solution
    A solution of AuCN₂^- was prepared by dissolving solid NaAuCN ₂ in NaOH solution at pH 11 to simulate the industrial gold cyanide leach solution. Metal biosorption by biomass is usually taking place at pH values less than pH 6 since some biomass types or their constituents could seriously hydrolyze at elevated pH levels [5]. In order to make a full use of the biomass potential to concentrate gold from the cyanide solution, the pH of the gold cyanide solution needs to be adjusted for adequate biosorption. Since toxic hydrogen cyanide gas released at pH lower than 9.3 [6] is extremely dangerous, the conventional AVR process (Acidification, Volatilization and Reneutralization of cyanide ) was employed [7]. The only difference from the standard AVR process in the current experiments was stripping of cyanide gas by nitrogen instead of air to avoid any oxidation of HCN. Total cyanide analysis was done by standard cyanide distillation followed by the titrimetric method for free CN^- in the alkaline solution [8].

2.3. Cysteine adsorption by biomass
    Approximately 40 mg dried protonated biomass contacted with 20 ml cysteine solution with certain initial cysteine concentration 0~ 1.2 mmol/l in 150 ml Erlenmeyer flasks. The solution was mixed and left to equilibrate for 4 hrs. The cysteine uptake was determined from the difference of cysteine concentrations in the initial and final solutions. Cysteine was analyzed by a UV-visible spectrophotometer (Cary 1).

2.4. Equilibrium sorption experiments
    Approximately 40 mg dried protonated biomass was combined with 20 ml sodium gold cyanide solution with or without L-cysteine in 150 ml Erlenmeyer flasks. The solution was gently mixed and equilibrated for 4 hrs. Uptakes of chromium were determined from the difference of metal concentrations in the initial and final solutions. The pH of the solutions before and during the sorption experiments was adjusted with 0.1M NaOH or HNO₃. The ionic strength was controlled by adding NaNO₃ . All reagents were ACS reagent grade quality. Au concentration was determined by a sequential inductively-coupled plasma atomic emission spectrometer (Thermo Jarrell Ash, Trace Scan).
 

3. RESULTS AND DISCUSSION
 
3.1. Effect of L-cysteine on Au biosorption
    The effect of L-cysteine on Au biosorption by Bacillus , Penicillium and Sargassum biomass was examined by varying L-cysteine concentration in the Au cyanide solution from 0 ~ 0.6 mmol/l at pH 2.0, with the initial Au concentration of 0.1015 mmol/l. No cyanide was released during the process. The results are shown in Figure 1. The final cysteine concentration around 0.5 mmol/l enhanced Au uptakes by Bacillus , Penicillium and Sargassum biomass up to 250% , 200% and 148%, respectively.
    L-cysteine biosorption isotherms for Bacillus , Penicillium and Sargassum biomass in Figure 2 show encouraging uptakes by Bacillus and Penicillium biomass, while Sargassum biomass sorbed very little. Under the experimental conditions, the sequence for the cysteine uptake by the three biomass types is Bacillus > Penicillium > Sargassum , which agreed with the sequence of increased Au uptake in the presence cysteine. Enhancement of Au biosorption in the presence of cysteine apparently relates to the "bridging" function provided by cysteine between the Au-cyanide complex and biomass. The main active sites on the cysteine molecule are sulfhydryl, amino and carboxyl groups [4]. The dissociation constants (pK) of those groups are respectively 8.12, 10.36 and 1.90 [9]. At low pH 2.0, the carboxyl group is more active than the other two groups of cysteine and tends to combine with positively charged groups on biomass.
 

   
Figure 1:    Effect of cysteine on Au-cyanide uptake
                  0.04 g biomass, initial Au concentration 0.1015 mmol/l,
                  20 ml solution, pH2.0, 4hrs, room temperature
 
 

  Figure 2: Cysteine biosorption isotherms
                0.04 g biomass, 20 ml solution, pH2.0, 4hrs, room temperature

3.2. Effect of pH
    The effect of pH on Au biosorption in the presence of cysteine was examined by varying pH from 2~6. The initial ratio of Au : cysteine was 1:5. During the process of acidifying the Au cyanide solution and Au biosorption equilibration, there was no cyanide released from the solution. Figure 3 shows that in the presence of cysteine Au adsorption by Bacillus, Penicillium or Sargassum biomass was strongly affected by pH. The equilibrium uptakes of Au at pH 2 were greater than those at pH>2. The pH had a tendency to increase during the equilibration, hence 0.1 N HNO₃ was used to adjust the pH. This observation is opposite to that reported for biosorption of Zn, Cd, and Pb(NO₃)₂ by cysteine alone [4]. Divalent ions of these metals complexed with the cysteine sulfhydryl (-S^- ) and amino groups. During the adsorption process, the hydrogen of sulfhydryl was dissociated, accounting for the pH drop. However, in the present case of AuCN₂^- uptake, it was hard to dissociate Au from the cyanide complex at room temperature. The present Au cysteine-aided biosorption probably still involved anionic AuCN₂^- complex adsorption. A similar general behavior was reported for biosorption of anionic Cr(VI) [12], whereby lowering of the equilibrium pH from neutral to acidic yielded an increase in Cr uptake by Sargassum. This was also the case for AuCN₂^- uptake by Bacillus, Pencillium and Sargassum biomass without cysteine addition [1]. Basically, sulfhydryl and amino groups on cysteine behaved like weak bases which could be protonated at low pH, the same as those found in biomass. When cysteine is encountered in aqueous solution, there exists a surface charge on ionizable functional groups. As the concentration of protons is increased at pH 2.0, more and more weak base groups either on cysteine or in biomass become protonated and many acquire a net positive charge. These charged sites become available for binding anionic AuCN₂^- . Meanwhile, some carboxyl groups on cysteine may still be dissociated as the solution pH was higher than the dissociation constant (pK=1.9) of the carboxyl group on cysteine. This allows binding on biomass through the combination with some positively charged biomass function groups. As biomass bound cysteine, the amount of weak base groups of AuCN₂^-.on biomass increased resulting in enhanced Au uptake.

 
   Figure 3: Effect of pH on Au uptake
                0.04g biomass, 20 ml solution, initial Au concentration 20 mg/l,
                initial cysteine concentration 75 mg/l, 4hrs, room temperature

 
3.3. Ionic strength effect
    Ionic strength of experimental solutions was changed from 0.005 M to 0.15 M by adding NaNO₃ at pH 2. During the process, there was no cyanide released from the solution just as the case was in the pH effect experiments, indicating that sodium nitrate can not assist in dissociating the gold-cyanide complex. The effect of ionic strength is illustrated in Figure 4. Increasing ionic strength reduced the Au biosorption. As the concentration of NaNO₃ increased to 68mM, the uptake of Au by Bacillus and Penicillium biomass was respectively reduced to 70% and 50% of that without NaNO₃ in the solution. The Au uptake by Sargassum decreased almost to zero at 20mM NaNO_3. Changing ionic strength (i.e. the background electrolyte concentration) influences adsorption in at least two ways: (a) by affecting the interfacial potential and hence the activity of electrolyte ions and adsorption; (b) by affecting electrolyte ions and adsorbing anions competition for available sorption sites.
 

   
Figure 4: Effect of ionic strength on Au uptake
                0.04 g biomass, 20 ml solution, pH 2.0 , initial Au concentration 20 mg/l,
                cysteine concentration 75 mg/l, 4hrs, room temperature

 
   Figure 5: Schematic representation of the structure of Three Layer Mechanism of
                aurocyanide adsorption on cysteine-loaded biomass in the presence of
                electrolyte NaNO₃.

3.4. Desorption of Au-loaded biomass
    The possibility of desorbing Au from biomass with sodium hydroxide was examined by first sorbing Au onto biomass in the presence of cysteine at pH 2 and then desorbing Au with 0.1 M NaOH at pH 3, 4 or 5. The initially Au-loaded Bacillus biomass contained 20.5 mmol Au /g of dry biomass, Penicillium biomass 14.2 mmol/g, and Sargassum biomass 4.7 mmol/g. The percentage of Au recovery, represented by the ratio of the amount of Au released per gram of the biosorbent during desorption and the equilibrium sorption uptake, was calculated for desorption experiments lasting for 4 hours. Figure 6 shows that more than 90% of Au was recovered at pH 5 with the solid-liquid ratio S/L=4 for all of these three biomass types, which indicated that Au could be easily eluted. These results further confirmed the outer-sphere complexation (FH-H⁺-AuCN₂^-) postulated. When the "bridge" was broken up by OH- combining with H+, Au was dissociated from solid phase. Basically, the outer-sphere complex is not stable probably because of the relatively long distance between sorbent functional groups (on o-plane) and the sorbate (on b-plane). From the view point of adsorption reaction equilibrium, increasing the pH pushed the reaction of anionic gold cyanide complex adsorption to the left, allowing the Au elution. However, in the case of biomass, the use of concentrated NaOH leads to massive leaching of a variety of compounds from the biomass and to the destruction of the biomass cellular structure. Therefore, Au elution was limited to pH no higher than 6.
 

   
Figure 6: Effect of pH on Au elution efficiency
                0.02 g biomass, 5 ml solution, initial Au loading was 20.5 mmol/g Bacillus
                biomass, 14.2 mmol/g Penicillium biomass and 4.7 mmol/g Sargassum biomass,
                4hrs, room temperature

F : functional group on biomass or cysteine
H: dissociable hydrogen on function group, the binding of which make
    cysteine-loaded biomass neutral
s_o: surface charge on o-plane
s_{b :} surface charge on b-plane
s_d : surface charge on d-plane
y_o: potential on o-plane
y_b: potential on b-plane.

   
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