Biosorption of lead(II), zinc(II) and nickel(II) from industrial wastewater by Stenotrophomonas maltophilia and Bacillus subtilis

Abstract The biosorption of Pb(II), Zn(II) and Ni(II) from industrial wastewater using Stenotrophomonas maltophilia and Bacillus subtilis was investigated under various experimental conditions regarding pH, metal concentration and contact time. The optimum pH values for the biosorption of the three metals were in the range 5.0-6.0, while the optimal contact time for the two bacterial species was 30 min. Experimental data was analyzed using Langmuir and Freundlich isotherms; the former had a better fit for the biosorption of Pb(II), Zn(II) and Ni(II). The maximum adsorption uptakes (qmax) of the three metals calculated from the Langmuir biosorption equation for S. maltophilia were 133.3, 47.8 and 54.3 for Pb(II), Zn(II) and Ni(II), respectively, and for B. subtilis were 166.7, 49.7 and 57.8 mg/g, respectively. B. subtilis biomass was more favorable for the biosorption of Pb (II) and Ni (II), while S. maltophilia was more useful for the biosorption of Zn (II).


INTRODUCTION
Pollution of the environment with toxic heavy metals is spreading throughout the world alongside industrial expansion; the resultant contamination of soils, groundwater, sediments, surface water and the air poses signifi cant problems for both human health and the environment 1 . Despite serious environmental concerns due to their toxicity even at low concentrations 1-5 , various industrial processes, such as electroplating, metal fi nishing, metallurgical work, tanning, chemical manufacturing, mining and battery manufacturing, result in the continuous introduction of heavy metal polluted wastewater to the environment (mainly Pb, Hg, Cu, Cd, Zn, Ni and Cr).
With regard to their impact on human health, each heavy metal imparts different effects and symptoms. For instance, in the case of minor Zn exposure, common symptoms are irritability, muscular stiffness, loss of appetite and nausea 6, 7 . Pb is extremely toxic and can infl ict damage to the nervous system, kidneys and reproductive system, particularly in children 4, 7 . The presence of Ni above a critical level might bring about serious lung and kidney problems, aside from gastrointestinal distress, pulmonary fi brosis and skin dermatitis 8 .
Conventional methods used to remove dissolved heavy metal ions from wastewaters include chemical precipitation, chemical oxidation or reduction, ion exchange, filtration, electrochemical treatment, solvent extraction, reverse osmosis, membrane technologies and evaporation recovery. These processes may be ineffective or extremely expensive, especially when the metals in solution are in the range 1-100 mg/l 8, 9 . Another major disadvantage of conventional treatment methods is the production of toxic chemical sludge and its subsequent disposal/ treatment being costly and not eco-friendly. Therefore, it is so signifi cant to fi nd a cost effective and environment--friendly method of removing toxic heavy metals down to a level considered environmentally safe 4, 10 .
One of the most promising technologies involved in the removal of toxic metals from industrial waste streams is biosorption, based on the ability of many algae, fungi, yeasts and bacteria to concentrate heavy metals from aquatic environments 8, 11, 12 . It offers the advantages of low operating costs, the possibility of metal recovery, regeneration of the biosorbent, minimization of the volume of chemical and/or biological sludge to be disposed of, and high effi ciency in detoxifying very dilute effl uents 13-15 . This complex process depends on the properties of metal ions, the cell wall composition of microorganisms, cell physiology, as well as physicochemical factors such as pH, temperature, contact time, ionic strength, and metal concentration 15 . It occurs through complexation, coordination, physical adsorption, chelation, ion exchange, inorganic precipitation or some combination of these processes 11, 16 . These processes involve the active participation of several anionic ligands present in the biomass, such as phosphoryl, carboxyl, carbonyl, sulfydryl and hydroxyl groups, to immobilize metal ions 17 .
The main objective of this work was to study differences in the adsorption of Pb(II), Zn (II) and Ni(II) between Stenotrophomonas maltophilia and Bacillus subtilis cells isolated from a wastewater treatment plant. Factors affecting biosorption (i.e. pH, reaction duration, metal concentration) were also studied. Biosorption isotherms and kinetics were determined from biosorption measurements.

Microorganisms
Lead, zinc and nickel resistant bacterial strains were isolated from a wastewater treatment plant located in Głubczyce (Poland). Samples were diluted 10-10.000 fold in sterile distilled water and plated on Nutrient Agar (Merck). To isolate resistant strains, the media were amended with 100 mg/l Pb(II), 50 mg/l Zn(II) and 50 mg/l Ni(II) and a standard spread plate method was performed. The inoculated plates were incubated for 48 h at 30 o C after which larger identical colonies from each plate were isolated. The most effective bacterial strains for the biosorption of Pb(II) and Ni(II) were identifi ed according to Bergey's Manual of Systematic Bacteriology 18 . Morphological, physiological and biochemical characteristics of the isolated bacterial species (S. maltophilia and B. subtilis) are given in Table 1.
Whatman fi lter (pore size 0.45 μm) and then diluted with deionized water. The initial and the fi nal concentrations of heavy metals used in batch mode studies were estimated spectrophotometrically. The removal effi ciency of the microorganisms was calculated from the difference between initial and fi nal concentrations.

Biosorption experiments
Parameters of Pb(II), Zn(II) and Ni(II) sorption by S. maltophilia and B. subtilis are presented in Table 3.
Experimental tests were conducted in a BIOSTAT A-plus bioreactor containing 1.0 l of wastewater at constant level of biomass (1.0 g/l) at 30 o C and agitation of 200 rpm. Biosorption experiments were carried out to investigate the effects of pH, contact duration and initial metal concentration. The pH values were adjusted between 2.0-7.0 by adding 0.1 M NaOH or 0.1 M HNO 3 . The contact durations ranged from 0-60 min. The initial Pb(II), Zn(II) and Ni(II) concentrations varied from 15.9 to 325.3 mg/l, 11.2 to 172.3 mg/l and 14.3 to 245.8 mg/l, respectively. The metal uptake (mg metal/g dry biomass) was calculated according to: (1) Where C 0 and C e are the respective initial and equilibrium metal concentrations in the solution (mg/l), V is the volume of the solution (l), and M is the dry weight of the biomass (g). The metal sorption ability of the biomass was determined by the above-mentioned procedure in all the following experiments unless stated otherwise.

Adsorption isotherms
Heavy metals biosorption isotherms were obtained at constant pH and ionic strength. To test the fit of data, Langmuir and Freundlich isotherm models were applied

Preparation of biomass
Bacterial strains of S. maltophilia and B. subtilis were cultivated aerobically at 30 o C in Nutrient Broth (Merck) constantly agitated at 150 rpm in glass fl asks. After inoculation, cells were harvested by means of centrifugation for 20 min at 3000 rpm. The cell pellet was rinsed three times with sterile deionized water, then freeze dried using a lyophilizer (Alpha 1-2 LD plus, Christ, Germany). For the purpose of the biosorption experiments, 1 g portions of bacterial cell mass (separately for S. maltophilia and B. subtilis) were suspended in 1 l of deionized water.

Wastewater sample
Wastewater was obtained from a chemical manufacturing plant in Głubczyce (Poland). The composition of the wastewater is given in Table 2. The concentrations of the other metal ions present in the wastewater were so minimal that they would not effect the removal of lead, zinc and nickel ions from the wastewater. If necessary, the effl uent was diluted with deionized water to an appropriate concentration of heavy metals.

Heavy metal assay
The concentrations of Pb(II), Zn(II) and Ni(II) in the biosorption experiments were determined spectrophotometrically (Photolab Spectral, WTW, Germany). Before measuring the samples were passed through a to this study. The Langmuir isotherm model is valid for monolayer sorption onto a surface and a finite number of identical sites, and is given by: (2) or presented in linear form as follows: Where q max is the maximum amount of the metal ion per unit weight of the cell to form a complete monolayer on the surface bound at a high C eq (mg/l) and b, a constant related to the affi nity of the binding sites, q max represents a practical limiting adsorption capacity when the surface is fully covered with metal ions and assists in the comparison of adsorption performance, particularly in cases where the sorbent did not reach full saturation in experiments. Another essential factor of the Langmuir isotherm is R L , which can be calculated according to the following equation: Where C 0 is the highest metal concentration (mg/l). The empirical Freundlich isotherm model based on a heterogeneous surface is given by: Where K f and n are Freundlich constants characteristic of the system, K f and n are indicators of adsorption capacity and intensity, respectively. Freundlich parameters can be determined from the linear form of the eq. (5) by plotting the lnq eq versus lnC eq the slope is the value of 1/n and the intercept is equal to lnK f . The Freundlich isotherm is also more widely used and provides information on the monolayer adsorption capacity, in contrast to the Langmuir model 16 . All data shown are the mean values of three replicate experiments, and error bars are indicated wherever necessary.

Characteristics of biosorbents
In this study the bacterial strains of Stenotrophomonas maltophilia and Bacillus subtilis were identified according to Bergey's Manual of Systematic Bacteriology 18 . Their biochemical and microscopic characteristics are given in Table 1. B. subtilis is a gram-positive aerobic rod-shaped bacterium ubiquitous in soils and waters, with a well--known parietal structure 19 . S. maltophilia is common in water and soil environments; many reports have indicated its potential wide application in biotechnology, including the biological control of plant pathogens, bioremediation and biosorption 20 .

Effect of pH
The effect of hydrogen ion concentrations on the biosorption of heavy metals has been the subject of many studies, which shows the importance of this parameter on the solubility of the metal ions as well as on the ionization of the fixing sites 5, 17, 21 . In this work, pH was varied in order to determine its optimum value for maximum biosorption of lead, zinc and nickel ions. It can be seen from Figure 1 and Figure 2 that the biosorptive capacity of Pb(II), Zn(II) and Ni(II) by the two bacterial strains was very low at a low pH value and it increased with pH until reaching an optimum between 5.0 and 6.0. At levels higher than 6.0 the heavy metals begin to precipitate. S. maltophilia demonstrated a maximum capacity (q e ) of 71.4 mg/g for Pb(II) and 29.8 mg/g for Zn(II) at pH 5.0 while B. subtilis demonstrated 78.8 and 30.0 mg/g respectively. The maximum biosorption of Ni(II) by S. maltophilia was 39.8 mg/g at pH 6.0, and 40.1 mg/g for B. acids feature carboxyl groups both of which contribute to the negative charge of the biomass and enable ion exchange. Gram-negative bacteria have a much thinner (only 1-3 molecules thick) PG layer which makes up about 10% of the weight of the total cell wall, which can be 30-80 nm thick. The PG layer of gram-negative bacteria does not contain TA or TUA, therefore they offer less negatively charged carboxyl groups, which is a reason for their lower biosorptive capacity 23 . On the other hand, a characteristic of these bacteria is an outer membrane which contains lipopolysaccharides (LPS) and phospholipids. Their phosphonate groups create a negative surface charge conducive to cation binding. As the pH increases, the competing effect of H 3 O + ions decreases. More functional groups such as carboxylic, phosphate and amino acid groups carrying negative charges are exposed 17, 24 . The degree of ionization of these negative groups also increases; leading to electrostatic attractions between the positively charged cations such as Pb(II), Zn(II) and Ni(II) and the negatively charged binding sites, thereby promoting the binding of heavy metals 9, 25 . This suggested that the biosorption of metals from wastewater is based on ion exchange. This fi nding also indicated that Pb(II), Zn(II) and Ni(II) removed was mainly bound to the cell walls and external surfaces of the biomass. Lead biosorption was maximal at pH 5.0, a value in agreement with results obtained by Veglió et al. 26  Maximal pH for zinc biosorption for the presented two bacterial species was 5.0. This is in agreement with Li et al. 30 and Chen et al. 16 , who also reported the optimum pH for Zn(II) removal by Pseudomonas putida CZ1 was 5.0. Other studies like Joo et al. 17 and Aston et al. 31 reported that maximal pH for zinc biosorption for Pseudomonas aeruginosa and Acidithiobacillus caldus BC13 were 6.0 and 4.0, respectively.
The maximal pH for Ni(II) biosorption for the presented bacterial biomass was 6.0. Pahlavanzadeh et al. 10 and Liu et al. 22 reported maximal removal of nickel in the pH range 5.0-6.0. In contrast, Gabr et al. 15 and Lopez et al. 32 found the maximal pH for nickel biosorption for Pseudomonas aeruginosa ASU 6a and P. fluorescens 4F39 were 7.0 and 8.0, respectively. A decrease in the removal of Ni(II) at a pH above 6.0 is due to the formation of Ni(OH) 2 . Substantial precipitation of nickel and nickel hydroxide occurs at high pH values. The formation of hydroxide precipitate reduces the amount of free nickel ions 13 .

Effect of contact duration
The kinetics of metal ion sorption is an important parameter for designing sorption systems and is required for selecting the optimum operating conditions for fullscale batch metal removal process 22 . The effect of contact duration on the extent of biosorption of Pb(II), Zn(II) and Ni(II) by bacterial biomass is shown in Figure 3 and   . The rate of Pb(II) biosorption by S. maltophilia and B. subtilis was very rapid, reaching almost 72.5% and 74.3% of the maximum adsorption capacity within 5 min of contact, respectively. However, it took longer for Zn(II) and Ni(II) to be adsorbed by S. maltophilia and B. subtilis, which reached approximate 67.4-73.5% and 63.9-78.9% of the maximum biosorption capacity within 20 min, respectively. The initial fast uptake was likely due to the high initial Pb(II), Zn(II) and Ni(II) concentration and empty metal binding sites on the microbes. The slower subsequent phase was likely due to the saturation of metal binding sites. Therefore, one can conclude that the appropriate equilibrium time for measurements was reached at 30 min. This represents the equilibrium time at which an equilibrium metal ion concentration is presumed to have been attained. This short time required for biosorption is in accordance with the results given by other authors 10, 15-17, 21, 30 . Gabr et al. 15 , Joo et al. 17 and Pahlavanzadeh et al. 10 showed that the maximum biosorption of lead, zinc and nickel was reached after 30-40 min. A rapid metal sorption is also highly desirable for successful deployment of biosorbents for practical applications 16 .

Biosorption isotherm
The biosorption isotherm curve represents the equilibrium distribution of metal ions between the aqueous and solid phases. The equilibrium distribution is important in determining the maximum biosorption capacity. Several isotherm models are available to describe this equilibrium distribution. Langmuir and Freundlich models are widely applied in equilibrium analysis to understand sorption mechanisms 9, 15-17, 21 . The Langmuir model considers sorption by monolayer type and supposes that all the active sites on the sorbent surface have the same affi nity for heavy metal ions 33 . The Freundlich isotherm is an empirical equation which assumes a heterogeneous biosorption system with different active sites 30 . The linearized Langmuir adsorption isotherms of each metal for S. maltophilia and B. subtilis are shown in Figures 5 and 6.   The values of q max , b and R 2 are given in Table 4. High coeffi cients of determination (R 2 = 0.990-0.998) for the Langmuir isotherm were obtained for all heavy metal biosorption with the bacterial isolates. A good fi t of the Langmuir model indicates that the biosorption of Pb(II), Zn(II) and Ni(II) could be characterized by a monolayer formation of metal ions on the surface of the biomass and belongs to a single type phenomenon with no interactions between sorbed metals. This result was consistent with a number of earlier studies focusing on the adsorption of lead, zinc and nickel ions 15, 16, 27, 34 . The Langmuir q max represents the saturation level of sorbed metal ions at high solution concentrations 30 . In the experiment of Pb(II) and Zn(II) biosorption, the q max values for S. maltophilia were respectively 133.3 mg/g and 47.8 mg/g, compared to 166.7 mg/g and 49.7 mg/g for B. subtilis. It was also found that the value of Ni(II) biosorption by B. subtilis was higher (57.8 mg/g) than that of S. maltophilia (54.3 mg/g). The data show that at a high concentration of metal ions in wastewater the B. subtilis biomass showed a higher level of saturation with Pb (II), Zn (II) and Ni (II) than S. maltophilia.
It is known that b is the constant related to the affinity of the binding sites, which allows us to make a comparison of the affi nity of the biomass for metal ions. As shown in Table 4 the affi nity of B. subtilis to Pb(II) and Ni(II) (0.019 and 0.047 l/mg, respectively) was higher than that of S. maltophilia (0.016 and 0.036 l/mg, respectively). However, the values obtained for Zn(II) indicate that S. maltophilia possesses a higher adsorption affi nity for Zn(II) as compared to B. subtilis. As reported by Li et al. 30 , the parameter indicates the shape of the isotherm and nature of the biosorption proces (R L > 1 unfavorable; R L = 1 linear; 0< R L <1 favorable; R L = 0 irreversible). The values shown in Table 4 indicate that the use of B. subtilis biomass was more favorable for the biosorption of Pb (II) and Ni (II), while S. maltophilia was more useful for the biosorption of Zn (II).
The Freundlich isotherm equation was originally empirical in nature, but it was later interpreted to be used in the case of sorption on heterogeneous surfaces or surfaces supporting sites of different affi nities. The linear plots of lnq eq versus for the two isolates are displayed in Figures 7 and 8.
The values of K f , n and R 2 are shown in Table 4. The Freundlich isotherm represents the amount of metals sorbed when the solution concentration in the equilibrium is unity 9, 15, 30 . In Table 4, the magnitude of K f shows a higher uptake of Pb(II), Zn(II) and Ni(II) using B. subtilis compared to S. maltophilia. The values of K f were found to be 52.   n which is related to the distribution of bonded ions on the sorbent surface, represents beneficial adsorption 35 . Larger values of n imply stronger interactions between the biosorbent and the heavy metals 30 . In this study, the n values for B. subtilis were 3.61-5.71 while those for S. maltophilia were 3.91-5.38, from which it could be derived that the effect of lead and nickel ions on B. subtilis was stronger than that on S. maltophilia biomass. Values of the correlation coeffi cient (R 2 = 0.789-0.938) are lower than the Langmuir model in the studied concentration range. Generally it can be stated that the sorption of lead, zinc and nickel by the analyzed bacterial strains depended on the initial concentration of the metal ions in wastewater. B. subtilis had high K f and q max values, indicating high sorption capacity, especially with regard to Pb(II), over the entire range of heavy metal ion concentrations in wastewater. The values obtained for S. maltophilia suggest its potential usefulness for the removal of Pb(II), Zn(II) and Ni(II) from wastewater containing low concentrations of these metals. Figure 9 shows the effi ciency of S. maltophilia and B. subtilis in removing of Pb(II), Zn(II) and Ni(II) from industrial wastewater (wastewater composition is shown in Table 2). Regardless of the type of used bacteria biomass, the effi ciency of Pb(II), Zn(II) and Ni(II) removal was over 96% when the concentration in the wastewater did not exceed, 39.6, 20.6 and 31.5 mg/l, respectively.
Effi ciency decreased with the increase of metal concentration in the wastewater, especially with regard to zinc and nickel ions. Only for lead ions did it remain above 80%, despite high concentrations of metals in the wastewater; 162.7, 104.3 and 159.4 mg/l, respectively for Pb(II), Zn(II) and Ni (II). In raw wastewater, removal effi ciency was less than 49% of lead ions, and less than 33% zinc and 25% nickel ions. The differences in the results obtained after the application of S. maltophilia and B. subtilis were around 10-12%. The high affi nity of living cells of genera Pseudomonas and Bacillus for Pb(II) is also confi rmed in other studies by this author 36 .
The descending order of selectivity of metal ions by the biomasses was Pb>Zn>Ni. This preferential type of adsorption may be ascribed to the difference in ionic radii and the electro-negativity of the metal ions. The ionic radius of Pb(II) is 1.20 A o , while that of Zn(II) and Ni(II) is 0.9 A o . The smaller the ionic radius, the greater its tendency to be hydrolyzed, leading to reduced biosorption. The electro-negativity of Pb(II) (2.33 Pauling) is greater than that of Ni(II) (1.91 Pauling) and Zn(II) (1.60 Pauling). Both the aforementioned factors contributed to the bacterial biomass had a greater affi nity for lead than for nickel or zinc 36 . A comparison between the results of this work for S. maltophilia and B. subtilis and other studies found in literature is presented in Table 5. Thus, the comparison of adsorption capacities shows that the studies species of bacteria were effi cient biosorbents of Pb(II), Ni(II) and Zn(II).

CONCLUSIONS
In this study, the live bacterial biomasses of S. maltophilia and B. subtilis were used as effective biosorbents of Pb(II), Zn(II) and Ni(II) from wastewaters. The biosorption performances were strongly affected by parameters such as pH, contact duration and heavy metal concentration. The optimum pH for the biosorption of Pb(II), Zn(II) and Ni(II) by the two bacterial species was achieved at pH 5.0-6.0 for 30 min. The uptake of metals was very fast. Adsorption equilibrium was reached