Removing Lead from Iranian Industrial Wastewater

. Metals and chemicals have been increased in industrial processes which they contain a high level of toxic heavy metals and cause a lot of disadvantages for the environment and human health .Biosorption of Pb (П) ions has been studied from aqueous solutions in a batch system by using a bacterial strain isolated from petrochemical wastewaters. Strain 8-I was selected to study the impact of different factors on removal rate. According to morphological, physiological and biochemical characterizations of the strain and in comparison with other studies the strain was tentatively identified as Bacillus sp strain 8-I. The maximum Lead biosorption capacity of 8-I isolate was determined to be 41.58 % at pH 4.0 with 80 mg/l concentration in 48 hours equilibrium time. The comparison between the biosorption capacity of live (45.50 mg/g), heat inactivated (30.23 mg/g) and NaN 3 pretreated biomass (26.86 mg/g) were indicated that the ability of live biomass for both of active and passive uptake of lead.


INTRODUCTION
Increased use of metals and chemicals in the industrial process has resulted in the generation of large quantities of effluent that contain the high level of toxic heavy metals and their presence poses environmental-disposal problems due to their non-degradable and persistence nature [1]. Furthermore, their accumulation throughout the food chain leads to serious ecological and health problems. Lead is extremely toxic and can damage the nervous system, kidneys, and reproductive system, particularly in children. Lead can contaminate the environment from anthropogenic sources as well as natural geochemical processes [2]. The severe toxic effects imposed by heavy metals on living tissues and environment directed the research at investigating alternative technologies for wastewater purification systems. Conventional separation techniques applied to the treatment of industrial effluents include chemical precipitation, chemical oxidation or reduction, filtration, ion exchange and electrochemical processes [3,4]. However, technical and economic constraints encountered in the application of these traditional methods have directed attention to the search for new technologies involving metal removal from waste streams [5,6]. As a result of development in the field of environmental microbiology, recent studies have focused on the use of microbial-based potential bio-sorbents such as yeast, bacteria and fungi [7]. Dead as well as living cells are used in the removal of metal ions [8]. This interaction includes both bioaccumulation and biosorption. Biosorption is a term that describes the removal of heavy metals by the passive binding to non-living biomass from an aqueous solution. This implies that the removal mechanism is not metabolically controlled. In contrast, the term bioaccumulation describes an active process whereby removal of metals requires the metabolic activity of a living organism [9,10].
The biosorption process includes physicochemical interactions between metal ions and several anionic ligands present on the biomass like carboxyl, phosphoryl, carbonyl and sulfhydryl. The efficiency of this process depends on various factors varying from the type of metal ion being studied to the type of biosorbent material used as well as pH [7]. The objective of this study was to isolate microbial strains from petrochemical waste water which contaminated with lead ions, study tolerance ability of Pb (П) of different bacterial isolates, identify and study the biosorption potential of the appropriate strains. The metal loading capacity of bacterial biomass was determined as a function of initial pH, contact time and initial metal ion concentration and the ability of selected isolate for active uptake was investigated [11][12][13][14][15]. Biosorption of Pb (П) ions has been studied from aqueous solutions in a batch system by using a bacterial strain isolated from petrochemical wastewaters.

Isolation and screening of microorganism
Three different wastewater samples from one of Iranian petrochemical industry were collected. Each sample was placed in sterilized tubes and 5 ml of each sample centrifuged (3500 rpm) for 20 min, then supernatant and pellet inoculated to 10ml nutrient broth and after 5 days growth in 37 °C, then 10 µl of solution inoculated to nutrient agar medium and finally 99 bacterial isolates by using of nutrient agar (pH adjusted in 7) were obtained. The agar dilution method was used to determine the tolerance of bacterial isolates. Examination of these isolates for Pb 2+ tolerance were carried out using PYT agar medium (it contained 10 gr peptone, 2 gr yeast extract, 0.8 gr tryptone, 15 gr agar per liter and MES buffer 10 mM, pH was adjusted to 7) supplemented with different concentrations of Pb (NO 3 ) 2 (4,5,6,6.5,7,8,10,12 and 15 mM). Volumes of 20 ml of this medium was poured into 8 cm plates. Then 10µL of bacterial suspensions (1.5 × 10 8 CFU ml -1 ) were inoculated on each plate and incubated at 37 °C for 48 h. Bacterial isolates were able to grow in a high concentration of Pb (NO 3 ) 2 and were selected (one isolate from each group) [16,17].

Estimation of Lead uptake by tolerant isolates
To 20 mL of PYT-broth medium supplemented with 80 mg/l of Lead, 200µL of bacterial suspension (1.5 × 10 8 CFU/ml) from each of tolerant isolate, was inoculated and incubated at 34 °C and 150 rpm for 48 h and 96 h. Additionally, a control without bacterial isolate was supplied. At the end of incubation, bacterial cultures were centrifuged at 5000 rpm for 20 min, and the supernatant was analyzed by AAS to determine residual Pb (П) concentrations [18,19].

Characterization of tolerant isolates
Microscopic and biochemical tests were applied to effective bacterial isolate (strain 8-I) according to Bergey's Manual of Systematic Bacteriology. The genus to which this isolate belongs was detected [20].

Preparation of wet biomass
PYT-broth medium containing no Pb ions was used for the production of isolated 8-I biomass. After 48h incubation at 37˚C and 150 rpm, viable biomass was harvested by centrifugation at 5000 rpm for 20 minutes at 4 °C and washed twice with deionized water. The experiments were carried out with wet biomass of the selected strain, but results were calculated using a dry biomass basis. According to Volesky, for scientific interpretations, the sorbent material dry-weight basis is preferred [18].

Preparation of dry cells
Washed biomass (wet) from a measured amount of whole-cell broth was placed in a previously weighed Glass cup and dried in an oven at 80 °C overnight. It was weighed again, the weight of the dry cell mass was calculated by finding the difference [18].

Bio-sorption experiments
The batch bio-sorption experiments were carried out with 20 g wet cells l−1 (3g/l dry weight) of bio-sorbent at 34 °C to elucidate the optimum operating conditions which enhance the Pb (П) uptakes. The effect of pH on the biosorption capacity of the bacterial biomass for Pb (П) was investigated in the pH range of 2.0-6.0 by using solutions containing 100 mg l −1 of metal ions. The solutions were adjusted to required pH values using 1N HCl and 1N NaOH. Biomass of selected strain was added (2%) to medium and the reaction mixture was shaken at 150 rpm and was incubated at 34˚C for 90 min. Experimental control tests were also carried out in the absence of the biosorbent. Similarly, metal solutions with varying concentrations of Pb ion (ranging from 50 to 200 mg l −1 ) were used to assess the effect of initial metal ion concentration. In these experiments, the pH of the Pb solutions was adjusted to 4.0 (optimum pH value) [7,18]. The effect of contact time on the bio-sorption was investigated in the time range of 30-360 min with metal solutions containing 150 mg/l of Pb (П) ions and pH was adjusted to 4.0. These samples were incubated at 34˚C and shaken at 150 rpm. For this purpose 10ml of samples were taken from the bio-sorption media at different time intervals (30, 60, 120, 240, 360 min) and tested for residual lead-ion concentration [7,8,18].

Metal analysis
The concentrations of unabsorbed Pb (П) in solutions were determined after the separation of biosorbent by centrifugation at 5000 rpm for 20 min using flame atomic absorption spectrophotometer.
All the experiments were carried out in duplicate and the arithmetical average values were used in calculations. The amount of adsorbed metal ions [2] per gram biomass was calculated using the general definition (Eq. 1): Where q is the metal uptake (mg metal per g bio-sorbent), V the liquid sample volume (l), C i the initial concentration of the metal in the solution (mg/L), C f the final (equilibrium) concentration of the metal in the solution (mg/L) and M the amount of the added bio-sorbent on the dry basis (g) [7,21]. The quantity of heavy metal ion uptake was determined by comparing the amount of heavy metal ion sequestered by the biosorbent (q from Eq. 1) with the initial concentration of heavy metals in the refinery effluent before bio-sorption studies. The percentage was calculated as follows (Eq. 2): where Q is the adsorption efficiency (%), q max is the maximum uptake capacity (mg/g dry wt) and Co is the concentration of the metal ions before bio-sorption (mg/l) [22].

Pretreatment experiments
An amount of live biomass (0.5 g wet weight) was subjected to pretreatment with two different methods as listed below in an effort to study the effect of pretreatment on Pb (П) uptake capacity of isolated 8-I biomass: autoclaved for 15 min at 121±2 C, biomass was added to 0.4 g/l solution of sodium azide (NaN 3 ) for 210 min and was shaken at 150 rpm. After 210 min, biomass was collected by centrifugation (5000 rpm, 20min). The subsequent bio-sorption experiments using pretreated biomass were performed in initial Pb (П) ion concentration of 150 mg/l under the optimum pH of 4.0 and contact time of 240 min [21].

RESULTS
A total 78 Gram-positive and 18 Gram-negative bacterial cultures were recovered from the petrochemical waste water. The results of tolerance experiments showed that 63, 29, 2, 3, 2 and 1 percent of strains were resistant in order to less than 4 , 4, 5, 6, 6.5 and 15 mM of Pb (NO 3 ) 2 (        (Table 5), but percent adsorption increased first with increasing in concentration (range 50-150 mg/L), then decreased to a small extent (range 150-200 mg/l) ( Table 5) due to rapid saturation of the metal binding sites of the biosorbent. The observed enhancement of metal uptake could be due to an increase in electrostatic interactions (relative to covalent interactions) which involve sites of progressively lower affinity for metal ions [18,21]. Effect of time Figure 1 shows the effect of time course profiles for the adsorption of Pb (П) by biomass of Bacillus sp. 8-I. The figure showed that rapid sorption rate (35 mg/g) was observed within the first 30 min, then Pb (П) sorption increased with contact time and reached equilibrium at about 240 min (48.25 mg/g).

International Letters of Natural Sciences Vol. 57
In this study, long contact time of 240 min was required for equilibrium and this can be related to active uptake of Pb that need longer time (Figure 1) of metal loading capacity for Pb (П) which it was found as 48.25 Pb (П) mg/g biomass at the initial concentration of 150 mg/l with pH 4.0 after 240 min.

Effect of biomass pretreatments on Pb sorption
The subsequent bio-sorption experiments using pretreated biomass were performed in initial Pb (П) ion concentration of 150 mg/l under the optimum pH of 4.0 and contact time of 240 min. It has been determined that the amount of Pb (П) removal by the living biomass was higher than the amount removed by the dead biomass (Table 6). Significant change was not observed between the biosorption capacity of autoclaved biomass and NaN 3 pretreated biomass which was 30.23 mg/g and 26.86 mg/g, respectively. Regard to the results of experiments, equilibrium was achieved after 4 hours of contact time and Pb (П) uptake capacity of inactivated biomass was lesser than active biomass, thus the ability of live biomass was demonstrated for both of active and passive uptake. The obtained results showed that is higher than many of corresponding bio-sorbents reported in the literature (Table 7).

DISCUSSION
Both Gram-positive and Gram-negative bacteria are ubiquitous in nature with highly resilient cell walls that are anionic. These anionic cell walls can fix metals, and provide sites for nucleation and growth of minerals. This property has been the basis for many heavy metal removal studies using bacterial biomass [9]. The walls of gram-positive bacteria are efficient metal chelators. Bacillus sp. incubation conditions [10]. The present results showed a high incidence of Pb (П) resistance as compared to other research [10,16,17,[36][37][38][39]. Bacterial cell metabolic process involved in the uptake of Pb (П) heavy metal into the cells. Biosorption results of Pb (П) reported by the other researchers in the literature by various bio-sorbents and operating conditions are summarized in Table 3. This rapid initial uptake is similar to the previous reports on the biosorption of heavy metals by different biosorbents [7,10]. The uptake values obtained in this study are comparable and were found to be higher than that of many corresponding biosorbents. We selected this isolate to study the effect of different factors on removal rate. The most effective strain was identified according to Bergey's Manual of Systematic Bacteriology [20]. Earlier studies on heavy metal biosorption showed that pH was an important factor affecting the biosorption process [7]. The interaction of metal cations with the electron-rich functional groups located on the biomass may be strongly sensitive to the pH value of the environment. In this study, biosorption was not observed at pH 2.0, the greatest uptake capacity was obtained at pH 3.0 and 4.0, with an increase of pH (4-6), bio-sorption decreased but generally, there was no significant difference in Pb uptake capacity. The pH of the adsorption medium affects the solubility of metal ions and the ionization state of the functional groups on the cell wall of biosorbent. Metal biosorption depends on the protonation or deprotonation of the functional groups on the cell wall. Because of high proton concentrations at extremely acidic conditions, cell wall functional groups are closely associated with the protons and it restricts the approach of metal ions as a result of increasing positive charge density of binding sites. Indeed, metal cations and protons compete for binding sites on the cell wall, which results in a lower metal uptake [7,17]. Metal loading capacity greater than 15% of biomass [2] could be used as an economic threshold for practical applications of bio-sorption as compared with alternative techniques [22]. The quantity of Q was autoclaved for living and NaN 3 pretreated biomass was 32.16, 21.13 and 18.87%. Thus, the both of active and inactivated biomass of Bacillus sp. 8-I can be used for removal of lead from industrial waste waters.
In this study, about 62% of Pb ions uptake was related to bio-sorption process and 35 mg/g of uptake was observed in 30min of contact time. This rapid initial uptake of metals may be an important parameter for a practical application of bio-sorption in industrial wastewater treatment.

CONCLUSION
A total 99 bacterial strains were isolated from Iranian petrochemical waste waters. Strain 8-I had the highest resistance ability. The bio-sorption properties of Bacillus sp. (8-I) were studied for Pb ions in the present work. This result indicated that this strain may be used as an inexpensive, selective, effective and easily cultivable bio-sorbent for the removal of Pb (П) ions from aqueous solutions. The bio-sorption process of this metal ions on Bacillus sp. (8-I) was found which can be dependent on experimental conditions such as the initial pH, initial metal ion concentration and contact time.
Bacillus sp. (8-I) can remove Pb from an aqueous solution through both bioaccumulation by living cells, and bio-sorption by dead cells.