BIOSORPTION OF COPPER AND LEAD USING BACTERIAL BIOMASS OF BACILLUS CEREUS AND BACILLUS SUBTILIS ISOLATED FROM EL-MANZALA LAKE, EGYPT

Shawky Z. Sabae. Two tolerant bacterial strains isolated from El-Manzala Lake, Egypt and identified using biochemical tests and confirmed by 16S r RNA gene as Bacillus cereus and Bacillus subtilis.Then the optimum conditions for biosorption of copper and lead were investigated by using two bacterial strain, the equilibrium time for copper were 25 minutes at Bacillus cereusand 30 minutes at Bacillus subtilis while the equilibrium time for lead were 40 minutes at Bacillus cereusand 50 minutes at Bacillus subtilis, the optimum pH for copper and lead biosorption at Bacillus cereus and Bacillus subtilis was pH 6. The experimental biosorption data mostly were fitted towards the models postulated by Langmuir and Freundlich isotherm equations. The maximum biosorption capacities (q max) for copper and lead obtained by usingBacillus cereuswere 47.6 and 250 mg/g while by usingBacillus subtiliswere 166.7 and 250 mg/g , respectively. Biosorpitve mechanism was confirmed by IR analysis and from the identification nature of acidic and basic sites. Moreover, the postulated mechanism was depended mainly on ionic interaction and complex formation. The results demonstrated that the two bacterial isolates of Bacillus cereus and Bacillus subtilis could be used as a promising biosorbents for the removal of copper and lead ions from aqueous solutions.

Two tolerant bacterial strains isolated from El-Manzala Lake, Egypt and identified using biochemical tests and confirmed by 16S r RNA gene as Bacillus cereus and Bacillus subtilis.Then the optimum conditions for biosorption of copper and lead were investigated by using two bacterial strain, the equilibrium time for copper were 25 minutes at Bacillus cereusand 30 minutes at Bacillus subtilis while the equilibrium time for lead were 40 minutes at Bacillus cereusand 50 minutes at Bacillus subtilis, the optimum pH for copper and lead biosorption at Bacillus cereus and Bacillus subtilis was pH 6. The experimental biosorption data mostly were fitted towards the models postulated by Langmuir and Freundlich isotherm equations. The maximum biosorption capacities (q max ) for copper and lead obtained by usingBacillus cereuswere 47.6 and 250 mg/g while by usingBacillus subtiliswere 166.7 and 250 mg/g , respectively. Biosorpitve mechanism was confirmed by IR analysis and from the identification nature of acidic and basic sites. Moreover, the postulated mechanism was depended mainly on ionic interaction and complex formation. The results demonstrated that the two bacterial isolates of Bacillus cereus and Bacillus subtilis could be used as a promising biosorbents for the removal of copper and lead ions from aqueous solutions. As potentially harmful and non-biodegradable pollutants that may accumulate through the food chain, heavy metals can threaten ecosystem and human health (Liu et al., 2013).
Conventional methods for removing metals from aqueous solutions include chemical precipitation, chemical oxidation or reduction, ion exchange, filtration, electrochemical treatment, 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 of 1-100 mg/l, the production of toxic chemical sludge and its disposal/treatment becomes a costly affair and is not ecofriendly (Nourbakhshet al., 1994).
Therefore, removal of toxic heavy metals to an environmentally safe level in a cost effective and environment friendly manner was of great importance. Biological treatment, based on living or nonliving microorganisms or plants, offers the reduction of toxic metal levels to environmentally acceptable limits in a cost-effective and environmentally friendly manner (Volesky, 1994).
]Biosorption has emerged as an alternative solution for the removal of toxic metals from water/wastewater. It shows superiorities in low cost, high efficiency, wide adaptability, no secondary pollution, and stable performance especially for low metal concentration effluents (Wang and Chen, 2009).
Nowadays, the use of microbial approaches for heavy metal removal has received much attention .Bacteria, algae, fungi, and yeasts constitute a wide range of biosorbents with different adsorption capacities. These capacities depend on the cell wall structure and the affinity of surface ligands to specific metal ions. Different parameters such as tendency toward the metal ions, the maximum sorption capacity, as well as the rate of the metal sorption on the surface of the biosorbents are the major criteria for comparing and choosing the best type of biosorbents for specific purposes. Using the equilibrium isotherms and kinetic studies are common for the calculation of these parameters. (Jooet al., 2010).
The bacteria make excellent biosorbents because of their high surface-to-volume ratios and a high content of potentially active chemo sorption sites such as on teichoic acid in their cell walls (Beveridge, 1989).
The main objective of this work is to study biosorption processes of copper Cu (II) and lead Pb (II) used Bacillus cereus and Bacillus subtilis. The conditions of these processes were optimized by selection of both the pH and time postulated at room temperature. Also study the biosorption isotherm and the mechanism of the process was discussed.

Materials and Methods:-
Isolation of heavy metal resistant bacteria:-Heavy metal resistant bacteria were isolated from heavy metal polluted water samples collected from El-Manzala Lake, Egypt , In order to minimize the complexation of heavy metals, the isolates were grown in Tris minimal medium (Tris-HCl-100 (pH-7.2), Glucose-11, NH4Cl2-4 , MgCl2-10, CaCl2-0.1, KH2PO4-0.1, in millimoles per liter of deionized water and Agar-15g/L) (Mergeay, 1995). For isolation of bacteria by agar dilution method, plates were inoculated with 200 µl from sample by spread technique on Tris minimal medium supplemented with different concentration of heavy metals from Cu (II) and Pb (II), one metal at a time. Plates were incubated for 72 hrs at 37°C.After the incubation, the plates were examined and the bacterial isolates were picked and transferred to agar slants and then purified by streaking several times on nutrient agar media, until pure single colonies were obtained. Isolates were maintained in slope culture and stored at 4 o C for further studies.
The chemicals used for this study were of analytical grade and they were supplied by Sigma Aldrich (Sigma Aldrich, St.louis, Mo) .The heavy metals were sterilized by filtration method. The solution metals pass through a membrane filter (pore size 0.45 μg). for rapid analysis and providing phylogenetically meaningful information. To determine the 16S rRNA gene sequence of the strain, cells were lysed according to Hiraishiet al. The 16S rDNA fragment was amplified by PCR using the following universal primers: forward, 59-AGAGTTT-GATCATG GCTCGA -39 ; and reverse, 59-GGCTACC-TTGTTACGACTT-39 (positions 1510 -1492). The sequence of the amplified 16S rDNA fragment was analyzed using gene bank and compared with the National Center for Biotechnology Information (NCBI) database.

Identification of the metal resistant bacterial isolates:-
Biosorbents preparation:-Nutrient medium was prepared and sterilized. A loop full of bacterial culture was taken and streaked on the agar plate to obtain more colonies. They are later transferred to nutrient broth for subculture. 100 ml of sterilized culture media was transferred to 250 ml Erlenmeyer flask. The media was allowed to cool and then the 100µ microbial solution was inoculated into the medium in laminar air flow chamber.
The inoculated flasks were incubated in an orbital shaker at 250 rpm at 32 0 C for 2 days to obtain the biomass. Biomass was harvested from the medium by centrifugation at 9000 rpm for 10 min. The supernatant was discarded and the cells pellet was rinsed three times with sterilized water to make sure that no media remain on the cell surface. Then using a lyophilizer to freeze and dry bacterial biomass which was used for the sorption experiments. (

Data evaluation:-
The effect of contact time, initial concentration and pH on metal adsorption was calculated using the following equation: Amount of metal adsorbed by bacterial biomass was calculated from the differences between the metal quantity added to the biomass and the metal content of the supernatant.
The specific metal biosorption q was calculated using the following equation: Where q e is the specific metal biosorption (mg metal / g biomass), C i and C e are the initial and equilibrium concentrations of metal (mg metal /l) respectively, V the volume of metal solution (l) and the M is the dry weight of biomass (g) in grams.
Mathematical formula for Freundlich model can be expressed as: Where K f and n are the distribution coefficient and a correction factor, respectively .By plotting the linear form of Eq. (1) ln q = 1/n ln C e + ln K f The slope is the value of 1/n and the intercept is equal toln K f .
And mathematical formula for Langmuir model can be expressed as And it's linear form is represented by the following equation: Where q max is the Langmuir constant (mg/g) reflecting the maximum adsorption capacity of the metal ion per unit weight of biomass to form a complete monolayer on the surface bound at high C eq . The value of Langmuir constantb (l/mg) represents a ratio of adsorption rate constant to desorption rate constant, which also gives an indication of the affinity of the metal for binding sites on the biosorbents.q max andb can be determined from the linear form of Langmuir equation (3) by plotting vs. .

FT-IR analysis:-
Samples were analyzed using Fourier transform Infrared (FT-IR) spectroscopy to give a qualitative and preliminary characterization of the main functional chemical groups present on the bacterial biomass, which are responsible for heavy metal biosorption. A raw sample of bacterial biomass and biomass loaded with different heavy metals were analyzed using FT-IR (Perkin Elmer, FT-IR system, Spectrum BX) adopting KBr disk technique.

Results and discussion:-
Characteristics of biosorbents:-Bacillus cereus and Bacillus subtilis used in this study were previously isolated from polluted water samples collected from El-Manzala Lake, Egypt. Then two bacterial strains were characterized by microscopic examination and biochemical tests as well as identified by a matrix of API 20E strip and the API 50 CHB strip (bioMérieux, France).
Identifications were confirmed by16S r RNA gene sequencing ,nucleotide sequence coding for 16SrDNA gene has been submitted to GenBank and the strains is closely related to Bacillus cereus and Bacillus subtilis (with similarities of 98% and 98% respectively) as shown in figures (1,2) phylogenetic tree based on 16S rRNA gene sequences .    Metal uptake by the biomass increases with increasing pH till it reaches a maximum after which the metal uptake decreases. The optimal pH values for Cu (II) and Pb (II) by Bacillus cereus and Bacillus subtilis adsorption were pH 6 , these results suggest that the adsorption of metals on the biomass surface is controlled by ionic attraction. At low pH values, the inactivated cell surface becomes more positively charged, leading to reduce the attraction between metal ions and functional groups at the cell wall. In contrast, when the pH increases, the cell surface is more negatively charged and the process of retention is favored (Pardoet al., 2003; Volesky and Holan, 1995) until a maximum is reached around pH 6 However for values of pH higher than the optimum, the formation of hydroxylated complexes of the metal will also compete with the active sites and as a consequence, the retention will decrease again. Copper and Lead biosorption is maximized at pH 6 ,this is in agreement with the results obtained by Pardoet al. (2003), who found that the maximum pH for lead by P. putida is pH 6. Moreover, Seki et al. (1998) studied the function of pH on biosorption of lead by Rhodobacter sphaeroides and reported that the maximum pH is around 6, the variation in biosorption of heavy metals by microbial biomass at different pH could be due to the differences in the sensitivity of cell wall molecules of the bacterial cells to pH. For instance, at a low pH, cell wall ligands tightly bind with the hydronium ions H3O_ and hence restrict the approach of metal cations due to repulsive force. On the contrary, at higher pH values, more ligands like carboxyl, phosphate, imidazole and amino group would be exposed and carry negative charges with a subsequent attraction of metallic ions with positive charge and biosorption onto the cell surface (Pardoet al., 2003).

Effect of initial metal concentration on biosorption:-
The effect of initial metal concentration on metal biosorption by dry biomass of B.cereus and B.subtiliswere evaluated as shown in Figures (7,8)that's indicate the rate of biosorption decreased with an increase in metal ion concentration. The maximum biosorption percentage of metal was recovered at a low initial metal ion concentration; the decrease in the percentage of biosorption may be attributed to the lack of sufficient free sites for metal biosorption. At lower concentrations, all metal ions present in the solution however, could interact with the binding sites and thus the biosorption percentage is likely to become higher than that at higher ion concentrations as found in this study .At higher concentrations, a lower adsorption yield is due to the saturation of adsorption sites. Similar  Biosorption isotherm:-The biosorption isotherm models described the biosorption data at equilibrium and showed the correlation between the mass of solute adsorbed per unit mass of sorbent at equilibrium .The biosorption isotherms were calculated using two different isotherms models including the Langmuir, and Freundlich, Figures (9, 10, 11 and 12)The equilibrium adsorption isotherm obtained showed that metal uptake by bacterial biomass was a chemically equilibrated and saturabled mechanism. Thus, there was an increase in metal uptake as long as binding sites were free. Values of Freundlich and Langmuir parameters are calculated and listed in Table (1), These data showed that the q max values obtained for lead uptake using lyophilized biomass of Bacillus cereus and Bacillus subtilis were 250 mg metal/g biomass, which were higher than those obtained for copper : 47.6 ,166.7 mg metal/g biomass respectively. However, the b values obtained were found to be 0.003, 0.004respectively, in the case of lead biosorption, where copper biosorption b values were recorded 0.018, 0.002respectively, which indicate that Bacillus cereus possesses a high adsorption affinity for copper as compared to that for lead, in contrast of Bacillus subtilis which possesses a high adsorption affinity for lead as compared to that for copper .The values of Freundlich parameters show that the adsorption capacity K f for copper usingBacillus cereus and Bacillus subtilis were 10.01 and 0.88 mg metal/g biomass, respectively, where The values obtained for lead, 3.59, 2.83, mg metal/g biomass respectively, However, the small K f values for copper ions atBacillus subtilisindicate a lower extent sorption, while more sorption was observed for copper ions atBacillus cereus because of their larger K f values .that's also observed in lead adsorption by the two bacterial strains .Here it is worth mentioning that the correlation coefficients for all the copper and lead by Bacillus cereus were found to be 0.948 and 0.986 respectively ,while the correlation coefficients for copper and lead by Bacillus subtilis were found to be 0.980 and 0.922 respectively , In general, these data indicated that the sorption capacity increased with increasing the initial metal ion concentration for both metals on the biomass surface for the two organisms . This sorption characteristic indicates that the surface saturation is dependent on the initial metal-ion concentrations. At low concentrations, adsorption sites took up the available metal more quickly. However, at higher concentrations, metals need to diffuse into the biomass surface by intra particular diffusion and greatly hydrolyzed ions will diffuse at a slower rate (Gaberet al., 2008, Sang et al., 2009).The overall observation from bothLangmuir and Freundlich isotherms for metals biosorption were in agreement with many previous studies (Pardo et al., 2003, M. Acosta et al., 2005,Saret al., 1999).
This preferential type of adsorption may be ascribed to the difference in their ionic radii (Gaberet al., 2008 )

FTIR Analysis:-
It is essential to identify the functional groups on the biomass involved in adsorbing process with FTIR, which is helpful to understand the surface-binding mechanism. FTIR spectrum in the absence and presence of metal revealed the changes in the peaks of functional groups at bacterial biomass, for example FTIR spectrum for Bacillus cereus biomass before and after adsorption of copper were occurred , Figure (13) showed FTIR spectrum in the absence and presence of Cu (II) which revealed the changes in the peaks of functional groups , such as a shift from 3402 cm -1 to 3435 cm -1 indicating hydroxyl O-H stretch, H-bonded , also the absorption bands characterizing alkyl chains and CHO have abroad band within the range 2932-2924 cm -1 , c=o of amide group at 1638 -1649 cm -1 ,COOof the carboxylate groups appeared at 1406-1404 cm -1 , That vibrations from1077 cm -1 (before Cu (II) adsorption) to 1110 cm -1 ( after Cu (II) adsorption) could be caused by C-N stretch, The peak at 555 cm cm -1 shifted to 617 cmafter Cu (II) adsorption could be assigned to the stretching of C-O (carboxyl).The overall FTIR spectra analysis implied that the functional groups like hydroxyl, carbonyl and carboxyl may be involved in Cu (II) adsorption. Therefore, infrared spectra of B. cereusbiomass showed the presence of amine R-NH2 (amino acids, proteins, glycoproteins, etc.), carboxylic acid (fatty acids, lipopolysaccharides, etc.), hydroxyls, and phosphates .In general the transmittance of the peaks in the loaded biomass is substantially lower than those in the raw sample of the bacterial biomass , this indicated that bond stretching occurs to a lesser degree due to the presence of metals and following peak transmittance is reduced ,these result s are in agreement with

Conclusions:-
The present work was designed to investigate the biosorption behavior of Cu (II) and Pb (II) to the gram positive bacteria Bacillus cereus and Bacillus subtilis. The optimum pH for copper biosorption is 6 , at temperature 30ᵒ C ,equilibrium time 25 minutes for B. cereus, and 30 minutes for B. subtilis, while the optimum pH at Bacillus cereus and Bacillus subtilis for lead biosorption is 6 , at temperature 30ᵒ C ,equilibrium time 40 minutes forB. cereus, and 50 minutes for B. subtilis. The maximum biosorption capacities for Bacillus cereus and Bacillus subtilis were 250 mg metal/g biomass for Pb at optimum operating conditions, while for Cu were 47.6, 166.7 mg metal/g biomass respectively. The experimental data revealed that Cu and Pb biosorption mostly were fitted to both Freundlich and Langmuir isotherms.
The mechanism of biosorption includes mainly ionic Interactions and formation of complexes between metal cations and acidic sites in the cell wall of bacterium, and this was confirmed by IR and pH experiments.IR spectroscopy result shows that the rod-shaped B.cereuscell mainly contains carboxyl, hydroxyl, phosphate, amino, and amide functional groups. Based on these results Bacillus cereus and Bacillus subtilis biomass can be used as an efficient low cost biomass for the removal of heavy metals from wastewater. Finally the results demonstrate that bacterial isolates of Bacillus cereus and Bacillus subtiliscould be used as a promising biosorbents for the removal of copper and lead ions from aqueous solutions.