Ø Isolation,culturing and biomass preparation of Blue green algae (Oscillatoria): Micro algalsamples were collected from various ponds of Surat district. The material socollected was identified under microscope and serial dilution was carried outfor isolation and then obtained algae were added to various synthetic medium inorder to check which media was able to support the best growth of alga. Themedium with below mentioned constituents showed maximum growth and thus wasfurther used for culturing and multiplication.
After required growth wasobserved it was filtered through whatman filter no.1 and oven dried at 60°C andthe powder was further used for biosorption process.Ø Marinealgae collection and preparation of biomass: Fresh and maturethalii of Sargassum and Ulva sp.
were collected from Veravalcoast in Junagadh district along the west coast of Gujarat. Algal material wasbrought to laboratory in polythene bags. Then it was cleaned, washed withdistilled water to remove dust and impurities.
After washing it was sun driedfor 24 hours and then kept in oven at 60°C for further drying until it attainedconstant weight. After complete drying it was ground and sieved to powder ofequal size. The powdered material was stored in plastic, air tight bottles atroom temperature. This biomass powder was used for biosorption experiments. Ø Preparationof metal solution: Metal solution was prepared using nitrate salt of themetal.
Heavy metal salt of analytical grade viz Pb(NO3)2 wasused to prepare stock solution of leadnitrate having concentration of 1000mg/L. Solutions of varying concentrationswere produced through dilutions of stock solution until desired concentrationswere reached. Ø Biosorptionexperiment: Batch experiment was carried out in 250 mlErlenmeyer flask containing weighed biomass and 50 ml stock solution. The flaskwere sealed with cotton plugs and kept on rotary shaker at room temperature.The metal solution was agitated at a constant speed of 120 rpm per minute.After a desired time interval the biomass was separated by filtering thecontents using whatman filter paper no.1.
The amount of metal adsorbed on tothe biosorbent was calculated from the difference between metal ionconcentration in solution before and after biosorption process. Initial andfinal concentration of metal was measured on Atomic Adsorption Spectroscopy.Process parameters related to biosorption such as pH, time of contact,concentration of metal, biomass dosage, temperature were varied and theiruptake was recorded. i. Effect of pH: Batch process was carried out with series of 250 mlErlenmeyer flask containing 50 ml of metal solution and 100 mg of biomass.Experiment was carried out at different pH values ranging from 2.0 to 6.
0 bykeeping all other parameters constant. The pH of the metal solution wasadjusted to the desired value by adding either 0.1 N NaOH or Hcl. The sampleswere run for 120 min and then filtered to determine the concentration of metal.Optimum pH giving maximum metal removal was determined by analyzing the metalconcentration using AAS. All the experiments were carried out in duplicates. ii.
Effect of contact time: In order to understand the effect of time on the processof biosorption the agitation period was varied from 0 to 120 min using 100 mgbiomass in 50 ml of metal solution, adjusted to optimum pH at which maximumbiosorption of metal ion occurred. The samples were removed after every 10 mininterval and filtered and analyzed for metal concentration on AAS. iii. Effect of metal ion concentration: Batch experiments were carried out in 250 mlErlenmeyer flask containing 50ml of selected stock solution. The metalconcentration of solution was ranging from 10 to 70 mg/L.
The experiment was set up at optimum pH using100 mg biomass and run for optimum time as occurred. Later the samples werefiltered and analyzed for residual metal content on AAS. iv. Effect of Biomass dosage: The weight of biomass added in each experiment was variedfrom 0.
1 to 2.0 gm. The optimum concentration of the metal was used and it wasmaintained at optimum pH.
The experiment was carried for optimum time intervalas occurred and the samples were filtered and the filtrate was analyzed formetal concentration on AAS. v. Effect of temperature: The effect of temperature on the sorption of metal ionwas studied at temperature range from 20°Cto 45°C. The pH, biomassconcentration metal concentration and contact time were maintained constant byvarying the temperature. The supernatants were analyzed for Pb uptake usingAAS. All the components were conducted I duplicates. Ø Determinationof Pb+2 ions in the solution:Biosorption experiments were carried out in duplicatesand average values were used in the analysis. The removal efficiency wasdetermined by computing the % sorption using the formula:% Sorption = Where Ci = initial metal concentration Ce = final metalconcentrationAtomic adsorption spectroscopy (AAS) was usedto determine amount of heavy metal in aqueous solution before and after theequilibrium was established.
Metaluptake during biosorption experiment was determined using above mentionedpercentage sorption formula, in order to calculate equilibrium concentrationfollowing formula is used:Where qe represents amount of adsorption at equilibrium(mg/g), M is mass of biosorbent (mg/g), C0 and Ce are initial and final(equilibrium) concentration of metal ion (mg/L), V is the volume of metal (ml).Ø Continuousflow column experiment: The adsorption experiment was carriedout in column made up of glass. The column was equipped with a stopper forcontrolling column flow rate. To enable a uniform inlet flow of solution intothe column glass beads of 1.5mm diameter were packed to attain a height of 2cm.
A known quantity of biomass of Sargassum,Ulva and Oscillatoria were placed in respective column to yield desiredsorbent bed height of 10 cm, maintaining constant flow rate of 1.5ml/ min. Leadsolution of different concentrations (10, 20, 30, 40, 50) ppm with pH: 5.0 werefed upward inside the column to get desired flow rate. Lead solutions at theexit of the column were collected at different time interval and were analyzedon AAS.
Ø Characterisationof biosorbent before and after metal sorption :· SEM/EDX: SEM is used for studying surface morphology of materials.The changes in the surface microstructures of the biosorbent due to chemicalsurface modification and biosorption are studied using SEM.Dried biomass of three different algae namelySargassum, Ulva and Oscillatoriawere coated individually with thin layer of gold under vacuum and examinedunder scanning electron microscope. To determine chemical composition andinorganic elements present on biosorbents energy dispersive X- ray spectrometer(EDX) is used which is compatible with SEM. The samples were prepared as perSEM and the compositions present on biosorbent were determined before and afterthe process of biosorption. Both the analysis was performed simultaneously. · FTIR: It is a technique which is used to determine vibrationfrequency changes in functional groups present on the biosorbent surface. Inorder to study the functional groups present on the dried biomass of algae (Sargassum, Ulva and Oscillatoria)FTIR was carried out.
The samples were first mixed with KBr and then ground inan agate motar to prepare a mixture. Then the mixture was pressed at 10 tonnesfor 5 min to obtain pellets. The pellets were used to record spectrum on IRspectrum within the range of wave number 400-4000 cm-1. IR analysisof all the 3 algae dried biomass was done before and after the biosorptionprocess in present study.Ø Desorptionstudies: For desorption studies, the column containing11 gm of algae was contacted with 120 ml of Pb+2 metal solution ofknown concentration.
After biosorption experiment, the biomass was treated fordesorption. Then 120 ml of disodium EDTA solution of 0.1 N was allowed to passthrough the column. Then at 30 min time interval for 3 times the filtrate wastaken and were analysed to determine the concentration of Pb+2 afterdesorption.
Desorption efficiency was calculated from the ratiobetween the amount of metal ion adsorbed on biomass and the final metal ionconcentration in desorption medium as shown in following equation:Ø Modellingof heavy metal biosorption: Sorption Isotherm: Several models of isotherms have been used.In this context two different isotherm models (Langmuir and Freundlich) wereused in order to determine the biosorption of lead.The Langmuir model describes quantitatively the formation of amonolayer adsorbate on the outer surface of the adsorbent, and after that nofurther adsorption takes place. The model assumes uniform energies of adsorptiononto the surface and no transmigration of adsorbate in the plane of thesurface.
(Vermeulan et al.,1966). Based on these assumptions, Langmuir represented thefollowing equation:Langmuiradsorption parameters were determined by transforming the above equation intolinear form. WhereCe is the equilibrium concentration of adsorbate (mg/L), qe is the amount ofmetal adsorbed per gram of adsorbent at equilibrium (mg/g), Qo is maximummonolayer coverage capacity (mg/g), KL is Langmuir isotherm constant(l/mg). The Langmuir plots obtained by plotting 1/qe Vs 1/Ce are linear showingthe applicability of Langmuir adsorption isotherm for metal adsorption using adsorbentsselected for this study.
The isotherm feasibility can also be expressed as abest fit by linear equation as seen from correlation coefficient values (R2).The values of qmax and KL were computed from the slopeand intercept of the Langmuir plot of 1/qe and 1/Ce. (13)Eventhe separation factor (RL) which is a dimensionless constant is anessential factor of Langmuir isotherm. It can be expressed I following way(Weber and Chakrabarti, 1974). WhereCo = initial concentration, KL = constant related toenergy adsorption. RL values indicate the adsorptionnature and feasibility of isotherm: RL values Type ofisothermRL >1 Unfavourable RL=1 LinearRL<1 FavourableRL=0 Irreversible Freundlich model can be applied to a multilayer adsorption ona heterogenous surface along with interaction between adsorbed molecules. These data fit the empirical equationproposed by freundlich:WhereKf is freundlich isotherm constant (mg/g), n is adsorption intensity; Ce is theequilibrium concentration of adsorbate (mg/L), Qe is the amount of metaladsorbed per gram of the adsorbate at equilibrium (mg/g). The linearized formof freundlich equation is: Ø Kineticsof biosorption:In order to evaluatethe kinetic mechanism which controls the biosorption process, the pseudo-firstorder (Lagergren, 1898), pseudo-second order (Ho and McKay, 1998) are explainedin this research.
The study of adsorptionkinetics is significant as it provides valuable insights into the reactionpathways and into the mechanism of the reactions. The adsorption process isusually demonstrated by four steps:1. Transport of adsorbate from bulksolution to the liquid film or boundary layer surrounding the adsorbent.2.
Transport of adsorbate from theboundary film to the external surface of the adsorbent (surface diffusion).3. Transfer of the adsorbate from thesurface to the intraparticle active sites (pore diffusion).
4. Adsorption of metals by the activesites of adsorbent.The first step is notinvolved with adsorbent and fourth step is a very rapid process and they do notbelong to the rate controlling steps. Therefore, the rate controlling stepsmainly depend on step 2 and step 3 either surface diffusion or pore diffusion(Theydan and Ahmed, 2012). Kinetic models are often used to explain the process ofmetal biosorption. They describe the solute uptake rate and this rate controlsthe residence time of adsorbate uptake at the solid solution interfaceincluding the diffusion process. The kinetic parameters which determinesadsorption rate, gives information for designing and modelling adsorptionprocess. This adsorption mechanism depends on the physical and chemicalcharacterization of the adsorbent as well as the mass transfer process.
In thepresent study two kinetic based models were applied to the experimental data tostudy the best fit. Ø Pseudofirst order:This model states that occupation ofadsorption site is proportional to number of unoccupied sites. It is alsocalled Lagergen’s pseudo first order and its equation is written as give (Hoand McKay, 1998). Whereqe (mg/g) is amount of solute adsorbed at equilibrium per weight of adsorbent,qt (mg/g) is amount adsorbed at any time and k1 is adsorptionconstant.Theintegrated form of above equation:Whereqe and qt are amount of adsorption (mg/g) at equilibrium and time t (min), k1rate constant of pseudo first order adsorption process. Theplot of Vs t should give a linearrelationship from which k1 and qe can be determined from the slopeand intercept of the plot.
Ø Pseudosecond order: Thismodel is proposed by Ho and McKay. The model assumes that the rate ofoccupation of adsorption sites is proportional to square of number ofunoccupied sites. The pseudo second order rate equation iswritten as:Thelinear form of pseudo second order rate equation is written as:Whereqe and qt (mg/g) are amount of metal that was adsorbed at equilibrium and attime t (min) respectively, K2 is pseudo second order rate constantof adsorption (g mg-1min-1).
The values of rateparameters K2 and qe can be directly obtained from intercept andslope of the plot t/qt Vs t.