Project Presentationon
ByASHA.S.HEGDE (1DS07BT010) DIVYA.G.KAMBI (1DS07BT014)KAVYA.S (1DS07BT021)
VIII Semester, B.E Biotechnology
DAYANANDA SAGAR COLLEGE OF ENGINEERINGDAYANANDA SAGAR COLLEGE OF ENGINEERINGSM. Hills, KS Layout, Bengaluru -560 078
Under the guidance of
Nagamani. S. K, (Guide)Lecturer, Department of BiotechnologyDayananda Sagar College of EngineeringSM. Hills, KS Layout, Bengaluru -560 078
Shwetha. N, (Co-Guide)Lecturer, Department of BiotechnologyDayananda Sagar College of EngineeringSM. Hills, KS Layout, Bengaluru -560 078
OBJECTIVES
To identify the effects of
• Initial copper ion concentration on the percentage removal of
copper
• pH on the percentage removal of copper
• Temperature on the percentage removal of copper
• Biomass loading on the percentage removal of copper
To statistically optimize these variables for maximum removal of
copper efficiently
Dept of Biotechnology 214-06-2011
INTRODUCTION Bioadsorption : Physiochemical process that occurs naturally in
certain biomass which allows it to passively concentrate and bind contaminants onto its cellular structure.
Biomass :
• Economical alternative for removing toxic heavy metals
Practiced in environmental cleanup Heavy metal characteristics
• Nondestructive • Toxicity • Bioaccumulation • Subsequent bio magnifications • Non-Biodegradable in nature
Dept of Biotechnology 314-06-2011
Sources of heavy metals•Mining operation
•Electroplating operations
•Metal processing
•Coal fire powered Generation
•Nuclear industry
•Other industrial operations
Metal plating industry• Discharges huge amounts of copper
• Causes acute and chronic disorders in humans
• Major concerns because of their toxicity and threat to human life and environment
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Heavy metal pollution Dangerous to health and environment (e.g. mercury, cadmium,
lead, chromium). Causes corrosion (e.g. zinc, lead). Carcinogenic in nature, affecting Central nervous system (manganese, mercury, lead, arsenic). Kidneys (mercury, lead, cadmium, copper). Liver (mercury, lead, cadmium, copper). Skin, bones, or teeth (nickel, cadmium, copper, chromium).
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Lead : Acute or chronic damage to the nervous system
Cadmium : Long term exposure - Renal dysfunction High exposure - Obstructive lung disease
Copper : High doses - Anemia, liver and kidney damage, stomach and intestinal
irritation
Chromium : Low-level exposure – Skin irritation (ulceration) Long-term exposure – Kidney dysfunction
Selenium and Mercury : Damage to brain, circulatory tissue and central nervous system
Effects of Heavy metals
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COPPER
Symbol : Cu
Atomic Number : 29
Atomic Mass : 63.546 amu
Melting Point : 1083.0°C (1356.15 K, 1981.4 °F)
Boiling Point : 2567.0°C (2840.15 K, 4652.6 °F)
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Continued…. Copper : Essential compound for plants ,animals
and humans in small quantities
Toxicity : Excess bioaccumulation
Cupric ion : Main ionic form of copper, highly toxic
Cupric ion in water : Bound with inorganic and organic compounds, Reduces cupric ion concentrations
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Physical and chemical properties
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Conventional TechniquesPrecipitationIon-Exchange MethodElectrochemical CellsReverse Osmosis
Biological MethodsBioremediationPhytoremediation
Advantages of Biosorbents: Cost EffectivenessHigh quality of treated effluentMinimization of chemical and or biological sludge No additional nutrient requirement Regeneration of bioadsorbent and Possibility of metal recovery Solid Phase Extraction (Adsorption) is an attractive technique based on the sorbent that retains analytes.04/08/23 Department of Biotechnology 10
Adsorbents used:• Peat • Marine algae • Clays• Maize cob• Bagasse
• Palm fruit bunch• Lalang leaf • Saraca • Indica leaf and• Nile rose plant
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Metal adsorption reactions:
1. Bulk partitioning relationships Freundlich Isotherms Langmuir Isotherms
2. Surface complexation model (SCM)Effects of changing pH, solution composition and ionic strength.
log[c]
max
R max is the max sorbate uptake under the given conditions
Adsorption Isotherm
Freundlich isothermAdsorption over a wider range of metal ion concentration.
The adsorption relationship is expressed as:
τ =K cNWhere, N is a fitting parameter.
log τ =log K +N log c
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Langmuir Isotherm Adsorbent contains a finite number of reactive site on the surface.
The metal ion sorption reaction can be expressed by a site-specific equilibrium reaction: [A- ]+[M+] [ AM]
An equilibrium constant can be determined from the law of mass action:
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MA
AMK
Kinetics of Bioadsorption Metal sorption by microbial biomass often involves two
distinct stages. The first stage – the passive adsorption to the cell surface ( a
rapid process). The second stage is slower and commonly involves diffusion-
controlled Accumulation.
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Mechanism of Bioadsorption Most organic functional groups are Amphoteric.
R-AH R-A- + H+
The ionization of functional groups in the cell wall provides an electrical charge at the cell’s surface.
Dissociation of functional group on the cell surface
• Organic acid:
R-COOH R-COO- + H+
• Hydroxyl group:
R-OH R-O- + H+
• Phosphate group:
R-PO4H2 RPO4H- + H+
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Neocuproine methodPrinciple: Copper in neutral or slightly acidic solution .
Reacts with neocuproine to form a complex in which 2 moles of neocuproine are bound by 1 mole of Cu+ ion.
The complex –extracted by chloroform-methanol (CHCl3-CH3OH) mixture,
to give a yellow solution at 457 nm.
The color follows Beer’s law up to a concentration of 0.2 mg Cu/25 ml solvent
Full color development -pH of aqueous solution is between 3 and 9.
The color is stable in CHCl3-CH3OH for several days.
MATERIALS AND METHODS
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Stock solution of copper• To 200.0 mg polished electrolytic copper wire or foil in a 250-ml conical
flask, add 10 ml water and 5 ml conc. Nitric acid.
• After the reaction has slowed, warmed gently to complete dissolution of the copper.
• Cooled & added 50 ml water, transferred to a 1litre Volumetric flask, and diluted to the mark with double distilled water; 1 ml = 200 µg Cu.
Source:www.uncp.edu/home/mcclurem/ptable/copper/Copper reacting with nitric acid
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•Standard copper solution:Dilute 50.00 ml stock copper solution to 500 ml with water;1.00 ml = 20.0 µ Cu
•Sulfuric acid
•Hydroxylamine-hydrochloride solution: Dissolve 50 g NH2OHHCl in 450 ml water.
•Sodium citrate solution:150 g in 400 ml.
•Ammonium hydroxide
•Neocuproine reagent
•Chloroform
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• Blank : 50 ml water was pipetted into a 125-ml separatory funnel for use as a reagent.
• 1.00ppm to 200ppm standard copper solution were prepared and taken into a series of separating funnels.
Calibration curve
Standardization values and graph Chromium Concentratio
n (ppm)
Optical Density
12
0.0030.005
3 0.0074 0.0145 0.0176 0.0227 0.0318 0.0319 0.038
10 0.03720 0.07330 0.11240 0.14950 0.19260 0.23570 0.28280 0.33190 0.381
100 0.44150 0.504 200 0.581
Calibration
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
1 2 3 4 5 6 7 8 9 10 11 12
Concentration(ppm)
Op
tic
al D
en
sit
y
Series2
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r² value = 0.9271
Department of Biotechnology 2104/08/23
Figures showing the samples prepared for calibration
Apparatus:
Colorimetric equipment: One of the following is required:1) Spectrophotometer: for use at 457 nm, providing a light path of 1 cm or longer.2) Filter photometer: providing a light path of 1 cm or longer and equipped with a narrow-band violet filter having maximum transmittance in the range 450 to 460 nm.
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Separatory funnels
Bioadsorbents
Tamarind fruit shell
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Tamarind fruit shell(Tamarindus indica)
Carrot peels (Daucus carota)
Preparation of Adsorbent1. Carrot (Daucus carota) Peels • Collected from Carrot pods.• Powdered and sieved to get uniform size (60-80) mesh
particle size.• Washed thoroughly with distilled water, acid, base and
dried in the oven for 2 hrs at 60ºC.
2. Tamarind (Tamarindus indica) fruit shells • Collected from tamarind fruit pods.• Powdered and sieved to get uniform size (60-80) mesh particle size.• Washed thoroughly with distilled water, acid, base and dried in the oven for 2 hrs at 60ºC.
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Temperature ( 30ºC - 50ºC )
Biomass loading ( 1g/l – 20g/l)
Batch studies: Following parameters were varied in a batch study
Initial metal ion concentration ( 1ppm – 100ppm)
pH (1 – 7)
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Batch StudiesBioadsorption studies:
•Carried out using a certain amount of carrot and tamarind biomass in copper nitrate solution.
•The operating conditions such as pH, adsorbent amount, contact time and metal concentration were investigated.
•For each of the investigation, the mixture was shaken in a rotary shaker at 180 rpm followed by filtration using Whatman filter paper.
•The filtrate containing the residual concentration of Cu (II) was determined spectrophotometrically at 457 nm using the method mentioned above. 04/08/23 Department of Biotechnology 26
Cu Ion Determination
A yellow colored complex was developed in the reaction between Cu(II) and 2,9-dimethyl-1,10-phenanthroline hemihydrate (Neocuprione) in Acidic condition.
The percentage of Cu removal due to Bioadsorption was calculated as,% Cu removed = [(Co – Ci) / Co] x 100%
Where, Co - Initial conc. of Cu solution (mg/l)
Ci - Equilibrium conc. of Cu solution (mg/l)
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Chemicals and Equipments
Chemicals Grade Suppliers
Hydroxylamine
hydrochloride
LR S.d.fine-Chem.Ltd
Sodium Citrate LR S.d.fine-Chem.Ltd.
Neocuprione LR S.d.fine-Chem.Ltd.
Chloroform LR S.d.fine-Chem.Ltd
Methanol LR S.d.fine-Chem.Ltd
Isopropyl alcohol LR S.d.fine-Chem.Ltd
Copper Foil
The chemicals and equipments used in the experimental work along with their specifications are given in the tables:
Specification of materials04/08/23 Department of Biotechnology 28
Name of the Equipment Name of the supplier/company
Weighing Balance AY220 - SHIMADZU
Shaker POOJA LAB EQUIPMENTS, MUMBAI
Hot air oven FATHA INSTRUMENT WORKS
UV double beam spectrophotometer UV VISIBLE 1700 - SHIMADZU
pH meter MICROPRO
Equipments Used
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Sieving machine UV spectrophotometer
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Shaking Incubator
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• The bioadsorption of metals using carrot peels (Daucus carota) and tamarind (Tamarindus indica) fruit shell powder in a batch process depends on the test variables considered.
• The effect of contact time on percentage removal of copper is studied by conducting the separate experiments designed using software minitab 14 and 15.
RESULTS AND DISCUSSION
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Software – Minitab version 15
Response surface methodology(RSM) – Empirical statistical technique employed for multiple regression analysis of quantitative data obtained from statistically designed experiment.In this study full factorial central composite design using response surface methodology was employed
pH Initial conc (ppm) Biomass loading (g/lt) Temperature (° C)
7 1.0 1.0 50
4 50.5 10.5 40
4 50.5 1.0 50
7 100 1.0 50
4 48.5 10.5 40
4 50.5 10.5 40
1 100 20.0 30
4 50.5 10.5 40
7 100 20.2 30
1 1.0 1.0 30
4 49.5 10.5 40
7 100 20.0 50
1 1.0 20 30
7 100 1.0 30
4 50.5 10.5 40
7 50.5 10.5 4
7 1.0 1.0 30
4 50.5 10.5 4004/08/23 Department of Biotechnology 34
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pH Initial conc (ppm) Biomass loading (g/lt) Temperature (° C)
1 100 20.0 50
4 50.5 10.5 50
7 1.0 20.0 50
1 1.0 1.0 50
1 100 1.0 50
4 50.5 10.5 40
4 50.5 8.5 40
2 50.5 10.5 40
7 1.0 20.0 30
4 50.5 20.0 40
1 100 1.0 30
1 1.0 20.0 50
4 50.5 10.5 40
Continued…
Temperature- 30°C, 40°C, 50°C.
Initial concentration- 1ppm, 48.5ppm, 50.5ppm, 100ppm
Biomass loading- 1g/l, 10.5g/l, 20g/l
Ph- 1, 2, 4, 7
Range of parameters designed using minitab software
Effect of Initial concentration- Tamarind
91
91.5
92
92.5
93
93.5
94
94.5
95
95.5
0 20 40 60 80 100 120
Initial concentration
%R
eco
very Series1
Series2
Series3
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Ph-4
Temperature- 30ºC
Biomass loading- 10.5g/l
Series 1- distilled water wash
Series 2- Acid wash
Series 3- Base wash
Effect of initial concentration- Carrot
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Ph-1
Temperature- 30ºC
Biomass loading- 10.5g/l
Series 1- distilled water wash
Series 2- Acid wash
Series 3- Base wash
Effect of Temperature(Carrot Distilled water wash)
92
92.5
93
93.5
94
94.5
95
95.5
0 10 20 30 40 50 60
Temperature('C)
%R
eco
very
Series1
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Effect of Temperature
Ph- 1
Initial conc- 50.5ppm
Biomass loading -20g/l
Effect of Temperature(Carrot HCl wash)
96.15
96.2
96.25
96.3
96.35
96.4
96.45
96.5
96.55
96.6
96.65
0 10 20 30 40 50 60
Temperature('C)
%R
eco
very
Series1
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Ph- 1
Initial conc- 50.5ppm
Biomass loading -20g/l
Effect of Temperature(Carrot NaOH wash)
94.4
94.5
94.6
94.7
94.8
94.9
95
95.1
0 10 20 30 40 50 60
Temperature('C)
%R
eco
very
Series1
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Ph- 1
Initial conc- 50.5ppm
Biomass loading -20g/l
Effect of Temperature- Tamarind
90.5
91
91.5
92
92.5
93
93.5
94
94.5
95
95.5
0 20 40 60
Temperature('C)
%R
eco
very
Tamarind Distilledwater wash
Tamarind HCl wash
Tamarind NaOH wash
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Ph- 4
Initial conc- 50.5ppm
Biomass loading -20g/l
Effect of pH
91.5
92
92.5
93
93.5
94
94.5
95
95.5
0 2 4 6 8
pH
%R
EC
OV
ER
Y
Series1
Effect of pH on copper adsorption using Tamarind Distilled water
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Effect of pH
Temperature- 40°C
Initial conc- 100ppm
Biomass loading- 10.5g/l
Effect of pH(Tamarind HCl wash)
92
92.5
93
93.5
94
94.5
95
95.5
0 1 2 3 4 5 6 7 8
pH
%R
eco
very
Series1
Effect of pH on copper adsorption using Tamarind Distilled water
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Temperature- 40°C
Initial conc- 100ppm
Biomass loading- 10.5g/l
Effect of pH(Tamarind NaOH wash)
80
82
84
86
88
90
92
94
96
0 1 2 3 4 5 6 7 8
pH
%R
eco
very
Series1
Effect of pH on copper adsorption using Tamarind NaOH wash
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Temperature- 40°C
Initial conc- 100ppm
Biomass loading- 10.5g/l
Effect of pH(Carrot Distilled water wash)
93.5
94
94.5
95
95.5
96
96.5
97
0 1 2 3 4 5 6 7 8
pH
%R
eco
very
Series1
Effect of pH on copper adsorption using Carrot Distilled water wash
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Temperature- 40°C
Initial conc- 100ppm
Biomass loading- 10.5g/l
Effect of pH(Carrot acid wash)
91
91.5
92
92.5
93
93.5
94
94.5
95
95.5
0 1 2 3 4 5 6 7 8
pH
%R
eco
very
Series1
Effect of pH on copper adsorption using Carrot acid wash
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Temperature- 40°C
Initial conc- 100ppm
Biomass loading- 10.5g/l
Effect of pH(Carrot NaOH wash)
90
90.5
91
91.5
92
92.5
93
93.5
94
94.5
95
95.5
0 1 2 3 4 5 6 7 8
pH
%R
eco
very
Series1
Effect of pH on copper adsorption using Carrot NaOH wash
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Temperature- 40°C
Initial conc- 100ppm
Biomass loading- 10.5g/l
Effect of Biomass loading on copper adsorption using Tamarind
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Effect of Biomass
Ph- 4
Temperature- 40°C
Initial conc – 48.5ppm
Effect of biomass loading on copper removal using carrot residues
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Ph- 4
Temperature- 40°C
Initial conc – 48.5ppm
Effect of time on percentage removal of copper using tamarind distilled water wash sample at 30°C
Effect of Time
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Effect of time on percentage removal of copper using tamarind distilled water wash sample at 30°C
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Effect of time on percentage removal of copper using tamarind acid wash sample at 30°C.
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Effect of time on percentage removal of copper using tamarind acid wash sample at 30°C
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Effect of time on percentage removal of copper using tamarind base wash sample at 30°C
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Effect of time on percentage removal of copper using tamarind base wash sample at 30°C
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Effect of time on percentage removal of copper using tamarind base
wash sample at 30°C
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Effect of time on percentage removal of copper using carrot distilled water wash sample at 30°C
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Effect of time on percentage removal of copper using carrot distilled water wash sample at 30°C
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Effect of time on percentage removal of copper using carrot acid wash sample at 30°C
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Effect of time on percentage removal of copper using carrot acid wash sample at 30°C
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Effect of time on percentage removal of copper using carrot base wash sample at 30°C
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Effect of time on percentage removal of copper using carrot base wash sample at 30°C
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Effect of time on percentage removal of copper using tamarind distilled water wash sample at 40°C
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Effect of time on percentage removal of copper using tamarind acid wash sample at 40°C
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Effect of time on percentage removal of copper using tamarind acid wash sample at 40°C
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Effect of time on percentage removal of copper using tamarind base wash sample at 40°C
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Effect of time on percentage removal of copper using tamarind
base wash sample at 40°C
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Effect of time on percentage removal of copper using carrot distilled water wash sample at 40°C.
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Effect of time on percentage removal of copper using carrot distilled water wash sample at 40°C.
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Effect of time on percentage removal of copper using carrot acid wash sample at 40°C
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Effect of time on percentage removal of copper using carrot acid wash sample at 40°C
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Effect of time on percentage removal of copper using carrot base wash sample at 40°C.
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Effect of time on percentage removal of copper using carrot base wash sample at 40°C.
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Effect of time on percentage removal of copper using tamarind distilled water wash sample at 50°C.
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It can be concluded from the laboratory trials both these biosorbent show their potential for commercialization since it is technically feasible, ecofriendly with good metal-binding capacity.
It was found that for tamarind distilled water wash and acid wash, initial concentration of 50.5ppm was found to have greater % removal of copper than tamarind base wash.
It was observed that tamarind acid wash has more efficient as bioadsorbent with 95% removal of copper.
SUMMARY AND CONCLUSION
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The adsorption capacity was found to be increasing with temperature for both tamarind and carrot biomass.
The adsorption capacity was found to be decreasing with increasing pH.
Optimum conditions pH 1 there is adsorptivity. biomass 10.5g/l the % removal of copper was more.
It was found that for carrot distilled water wash and acid wash, initial concentration of 100 ppm was found to have greater % removal of copper than tamarind base wash.
From this we observed that carrot acid wash has more efficient as bioadsorbent with 96.9% removal of copper.
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FUTURE PERSPECTIVES
Dynamic studies have to be carried out on different Biosorbents.
Surface analysis of the adsorbent. Disorption studies of the adsorbents. Optimising experiment by using software's Batch kinetic study
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K.M.S. Sumathi, S. Mahimairaja and R. Naidu, 2004 Use of low-cost biological wastes and vermiculite Solids, for removal of copper from tannery effluent. Bioresource Technology Raji, C. and T.S. Anirudhan, 1997. Copper (II) adsorption by sawdust: kinetics and equilibrium. Indian Journal of Chemical Technology, 4: 228-236. Deans, J.R. and B.G. Dixon, 1992. Uptake of Pb2+ and Cu2+ by novel biopolymers. Water Research, 26(4): 469-472. Veglio, F., F. Beolchini and A. Gasbarro, 1997. Biosorption of toxic metals: an equilibrium study using free cells of Arthrobacter sp. Process Biochemistry., 32: 99-105. Gadd, G.M. and C. White, 1993. Microbial treatment of metal pollution – a working biotechnology. Trends Biotechnology, 11: 353-359. Volesky, B., 1987. Biosorbents for metal recovery. Trends Biotechnology, 5: 96-101. Volesky, B. and Z.R. Holan, 1995. Biosorption of heavy metals. Biotechnology Program, 11: 235-250.
References
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ACKNOWLEDGEMENTWe consider it our privilege to express our gratitude and respect to all who guided us in the completion of this project.
We express our sincere gratitude to Dr. Netaji.S.Ganesan, Principal, for providing us with excellent infrastructure to complete our project work.
We would like to express our sincere thanks to our HOD, Dr. G.S.Jagannatha Rao for his continuous and never ending support, and without whose help this venture would not be complete.
We would like to express our sincere thanks to Dr.Kiran and Dr.Rajeshwari for their valueable suggestions
We are deeply indebted to our guide, Dr. Nagamani.S.Khandre and Ms. Shwetha.N, for their timely help, crisp advice, constructive criticism, masterly guidance, academic freedom, unparalleled support, valueable suggestions and for their keen interest in our project.
•We would like to thank Mr. Sudhanva madhava Desai, Mr.Sinosh skariyachan and Mr. Sameera for their timely help and valueable suggestions while doing our project.
•Last but not the least we would like to thank the department and all our friends.04/08/23 Department of Biotechnology 80
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