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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

04/08/23 Department of Biotechnology 4

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).


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


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)


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


Physical and chemical properties


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


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


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:


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.


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+


Department of Biotechnology 16

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

04/08/23


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

04/08/23


•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

04/08/23


• 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


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.


Separatory funnels

Bioadsorbents

Tamarind fruit shell


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.


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)


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)


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


Sieving machine UV spectrophotometer



Shaking Incubator


• 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

04/08/23


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


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


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


Ph-1

Temperature- 30ºC


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


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


Ph- 1



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


Ph- 1



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


Ph- 4



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


Effect of pH

Temperature- 40°C

Initial conc- 100ppm


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


Temperature- 40°C



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


Temperature- 40°C



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


Temperature- 40°C



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


Temperature- 40°C



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


Temperature- 40°C



Effect of Biomass loading on copper adsorption using Tamarind


Effect of Biomass

Ph- 4

Temperature- 40°C

Initial conc – 48.5ppm

Effect of biomass loading on copper removal using carrot residues


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




Effect of time on percentage removal of copper using tamarind acid wash sample at 30°C.


Effect of time on percentage removal of copper using tamarind acid wash sample at 30°C


Effect of time on percentage removal of copper using tamarind base wash sample at 30°C




Effect of time on percentage removal of copper using tamarind base

wash sample at 30°C


Effect of time on percentage removal of copper using carrot distilled water wash sample at 30°C


Effect of time on percentage removal of copper using carrot distilled water wash sample at 30°C


Effect of time on percentage removal of copper using carrot acid wash sample at 30°C




Effect of time on percentage removal of copper using carrot base wash sample at 30°C


Effect of time on percentage removal of copper using carrot base wash sample at 30°C










Effect of time on percentage removal of copper using tamarind

base wash sample at 40°C


Effect of time on percentage removal of copper using carrot distilled water wash sample at 40°C.


Effect of time on percentage removal of copper using carrot distilled water wash sample at 40°C.






Effect of time on percentage removal of copper using carrot base wash sample at 40°C.


Effect of time on percentage removal of copper using carrot base wash sample at 40°C.


Effect of time on percentage removal of copper using tamarind distilled water wash sample at 50°C.


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


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.


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


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


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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