REMOVAL OF COPPER(II) FROM

SYNTHETIC WASTE WATER USING

BIOREMEDIATION

UNDER THE GUIDANCE OF

SUBHAJIT MAJUMDER

PRESENTED BY:
SHITANSHU JAIN(2011A1PS552P)
VIJAY ARORA(2011A1PS540P)
1
 INTRODUCTION
Heavy Metal Contamination Problem
• Heavy metal describe those transition elements that have
potential to cause harm in the environment.
• Most heavy metal that enter the environment are as a
result of the industrial processes.
• The toxic level of the heavy metal depends on their
concentration. Below some critical level they have some
value but after that their toxicity increases.
• Bio remediation is the process of removing pollutants
from waste using microorganisms ,a field of research
with a lot yet to discover.

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 Copper(II)
 Copper exhibits a variety of compounds, many of which
are colored.
 The two principal oxidation states of copper are +1 and
+2 although some +3 complexes are known.
 The copper(II) ion is usually the more stable state in
aqueous solutions. Compounds of this ion, often called
cupric compounds, are usually colored.
 They are affected by Jahn Teller distortions and exhibit a
wide range of stereochemistry's with four, five, and six
coordination compounds predominating. The +2 ion
often shows distorted tetrahedral geometry.

3
Copper toxicity
 Too much copper in water may damage marine and
freshwater organisms such as fish.
 High levels of copper in human body leads to mental
illness and nervous breakdown.
 It also reduces the ability to cope normally with stress.
 It can contribute to the development of schizophrenia
and autism.

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LITERATURE REVIEW
o Novel biofiltration methods for the treatment of
heavy metals from industrial wastewater

 The various parameters of the biofiltration processes,

their mechanism for heavy metals removal along with
the kinetics of biofilters and its modeling aspects have
been discussed
 The applications of genetic engineering in the
modification of the microorganisms for increasing the
efficiency of the biofiltration process for heavy metals
removal have been critically analyzed.

5
o Effect of Copper on Activated Sludge Bacteria
Growth Kinetics

 Different concentrations of Cu(II) and Zn(II) were

introduced in reactor keeping other parameters constant.
 The metallic ions were mixed to determine the
combined effects.
 Data showed that Zn(II) was less toxic than Cu(II).
Moreover, bio kinetic parameters were not adversely
affected by the presence of Cu(II) up to a concentration
of 5 mg/L.

6
o Toxicity of Copper(II) ions to microorganisms in
biological wastewater treatment systems
 The objective of this study was to evaluate the inhibitory
effect of copper(II) towards various microbial trophic
groups responsible for the removal of organic
constituents and nutrients in wastewater treatment
processes.
 The results indicated that copper(II) caused severe
inhibition of key microbial populations in wastewater
treatment systems.
 Nonetheless, denitrifying and nitrifying bacteria showed
considerable recovery of their metabolic activity.

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GAPS IN LITERATURE
 Many papers compared the various carcinogenic
elements like Cu and Zn in their toxicity and other
parameters.
 There is a significant lack of focused bioremediation
research done in India.
 Most of the study is extremely general in nature and the
variation in results due to different regions has not been
accounted for.
 There have been many developments and efforts in
general heavy metal removal from waste water but not a
considerable portion of it talks about Copper in
particular.

8
Objectives
 To develop microbial cultures for bio remediation of Cu
(II) from waste water.
 To conduct a batch study and study the variation of
biomass and the absorbance with the help of U.V.
Spectrophotometer.
 To conduct a kinetic study and study the variation of
metal concentration as well as biomass concentration
with both time and absorbance

9
Chemicals Required
• Mixed Bacteria Strain from Sludge
• Distilled Water (H2O)
• Glucose(C6H12O6)
• Calcium Sulphate (CaSO4.2H2O)
• Magnesium Sulphate (MgSO4.7H2O)
• Potassium dihydrogen orthophosphate (KH2PO4)
• Iron(II) Sulphate (FeSO4)
• Ammonium Sulphate (NH4)2SO4
• Di-Potassium hydrogen orthophosphate (K2HPO4)
• Copper Sulphate(CuSO4.5H2O )
• Ethanol(C2H5OH)
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 APPARATUS
 Laboratory weighing Machine
 BOD Incubator
 Horizontal Laminar Air flow Chamber
 Orbital Shaking Machine
 Refrigerator
 Hot air oven
 UV Visible Spectrophotometer

Horizontal Laminar Air flow Chamber Orbital Shaking Incubator 11

Experimental Techniques
o UV-Vis Spectroscopy
 It refers to absorption spectroscopy in the ultraviolet-
visible spectral region by using U.V. light in the visible and
adjacent (near-UV and near-infrared (NIR)) ranges.
 The absorption or reflectance in the visible range directly
affects the perceived colour of the chemicals involved. In
this region of the electromagnetic spectrum, molecules
undergo electronic transitions.
 UV/Vis spectroscopy is routinely used in analytical
chemistry for the quantitative determination of different
analytes, such as transition metal ions,
highly conjugated organic compounds , and biological
macromolecules.

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o Atomic Absorption Spectroscopy
 It is used to determine the quantitative properties of
chemical elements using the optical radiation absorption
by free atoms within gaseous state.
 The basic principle of atomic absorption is that the
technique makes involve the absorption spectrometry to
know the concentration of an analyte within the sample,
the electrons within the atoms put to higher orbitals for
little time by making them absorb a great amount of
energy.

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MATERIALS AND METHOD
Preparation of Microbial Culture
 Take around 20 mL of activated sludge brought from the
sewage treatment plant in a beaker.
 Add an equal amount of distilled water to it . Stir it and
keep the apparatus at rest for 2 hours.
 Drain the upper portion and take the bottom half and
pour it in another beaker. Again, add same amount of
distilled water. Stir it and now keep the system at rest
for 2 minutes.
 This time take only top portion of the previous sample
and the culture is ready.

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Preparation of 1000 ppm Stock Cu(II) Solution
Dissolve 3.9322 g of Copper Sulfate in 1L of distilled
water in a conical flask to make 1000 ppm of stock Cu(II)
solution.

Diluting the Stock Solution to 10 ppm

Take 1mL of stock solution and add distilled water to it to
make 100mL solution. The resulting solution is 10 ppm.

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Preparation of MSM
To make 1L MSM(Minimal Salt Media) the following salts
are added to 1L of distilled water in a 1L conical flask :
 0.8 g K2HPO4
 0.2 g KH2PO4
 0.05 g CaSO4.2H2O
 0.5 g MgSO4.7H2O
1 g (NH4)2SO4
 0.01 g FeSO4
It is then AUTOCLAVED at 121˚C and 121 KPa for 15-20
min.

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Autoclaving
The flask is kept in a pressure cooker for around 20 min as
we wait for 2 whistles. After that we simmer the gas for 5
min. Then the flask is taken out and kept at room
temperature for few minutes.
The autoclaved MSM is stored in a BOD Incubator at
25˚C until further use.

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Batch Study

• 1 mL D-Glucose,100 ml MSM, 2ml

DAY 1 microbial culture

• 0.2 mL 10 ppm Cu(II), 0.8 mL D-

Glucose, 100 ml MSM, 2mL fist day
DAY 2 sample
• 0.4 ml 10 ppm Cu(II),0.6 mL D-
Glucose, 100 ml MSM, 2mL second
DAY 3 day sample

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Each day sample is prepared inside the Laminar
Air Flow Chamber. Then , kept in ORBITAL
SHAKING INCUBATOR for 24 hrs.

• 0.6 mL 10 ppm Cu(II),0.4 mL D-

Glucose,100 mL MSM, 2 mL third
DAY 4 day sample.
• 0.8 mL 10 ppm Cu(II), 0.2 mL D-
Glucose, 100 mL MSM, 2 mL fourth
DAY 5 day sample
• 1mL 10 ppm Cu(II), 0.05 mL D-
Glucose, 100mL MSM, 2 mL fifth
DAY 6 day sample

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Study of Growth Of Microorganisms

Note : Only Day 3 , Day 4, Day 5, Day 6 samples were used

for this study.
The following steps are performed :
 Take the weight of 4 empty filter papers.
 Now filter 50mL of each of the samples into some other
flasks using these filter papers.(Remember to number these
filter papers and the flaks according to their Day)
 Keep these wet filter papers in HOT AIR OVEN for around
15 min at 70℃ .
 Take them out and again weigh them.

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 Subtract the weight of empty filter paper from the new
measured weight and note the weight of the BIOMASS
in each of the samples.
 Now measure the Absorbance (Optical Density) of each
of the samples using a UV-Vis Spectrophotometer.
 For this study, MSM is used as the reference sample.
 Now, the absorbance of each of the samples is measured
by taking at least 3 readings.
 Plot the biomass against the absorbance for each of the
samples respectively.

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 Biomass(g) Absorbance

0.0044 0.337
0.005 0.057
0.0081 0.049
0.0049 0.0406

0.4

0.35

0.3

0.25

0.2
Absorbance

0.15

0.1

0.05

0
0 0 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01
Biomass

Absorbance vs. Biomass concentration

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KINETIC STUDY
A kinetic study is performed on the 6th DAY sample.
The objective is to study the variation of metal
concentration as well as the mass of biomass (in the
sample) with time. The following procedure is followed :
 Take five 250 mL conical flasks.
 Add 100 mL of autoclaved MSM and 2mL of 6th Day
sample to each of them.
 Now add 0.5 mL of 10ppm Cu(II) and 0.05 mL of D-
Glucose to each flask.
 Keep these samples in the BOD Incubator at 37℃ and
100 -110 rpm.

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Variation of Metal Concentration with Time
 For measuring the metal concentration in these samples
Atomic Absorption Spectrometer(AAS) is used.
 For reference sample used in AAS, three Cu(II)
samples : 1ppm , 2 ppm , 3 ppm are made from the stock
1000 ppm Cu(II) solution.
 The samples are taken out from the incubator at the
following time intervals :
1st sample – 3 hours
2nd sample – 15 hours
3rd sample – 18 hours

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 4th sample – 21 hours
5th sample – 24 hours
 The metal concentration as well as absorbance is now
measured in all the samples using the AAS.
 Graphs are drawn : Metal concentration versus time and
Absorbance versus Metal Concentration.
 The following result is obtained :

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 Time(
hrs) Concentration(ppm)
3 0.1259
15 0.1228
18 0.1216
21 0.1158
24 0.1259

0.13

0.13

0.12

0.12

0.12
Concentration
0.12

0.12

0.11

0.11

0.11
0 5 10 15 20 25 30
Time(hrs)

Metal concentration vs. Time

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 Concentration(p
pm) Absorbance
0.1259 0.0044
0.1228 0.0036
0.1216 0.0033
0.1158 0.0018
0.1259 0.0044

Absorbance
0.01

0
0.11 0.12 0.12 0.12 0.12 0.12 0.13 0.13 Concentration

Absorbance vs. Metal concentration

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Variation of Biomass with Time
The following steps are performed :
 Take the weight of 5 empty filter papers.
 Now filter 50mL of each of the samples into some other
flasks using these filter papers.(Remember to number
these filter papers according to the sample no.)
 Keep these wet filter papers in HOT AIR OVEN for
around 15 min at 70℃ .
 Take them out and again weigh them.
 Subtract the weight of empty filter paper from the new
measured weight and note the weight of the BIOMASS

28
in each of the samples.
 Now measure the Absorbance (Optical Density) of each
of the samples using a UV-Vis Spectrophotometer.
 For this study, MSM is used as the reference sample.
 Now, the absorbance of each of the samples is measured
by taking at least 3 readings.
 Plot the absorbance of each sample against the biomass
concentration.
 Also plot the variation of biomass concentration against
time.

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 Time(
hrs) Biomass(g)
3 0.0018
15 0.0038
18 0.0040
21 0.0041
24 0.0039

0
Biomass 0

0
0 5 10 15 20 25 30
Time(hrs)

Biomass concentration vs. Time

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 Biomass(g) Absorbance
0.0018 0.021
0.0038 0.04
0.004 0.067
0.0041 0.032
0.0039 0.041

0.08

0.07

0.06

0.05

0.04
Absorbance

0.03

0.02

0.01

0
0 0 0 0 0 0 0
Biomass

Absorbance vs. Biomass concentration

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CONCLUSION
 We started the study by doing literature survey.
 We brought the sludge from the sewage treatment plant
and prepared a microbial culture.
 After this our 6 Day batch study began. We calculated
the weight of biomass in each sample. Also we found
the absorbance by using UV-Vis Spectrometer.
 Then a plot of biomass against absorbance was made.
Ideally, this plot should have been a straight line but we
did not get the desired results. That may have been due
to the fact that our samples were kept in the refrigerator
for a long time before

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 performing the UV-Vis study because of which
microorganisms could have died.
 A kinetic study was performed to analyse the variation
of metal concentration as well as biomass concentration
with respect to time as well as absorbance of the
samples which were made from the Day 6 Sample of the
batch study. The variation of metal concentration with
time was appropriate with the exception of one sample.
And the biomass variation with time is, as expected,
increasing at first followed by a period of constancy and
then a slight decrease.

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REFERENCES
 Srivastava N.K., C.B. Majumder, Novel biofiltration methods
for the treatment of heavy metals from industrial wastewater,
Journal of Hazardous Materials, 151, 1: (2008) 1-8
 Cabrero Alberto, Ferenandez Sara, Mirada Fernando, Garcia
Julian, Effects of copper and zinc on the activated sludge
bacteria growth kinetics, Journal of Water Research, 32,5:
(1998) 1355-1362.
 Ochoa-Herrera V, León G, Banihani Q, Field JA, Sierra-
Alvarez R,Toxicity of Copper(II) ions to microorganisms in
biological wastewater treatment systems, Sci Total
Environ(Science of Total Environment) ,380,5:(2011) 412-
413.

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THANK YOU

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