A

Report
On

Biofiltration for Removal of Cu(II) From Industrial

Wastewater
Prepared in Partial Fulfilment of The

Study Project Course CHE F366

Submitted by

Shitanshu Jain (2011A1PS552P)and VijayArora(2011A1PS540P)

Submitted On

27th September, 2013

Under Guidance of

Mr. Subhajit Majumdar

Department of Chemical Engineering

 ACKNOWLEDGEMENT

We would like to thank our project advisor,Mr. Subhajit Majumdar, for giving us the
opportunity to work on this research project. Working under him has always been a learning
process as he ensures enhancement of knowledge and implementation of talent with hardwork.
We would also like to express our gratitude to the faculty and staff of Chemical Engineering,
BITS Pilani for their co-operation and support.

We are thankful to Dr. Suresh Gupta, Head of Dept., Dept. of Chemical Engg., Birla Institute of
Technology and Science, Pilani for inspiring us on various scopes of research and its benefits.

We would also like to thankJungveer Sir, SubodhSir as they were always there for us when we
needed their help.
 ABSTRACT

Copper (Cu), especially Cu(II) is of particular environmental concern due to its solubility,
bioavailability and toxicity. When discharged into wastewater in large quantities, it poses a
serious threat to the marine life and flora and fauna on land too. Thus, it is essential to remove
copper from the wastewater. In this study we tried to remove copper (II) with the help of
microbes. This process to remove pollutants with the help of microorganisms is called Bio
filtration. A batch study is performed to prepare the final sample by changing the volume of
glucose and 10ppm Copper Solution. Also the absorbance of each day sample is measured using
UV-Vis Spectrophotometer.
 TABLE OF CONTENTS

1.Introduction 1
1.1Heavy Metal Contamination
1.2Copper
1.3Toxicity of Copper 2
2.Physiochemical Methods 3
2.1Ion Exchange
2.2Nano filtration
2.3Electrocoagulation 3
3.Bioremediation............................................................................................................................4

4. Literature Review…………………………..................................................…………………5

5.Material and Methods 7

5.1Chemicals
5.2Apparatus and glassware
5.3Preparation
5.4Batch Study
5.5Study of growth of microorganism
6. Conclusion..............................................................................................................................13

7. References..........…………………………………………………………………………….14
 1. INTRODUCTION

1.1 HEAVY METAL CONTAMINATION

Heavy metalsare a term used to describe commontransition metals that have the potential to
cause harm in the environment.Heavy metal ions such as cobalt, copper, nickel, chromium and
zinc are detected in the waste streams from mining operations, tanneries, electronics,
electroplating and petrochemical industries, as well as in textile mill products. The release of
large quantities of heavy metals into the natural environment e.g. irrigation of agricultural fields
by using sewage has resulted in a number of environmental problemsand due to their non-
biodegradability and persistence, can accumulate in the environment elements such as food
chain, and thus may pose a significant danger to human health. 

1.2 COPPER
Copper has been known since prehistoric times. It has continued to this day to be an important
metal to human kind. The abundance of copper in the earth’s crust amounts to 68 ppm. It occurs
mainly as the sulfide, oxide, or carbonate. Its major ores are copper pyrite (chalcopyrite,
CuFeS2), copper glance (chalcocite), cuprite, and malachite. Copper is essential to all living
organisms as a trace dietary mineral because it is a key constituent of the respiratory enzyme
complex cytochrome c oxidase. In molluscs and crustacea copper is a constituent of the blood
pigment hemocyanin, which is replaced by the iron-complexed hemoglobin in fish and
other vertebrates. The main areas where copper is found in humans are liver, muscle and bone.
Copper compounds are used as bacteriostatic substances, fungicides, and wood preservatives.
 1.3 COPPER TOXICITY
Copperiedus refers to the consequences of an excess of copper in the body. Copperiedus can
occur from eating acid foods cooked in uncoated copper cookware, or from exposure to excess
copper in drinking water or other environmental sources. Initially, the copper will build up in the
liver, further impairing its ability to excrete copper. As copper retention increases, it will build
up in the brain, the joints and the lungs, adversely affecting the structure and functioning of the
tissues.

Copper toxicity reduces the ability to cope normally with stress and the inability to respond
adequately can provoke many fearful emotions, including anxiety and panic. It can also
contribute to the development of schizophrenia and autism in susceptible people. It also results in
slow metabolism and Zinc deficiency. Its symptoms include mental and physical conditions such
as depression, irritability, mood swings, poor concentration, dizziness, fatigue etc.
 2. PHYSIO-CHEMICAL METHODS

Various physio-chemical methods like ion-exchange, electrodialysis, photocatalysis, nano-

filtration, activated charcoal, chemical precipitation, chemical reduction, coagulation and
adsorption have been used for heavy metal removal from wastewater.

2.1. Ion Exchange 

Removal of copper from water and wastewater is obligatory in order to avoid water pollution.
Batch shaking adsorption experiments is done to evaluate the performance of cation exchange
resins in the removal of copper from aqueous solutions.The adsorption of copper on these cation
exchange resins follows the first-order reversible kinetics. The ion exchange resins investigated
in this study showed reversible uptake of copper and, thus, have good application potential for
the removal/recovery of copper from aqueous solutions.

2.2. Nano-Filtration

Nano-filtration (NF) was capable of chromium removals of over 70%. The recoveries ranged
between 15 to 20%. A recent study showed that the removal efficiency dropped significantly
during pilot-scale tests where the process was operated at more realistic recoveries. If Nano
filtration is used by small systems in the western U. S., water recovery will likely need to be
optimized due to the scarcity of water resources. The increased water recovery can lead to
increased costs for copper removal.

2.3. Electrocoagulation

The performance of electrocoagulation with iron and aluminum sacrificial anode for removal of
Cu(II) is studied.The process is successfully applied to the treatment of electroplating wastewater
where an effective reduction of Cu(II)concentration under legal limits was obtained just after 20-
60 min.
 3. BIOREMEDIATION

Bioremediation is the use of micro-organisms to break down toxic and hazardous compounds in
the environment. The main biological treatment of copper is:

 Biosorption.

Various physio-chemical methods like ion-exchange, activated charcoal, chemical precipitation,

chemical reduction and adsorption have been used for heavy metal removal but these
conventional methods have some limitations. These are cost-expensive and can themselves
produce other waste problems. Among the available treatment methods biological treatment is
more prominent due to the following reasons.

 Chemical requirement for the whole treatment process is reduced.

 Low operating costs.
 Eco-friendly and cost-effective alternative of conventional methods.
 Efficient at lower levels of contamination.

Biofilters and bioreactors are the most promising development in the field of bioremediation of
heavy metals contaminated industrial wastewater. Due to the developments in process
engineering more bioremediation techniques are emerging. Bioremediation also plays an
important role in concentrating metals and radioactive materials to avoid toxicity or to recover
metals for reuse. An added advantage of using microbes is that they can biodegrade organic
chemicals; purposeful enhancement of this natural process can help in pollutant degradation and
waste-site cleanup operations. It is important to note that microbes can’t breakdown the metals.
They just reduce or oxidize them converting them to less toxic form.
 4. LITERTAURE REVIEW

 Novel biofiltration methods for the treatment of heavy metals from

industrial wastewater.[1] 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 comparison of various physico-chemical
treatment and the advantages of biofiltration over other conventional processes for
treatment of heavy metals contaminated wastewater have also been discussed.
 Effect of Copper on Activated Sludge Bacteria Growth Kinetics [2]Different
concentrations of Cu(II) and Zn(II) (1, 5, 10 and 20 mg/l) were introduced singly in the
reactors keeping all environmental parameters constant (pH,Ta, basic nutrients). The
combined effects of Cu(II) and Zn(II) were determined by mixing these metallic ions
(5/5, 5/10, and 10/5 mg/l of Cu(II)/Zn(II) respectively). Experimental data showed that
Zn(II) was less toxic than Cu(II), as expressed by a slight stimulating effect for 1 mg/l
Zn(II). Moreover, biokinetic parameters were not adversely affected by the presence of
Cu(II) up to a concentration of 5 mg/l.
 Toxicity of Copper(II) ions to microorganisms in biological wastewater
treatment systems.[3] 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 of the
batch bioassays 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 after only several days
of exposure to high copper levels (up to 25 and 100mg Cu(II) L(-1) for DE nitrification
and nitrification, respectively). The recovery could be due to attenuation of soluble
copper or to microbial adaptation.
 Contribution of Copper Ion Resistance to Survival of E.Coli on Metallic
Copper surfaces.[4]The survival ofEscherichia coli on metallic copper surfaces has
been studied previously; however, the mechanisms underlying bacterial inactivation on
copper surfaces have not been elucidated. Data presented in this study suggest that
bacteria are killed rapidly on dry copper surfaces. Several factors, such as copper ion
toxicity, copper chelators, cold, osmotic stress, and reactive oxygen species, but not
anaerobiosis, influenced killing rates. Strains deleted in copper detoxification systems
were slightly more sensitive than was the wild type. Preadaptation to copper enhanced
survival rates upon copper surface exposure. This study constitutes a first step toward
understanding the reasons for metallic copper surface-mediated killing of bacteria.
 Comparative efficiency of algal biofilters in removal of chromium and
copper from waste water[5]The performance of a macroalgae(Sargassumsp.), a
laboratory-cultivated microalgae (Chlorococcumsp.)And a commercially available
granulated activated carbon (GAC) for the removal of copper (Cu) and
chromium (Cr) from aqueous solutions was evaluated using batch experiments. Kinetic
and isotherm experiments were done at the optimal pH of 4.5 ±0.1 for Cu (II)
and 2.0±0.1 for Cr (total). The equilibrium isotherms were determined and the
results were analyzed using the Langmuir and Freundlichmodels. The best Cu removal
performance was observed on Sargassumat a maximum removal of 87.3%obtained for
an initial concentration of 20mgL −1Cu. The maximum uptake capacities for Cu (II)
were71.4, 19.3 and 11.4mgg−1of SargassumChlorococcumand GAC,respectively.
 Summary Report on the Effects of Heavy Metals on the Biological
Treatment Processes [6] The effects of Copper,Chromium,Nickel and Zinc
individually and in combination on biological treatment processes have been studied at
the Robert A. Taft Sanitary Engineering centre. This study resulted from a suggestion by
the national technical task committee on industrial wastes that the centre study the
metallic waste from the plating industry from the stand point of their effects on biological
treatment.
 5. MATERIALS AND METHODS

5.1. Chemicals

Pure and analytical grade chemicals were used in all experiments. Chemicals required for the
experiment are-

 Mixed Bacteria Strain from Sludge

 Copper sulphate ( Make : Rankem, India )
 Distilled Water (H2O)
 Glucose(C6H12O6) ( Make: Qualigens, India )
 Calcium Sulphate (CaSO4.2H2O) ( Make : Central Drug House, India )
 Magnesium Sulphate (MgSO4.7H2O) ( Make : Merck, India )
 Potassium dihydrogen orthophosphate (KH2PO4) ( Make : s.d.fine chemical, India )
 Iron(II) Sulphate (FeSO4) ( Make : Merck, India )
 Ammonium Sulphate (NH2(SO4)2) ( Make : Merck, India )
 Di-Potassium hydrogen orthophosphate (K2HPO4) ( Make : Central Drug House, India )
 Ethanol(C2H5OH) ( Make : Merck, India )

5.2. Glassware and Apparatus

The glasswares and other materials used the study are:
 Conical Flasks

 Measuring Cylinder

 Eppendorf

 Beakers

 Pipette

 Cotton Plug

 Thread
  Paper

 Filter Paper

The apparatus used in the experiment:

 Laminar airflow (Make : Macro Scientific Works Pvt. Ltd )

 BOD Incubator and Shaker (Make : Macro Scientific Works Pvt. Ltd )

 Incubator

 Heater

 Laboratory Weighing Machine ( Make : Precisa 125A SCS )

 Refrigerator
 Hot Air Oven
 UV Visible Spectrophotometer

5.3 Preparation

5.3.1 Microbial Culture

The activated sludge sample that was used to prepare microbial culture was obtained from
secondary clarifier of the Municipal Sewage Treatment Plant Pilani.The sludge sample also
contained solid impurities, which were removed by the following procedure:

Take around 20 mL of activatedsludge 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
as the solid particles would have settled. 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.

5.3.2 Preparation of Stock Cu(II) Solution and Glucose

Take a 1L conical flask. Wash it properly to remove any impurities. Take 1L distilled water in
the flask and dissolve 3.9322 g of Copper Sulfate in it to make 1000 ppm of stock Cu(II)
solution. Then plug the flask with cotton.
To prepare D-Glucose, dissolve 10g of it in 1L of distilled water in a conical flask.

5.3.3 Dilution of Stock Solution to 10 ppm

Since we will use 10 ppm Cu solution in our study, the 1000 ppm stock solution is diluted. 1mL
of stock solution is added to 100mL distilled water to make the resulting solution 10 ppm.

5.3.4 Culture Media

The culture media also known as Minimal Salt Media MSM) is prepared using the following
salts:

 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

The salts are weighed on the electronic weighing machine in the lab. These salts are added to 1L
of distilled water in a conical flask and dissolved properly. The flask is then plugged with cotton
and wrapped with paper. After this AUTOCLAVING is done at 121˚C and 121 KPa. In
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.

Autoclaving is done to kill the microbes present in the media. MSM acts a culture medium or the
nutrient supply for the growth of micro-organisms.
5.4 Batch Study

The batch study is spread over six days to observe the bioremediation of Cu (II). After each day
we change the volume of glucose and Cu(II) added to the sample.

DAY 1: 1 mL D-Glucose and 2ml microbial culture prepared are added to 100 mL MSM in a
250 mL conical flask inside the LAMINAR FLOW CHAMBER. The flask is then plugged with
cotton and wrapped with paper and kept in the ORBITAL SHAKING INCUBATOR for 24
hours at 37 ˚C, 110-115 rpm. The sample becomes turbid which indicates the growth of
microbes.

DAY 2: 0.8 mL D-Glucose, 0.2 mL 10 ppm Cu (II) solution and 2mL of DAY 1 sample
prepared are added to 100 mL MSM in a 250 mL conical flask inside the LAMINAR FLOW
CHAMBER. The flask is then plugged with cotton and wrapped with paper and kept in the
ORBITAL SHAKING INCUBATOR for 24 hours at 37 ˚C, 110-115 rpm. The sample becomes
turbid which indicates the growth of microbes.

DAY 3: 0.6 mL D-Glucose, 0.4 mL 10 ppm Cu (II) solution and 2mL of DAY2 sample prepared
are added to 100 mL MSM in a 250 mL conical flask inside the LAMINAR FLOW CHAMBER.
The flask is then plugged with cotton and wrapped with paper and kept in the ORBITAL
SHAKING INCUBATOR for 24 hours at 37 ˚C, 110-115 rpm. The sample becomes turbid
which indicates the growth of microbes.

DAY 4: 0.4 mL D-Glucose, 0.6 mL 10 ppm Cu (II) solution and 2mL of DAY 3 sample
prepared are added to 100 mL MSM in a 250 mL conical flask inside the LAMINAR FLOW
CHAMBER. The flask is then plugged with cotton and wrapped with paper and kept in the
ORBITAL SHAKING INCUBATOR for 24 hours at 37 ˚C, 110-115 rpm. The sample becomes
turbid which indicates the growth of microbes.

DAY 5: 0.2 mL D-Glucose, 0.8 mL 10 ppm Cu (II) solution and 2mL of DAY 4 sample
prepared are added to 100 mL MSM in a 250 mL conical flask inside the LAMINAR FLOW
CHAMBER. The flask is then plugged with cotton and wrapped with paper and kept in the
ORBITAL SHAKING INCUBATOR for 24 hours at 37 ˚C, 110-115 rpm. The sample becomes
turbid which indicates the growth of microbes.

DAY 6: 0.05 mL D-Glucose, 1 mL 10 ppm Cu (II) solution and 2mL of DAY 5 sample prepared
are added to 100 mL MSM in a 250 mL conical flask inside the LAMINAR FLOW CHAMBER.
The flask is then plugged with cotton and wrapped with paper and kept in the ORBITAL
SHAKING INCUBATOR for 24 hours at 37 ˚C, 110-115 rpm. The sample becomes turbid
which indicates the growth of microbes.

5.5Study of Growth of Microorganisms

In order to see if the study performed is correct, a study is performed using UV-Vis
SPECTROPHOTOMETER. It is used to find the absorbance of each of the samples prepared
using MSM as a reference sample. The absorbance of each of the samples is measured by taking
at least 3 readings.

Also, we calculate the weight of biomass in 50mL of each of these samples by the following
procedure:

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 minat 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 in each of the samples.

The weight of biomass is plotted against the absorbance of each sample.

 6. CONCLUSION

We started the study by doing literature survey. Then we moved on to actually performing our
lab project. We brought the sludge from the sewage treatment plant and prepared a microbial
culture. Then, we made MSM and 10 ppm Cu (II) solution from 1000 ppm stock Cu (II) solution.
After this our 6 Day batch study began in which the amount of glucose added decreased each day
and the volume of Cu (II) solution increased each day by the same amount. Every day the sample
was kept in the ORBITAL SHAKING INCUBATOR for 24 hours and turbidity was observed in
the sample to confirm the growth of microbes. Then, we confirmed the growth of
microorganisms by doing a study using UV-Vis Spectrophotometer which is used to measure the
absorbance of samples using a reference sample (which is MSM here). We also calculated the
weight of biomass in each sample. Then a plot of biomass against absorbance was made.
 7. REFERENCES
[1Srivastava 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

[2]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.

[3] 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.

[4] Christophe Espírito Santo, Nadine Taudte, Dietrich H. Nies, and Gregor Grass,Contribution

of Copper Ion Resistance to Survival of E.Coli on Metallic Copper surfaces,Applied and
Environmental Microbiology,74,4:(2008) 977-986.

[5] Maria Lourdes J.A.J. Jacinto, Carlos Primo C. David, Teresita R. Perez, Benjamin R. De
Jesus, Comparative efficiency of algal biofilters in the removal of chromium and copper from
wastewater,Ecological Engineering 35 (2009) 856–860.

[6] E. F. Barth, M. B. Ettinger, B. V. Salotto and G. N. McDermott, Summary Report on the

Effects of Heavy Metals on the Biological Treatment Processes,Journal Water Pollution Control
Federation 37,1(1965) 86-96.