Al-Azhar University

Faculty of Science
Botany and Microbiology Department

BIOTECHNOLOGICAL APPLICATION OF
WASTEWATER TREATMENT USING FUNGI

A Thesis
Submitted for the Degree of Doctor of philosophy of
Microbiology

BY
Mohamed Said Mahmoud Mohamed

B.Sc. Microbiology Chemistry, Al-Azhar University (2004).

M.Sc. Microbiology, Al-Azhar University (2009).

Under Supervision of

Prof. Dr. H.H. El-Sheikh Prof. Dr. M.M. El- Shafei

Prof. of Microbiology,
Botany & Microbiology
Department, Faculty of Science.
Director of Regional Center for Mycology
& Biotechnology (RCMB),
Al-Azhar University.
Prof. of Sanitary & Environmental
Engineering,
Director of Sanitary &
Environmental institute (SEI),
Housing & Building National
Research Center (HBRC).

Dr. M.M. El-Tayieb

Lecturer in Sanitary & Environmental institute (SEI), Housing & Building
National Research Center (HBRC).

2012


( )2004
( )2009

()
( )

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1433 2012 -
ACKNOWLEDGEMENT

ACKNOWLEDGEMENT

First, and foremost, I must thank Allah, the Most

Grateful and Most Faithful.
I would like to express my deepest gratitude and
sincere thanks to Prof. Dr. H.H. El-Sheikh, Professor of
Microbiology, Botany and Microbiology Department,
Faculty of Science. Director of the Regional Center for
Mycology and Biotechnology, Al-Azhar University for
planning the work program, his kind supervision, guidance
and encouragement throughout the course of this thesis.
My deepest appreciation is also extended to Prof. Dr.
M.M. El-Shafei, Professor of sanitary and environmental
engineering. Director of Sanitary & Environmental institute
(SEI), Housing & Building National Research Center
(HBRC) for her help, suggestions and kind supervision
during the implementation of the work.
Thanks to Dr. M.M. El-Tayieb, Lecturer of inorganic
chemistry, Sanitary & Environmental institute (SEI),
Housing & Building National Research Center (HBRC) for
her supervisions, sincere advice, best efforts and
cooperation throughout this thesis.
My thanks and gratitude are also extended to Dr.
T.M. Abd El-Ghany, Assistant Professor of Mycology,
Botany and Microbiology Department, Faculty of Science,
Al-Azhar University, for his valuable guidance, help as
well as continuous encouragement.
I am also thankful to Prof. Dr. Samir El-Laboudy,
Professor and Head of Botany & Microbiology
Department, Faculty of Science and all staff members.
I am also thankful to all staff members at Sanitary &
Environmental Institute (SEI), Housing & Building
National Research Center (HBRC) and all staff members at
the Regional Center for Mycology & Biotechnology.
ACKNOWLEDGEMENT

ACKNOWLEDGEMENT

First, and foremost, I must thank Allah, the Most

Grateful and Most Faithful.

I would like to express my deepest gratitude and

sincere thanks to the management system in Scientific
Research and Technology Academy (SRT) and all staff
members for their financial support to my PhD thesis in
Title :
"Biotechnological application of wastewater
treatment using fungi ".
ACKNOWLEDGEMENT

.


:
" ".
DEDICATION

DEDICATION

To my beloved father, mother, brothers, sisters, wife and son,

To my doctors, friends and university,
To my beloved homeland,
Those give me the needed support and encouragement all the
time to complete this work.
DECLARATION

DECLARATION

I declare that this work has not been

submitted for any degree to this or any other
university.
ABSTRACT

ABSTRACT
Tannery wastewater sample from chromium stage,
Elmontaza tannery, Ein Elsira, Cairo, Egypt was selected
for this study. Tannery wastewater sample had high mixed
heavy metals pollutants and high organic loads.
Isolation of fungal species from tannery wastewater
sample had given up about fifteen fungal colonies at
wastewater sample diluted by ratio (1: 1) with doubled
distilled water. These fungal colonies represented five
genera. The most polluted ions (Cr6+, Pb2+, Cu2+ and Cd2+)
were selected for screening, tolerance and training studies.
A. niger strain was found to be the most active and
tolerated organism.
Training of A. niger strain increased the growth rate
as well as the removal efficiency of these metal ions than
the control strain and this removal had realized Langmuir's
and Freundlich's equations.
Also, batch experiments were studied to show the
effect of environmental conditions on the uptake of Cr6+ .
The maximum removal were obtained at pH 5.0 0.2,
contact time 2h, stirring rate 250 rpm and biomass weight
0.1g.
The high removal efficiency for the studied metal
ions at concentration 20 ppm in a single state was in the
following order Cr6+ (86%, 17.2 mg/g) > Pb2+ (70.5%, 14.1
mg/g) > Cu2+ (66.5%, 13.3 mg/g) > Cd2+ (55.5%, 11.1
mg/g).
The effect of physical and chemical pretreatments of
A. niger biomass on biosorption capacity of Cr6+ ions was
also studied indicated that alkali-treated biomass had high
removal efficiency.
Treatability of tannery wastewater (chromium stage)
has been applied using alkali-treated A. niger (free and
immobilized biomass) to remove toxic constituents. Batch
biosorption experiments were done at optimum conditions
ABSTRACT

by alkali-treated biomass showed decrease of all metal ions

present in tannery wastewater, it decreased chromium
(31%, 155 mg/g), cadmium (40%, 6.2 mg/g), copper
(57.4%, 1.55 mg/g), lead (46.1%, 7.1 mg/g), nickel (52.8%,
2.8 mg/g), iron (43.1%, 4.66 mg/g) and manganese ions
(61.9%, 1.3 mg/g). Also, it was effective in decreasing the
color absorbance and organic loads from tannery
wastewater; it decreased TDS, EC and salinity up to 32.1,
32.2 and 27.4 %, respectively. It also decreased COD up to
51.8 %, BOD up to 48.6 %, ammonia nitrogen up to 66.1
%, nitrate up to 63.0 % and phosphorous up to 43.3 %.
The immobilized biomass had gained several
advantages over freely suspended cells. It decreased
chromium (51%), cadmium (70.97%), copper (91.9%), lead
(79.9%), nickel (93.8), iron (58.3) and manganese ions
(87.6 %). Also, it was effective in decreasing the color
absorbance and organic loads from tannery wastewater; it
decreased TDS, EC and salinity up to 52.1, 52.2 and 47.4
%, respectively. It also decreased COD up to 71.8 %, BOD
up to 68.6 %, ammonia nitrogen up to 86.1 %, nitrate up to
83.0 % and phosphorous up to 63.3 %.
The availability of recycling of Ca-alginate and Ca-
alginate-biomass by eluting with 0.05N HNO3 was studied
for three subsequent cycles.
SEM examination with EDAX analysis and FT-IR
technique for free cell biomass and immobilized beads
were also investigated.
INTRODUCTION

INTRODUCTION
1. Water:-
Water is one of the most valuable resources on
Earth. The River Nile will remain by far the largest and
almost exclusive surface water resource in Egypt.
According to the 1959 Nile agreement, Egypts stable
share is 55.5 billion cubic meters per year. In addition to
this amount, the annual extraction of water from ground
water reservoirs is of the order of 4.8 billion cubic meters
(Hendy, 2006).
Water and sanitation have a great effect on human
health, food security and quality of life. Demands on water
resources for household, commercial, industrial and
agricultural purposes are increasing greatly. Yet water is
becoming scarcer globally with many indications that it
will become even scarcer in the future. More than one-third
of the worlds population roughly 2.4 billion people live
in water-stressed countries and by 2025 the number is
expected to rise to two-thirds (Natural Resources Defense
Council, 2006).
Growing demand for water due to the growing world
population and also an increase in the amount of water used
per capita is creating significant challenges to both
developed and developing countries (Prince of Orange,

1
INTRODUCTION

2002 and Ceres & Pacific Institute, 2009).

The use of wastewater is one of the most sustainable
alternatives to cope with water shortage. It would have a
number of advantages that include closing the gap between
supply and demand, stopping the pollution of fresh water
resources, providing sound solution to water scarcity and
climate change and helping to achieve millennium
development goals (Bahgat, 2009).
In the United States, according to California Gray
Water Law (1997) when water begins to become scarce,
one of the first sources of water will be gray water. Gray
water is defined as water from a household. In California it
is legal to use gray water from bathroom sinks, showers
and washing machines to irrigate landscape (Loutfy, 2010).
2. Wastewater :-
There are many types of wastewater including
domestic, commercial, industrial and agricultural. For
technical purposes wastewater can be divided into
industrial and urban sewage wastewater. Each type has a
different chemical makeup. The constituents of untreated
wastewater can be divided into three types: physical,
chemical and biological. Physical constituents are the
particles or solids in the effluent. Effluent is defined as
liquid waste. Chemical constituents include nutrients and

2
INTRODUCTION

heavy metals. Biological constituents include organisms

such as bacteria, protozoa, helminthes and viruses
(Derjicke & Verstraete, 1986).
2.1. Tannery Wastewater:-
Tanning is one of the oldest industries in the world.
During ancient times, tanning activities were organized to
meet the local demands of leather footwear, drums and
musical instruments. With the growth of population, the
increasing requirement of leather and its products led to
the establishment of large commercial tanneries
(Verheijen et al., 1996).
The tanning industry forms the backbone of the
Egyptian leather industry. The total numbers of tanneries
in Egypt are more than 300, of which more than 85%
adopt the chromium tanning process because of its
processing speed, low costs and greater stability of the
resulting leather along with simplicity of operation. In this
process about 60% - 70% of chromium reacts with the
hides. In other words, about 30%- 40% of the chromium
amount remains in the solid and liquid wastes.
Tanneries are typically characterized as pollution
intensive industrial complexes which generate widely
varying and high-strength wastewater. Major problems
are due to wastewater containing heavy metals, toxic

3
INTRODUCTION

chemicals, chloride, lime with high dissolved and

suspended salts and other pollutants (Uberoi, 2003).
During tanning process at least about 300 kg chemicals
are added per ton of hides. Tanneries generate wastewater
in the range of 30-35 L kg-1 skin/hide processed with
variable pH, high concentrations of suspended solids,
BOD, COD and tannins including chromium (Khan et
al., 1999 and Nandy et al., 1999).
In Egypt, the tannery wastewater is discharged
directly to the main domestic sewage pipeline without
proper treatment which adds difficulties to the sewer
system and to the wastewater treatment plants. Therefore,
the removal and reuse of the chromium content of these
wastewater is necessary for environmental protection and
economic reasons (Abdulla et al., 2010). Treatment of
tannery wastewater is carried out by physical or chemical
or biological or combination of these methods. Simplified
leather production chain and management of the effluents
associated are showed in Fig.(1) (Lefebvre et al., 2005
and Durai & Rajasimman, 2011).

4
INTRODUCTION

Fig.1: Simplified leather production chain and management

of the effluents associated (Lefebvre et al., 2005).
3. Wastewater Pollutants:-
Due to the industrial, day-to-day human activities
and technological advancements in this century, a large
amount of liquid wastes are produced and disposed into
the environment. The increased concern about the
environment and health aspects due to the discharge of
such liquid wastes containing toxic compounds have led
to stricter legislations concerning pollution control
measures (Barbose & Sant, 1989 and Faisal &
Hasnain, 2004).
5
INTRODUCTION

3.1. Heavy metal pollutants:-

Heavy metals are elements having atomic weight
between 63.545 and 200.5g, specific gravity greater than
4.0 and with an atomic density greater than 6.0 g/cm3.
Heavy metals are among the worst group of pollutants of
the environment. Heavy metals occur in immobilized form
in sediments and as ores in nature as natural phenomena
(volcanic activities or erosion) (Greenwood & Earnshaw,
1998 and Florea & Busselberg, 2006). However due to
various human activities like ore mining, milling, surface-
finishing industries and other industrial processes, the
natural biogeochemical cycles are disrupted causing
increased deposition of heavy metals in terrestrial and
aquatic environment (Neal et al., 1990; Faisal & Hasnain,
2004 and Malik, 2004).
Heavy metals are one of the most persistent
pollutants in water. Unlike other pollutants, they are
difficult to degrade, but can accumulate throughout the
food chain, producing potential human health risks and
ecological disturbances (Gardea-Torresdey et al., 2005).
It has also severe effects include reduced growth and
development, cancer, organ damage, nervous system
damage and in extreme cases death (Sandrin & Maier,
2003 and Horsfall & Spiff, 2005).

6
INTRODUCTION

Heavy metal pollution is a global issue, although

severity and levels of pollution differs from place to place.
At least twenty metals are classified as toxic with half of
them emitted into environment in concentrations that pose
great risks to human health. The common heavy metals that
have been identified in polluted water include arsenic,
copper, cadmium, lead, chromium, nickel, mercury and
zinc. The release of these metals without proper treatment
poses a significant threat to public health and the
environment because of their toxicity, bio-accumulating
tendency (biomagnifications), persistence and
accumulation in food chain processes. Therefore, it is
frequently desirable to measure and control the
concentration of these substances (El-Nady & Atta, 1996;
Inthorn et al., 1996; Nagase et al., 1997; Raji &
Anirudhan, 1998; Igwe & Abia, 2003; Tarley et al., 2004
and Ghorbani et al., 2008).
Among the toxic heavy metals, mercury, lead and
cadmium, called the big three are in the limelight due to
their major impact on the environment (Volesky, 1994).
Arsenic, chromium, copper and zinc are also toxic. Lead
and cadmium are potent neurotoxin metals (Puranik &
Pakniker, 1997).
Lead, cadmium and mercury are examples of heavy

7
INTRODUCTION

metals that had been classified as priority pollutants by the

US-EPA (Keith & Telliard, 1979 and US-EPA, 1998).
3.1.1. Chromium: It causes serious environmental
problems (Wang et al., 2007). Chromium in the aquatic
environment has been classified according to the US-
EPA as Group A of human carcinogens (Costa, 2003).
Chromate and dichromate extensively used in
electroplating, leather tanning, metal finishing, nuclear
power plants and textile industries (Barnhart, 1997).
Among its several oxidation states (e.g., divalent,
trivalent, pentavalent, and hexavalent), trivalent (Cr3+ and
CrOH2+) and hexavalent (HCrO4- and Cr2O72-) species of
chromium are mainly found in these industrial effluents.
Particularly, effluents from electroplating and leather
tanning facilities contain chromium at concentrations
ranging from tenths to hundreds of milligrams per liter
(mg/L) (Virendrakumar & Sharma, 1984; Xi et al.,
1996 and Faisal & Hasnain, 2004). Cr6+ is known to be
a strong oxidizing agent and a potential carcinogen, so, it
is very toxic to both plants and animals (Park & Jung,
2001 and Costa, 2003) whereas, Cr3+ is an essential
nutrient for plant and animal metabolism (Anderson,
1997). Although Cr3+ is less toxic than Cr6+ or nontoxic,
long-term exposure to a high concentration of Cr3+ may

8
INTRODUCTION

cause poisoning symptoms such as allergic skin reactions

(Rudolf & Cervinka, 2005). Also, the oxidation of Cr3+
would be produced during storage and sometimes through
the tanning process. When soluble Cr3+ is added to soil,
manganese oxides present in the soil may cause oxidation
to Cr6+. Chromium can replace other metals in biological
systems with toxic effects and its accumulation
throughout the food chain leads to serious ecological and
health problems (Ahalya et al., 2003).
Therefore, the discharge of Cr6+ to surface water is
regulated to < 0.05 mg/L, according to the US-EPA,
whereas; the total chromium (containing Cr3+, Cr6+, and
other forms of chromium) is regulated to be discharged at <
2 mg/L (Baral & Engelken, 2005 and Park et al., 2006).
The maximum permissible levels of Cr6+ in potable and
industrial wastewater are 0.05 and 0.1 mg/L, respectively
(James & Bartlett, 1984; Murti & Viswanathan, 1991;
Thyagarajan, 1992; Goyal et al., 2003; Krishna et al.,
2004 and Saifuddin & Kuruaran, 2005).
Due to the harmful effect of chromium on human
and living organisms, in addition to the cost of the
chromium metal it is suggested to be recovered from the
tanning wastewater (Kocaoba & Akin, 2002).

9
INTRODUCTION

3.1.2. Lead (Pb): Lead is a well known toxic metal which

is common in the soils and rocks of the earth. Its
widespread occurrence in the environment, especially in
soils comes from anthropogenic sources such as metal
smelting industrial emissions and vehicular exhaust gases
(Adeyemi, 2009). Lead is normally found in dyes,
pigments and tanning industries (Ademorati, 1992). The
maximum levels of lead could exert toxic effects on human
beings if consumed from the water or irrigated agricultural
products from the sites. Lead interferes with functions
performed by essential mineral elements such as calcium,
iron, copper and zinc. It also inhibits red blood cell
enzyme systems (Vasudevan & Streekumari, 2000).
Lead can displace calcium in bone to form softer denser
spots. It also inactivates cysteine containing enzymes,
allowing more internal toxicity from free radicals,
chemicals and other heavy metals. Moreover, hyper
reactivity and learning disorders have been correlated with
lead intoxication in children. Other defects include
decrease ability to follow instruction and poor learning
focus in children (Underwood, 2002).
3.1.3. Copper: Cu2+ is a ubiquitous metal present in the
environment and is the most common contaminant of
industrial effluents such as those produced by mining and

10
INTRODUCTION

metal processing (Anand et al., 2006). Cu2+ is also an

element essential for all living organisms as a co-factor for
a variety of enzymes; however, an excess of this element
can be mutagenic and can cause the appearance of highly
reactive oxygen radicals (Zapotoczny et al., 2006).
3.1.4. Cadmium: The sources of human exposure to Cd
include atmospheric, terrestrial and aquatic routes (Lopez
et al., 1994). Cadmium is used in dipped coatings on
metals-bearing, low-melting alloys, fire protection systems
and batteries (Ziagova et al., 2007). Cadmium is
transferred to humans via the food chain. The human body
has no specialized physiological route for cadmium
elimination (Nadig, 1990; Zhou & Kiff, 1991 and Say et
al., 2001). The most severe form of cadmium toxicity in
humans is itai-itai, a disease characterized by
excruciating pain in the bone (Yasuda et al., 1995). Other
health implications of Cd2+ in humans include; kidney
dysfunction, hepatic damage, hypertension and anemia
(Klaassen, 2001 and Bayramoglu et al., 2002). It had
been shown that Zn and Cu competitively inhibit Cd uptake
by cells (Endo et al., 1996; Kapoor et al., 1999 and Gadd,
2000).

11
INTRODUCTION

Table (1) heavy metals admissible concentrations and their

effects on plants (Olajire & Imeokpara, 2000 and Gardea-
Torresdey et al., 2005).
WHO Standard For
Metal Fresh Water Effects
MAC (mg/dm3)
Cadmium Decreases seed germination, lipid
0.005
(Cd) content and plant growth.
Decreases enzyme activity and plant
Chromium
0.05 growth; produces membrane damage
(Cr)
and root damage.
Copper Inhibits photosynthesis, plant growth
0.05
(Cu) and reproductive process.
Decreases photosynthetic activity,
Mercury water uptake and antioxidant
(Hg) ----- enzymes; accumulates phenol and
proline.
Reduces seed germination, dry mass
Nickel accumulation, protein production,
(Ni) ----- chlorophylls and enzymes; increases
free amino acids.
Reduces chlorophyll production and
Lead
0.05 plant growth; increases superoxide
(Pb)
dismutase.
Reduces Ni toxicity and seed
Zinc
5.0 germination; increases plant growth
(Zn)
and ATP/chlorophyll ratio.

WHO: World Health Organization; MAC: Maximum Admissible

Concentration (Safe limit).

The color of industrial wastewater is often a

significant characteristic especially in the textile, paper,
food and clothing industries. The coloring of industrial
wastewater is caused by metallic ions: yellow and green
colors are typical of chrome in its reduced form, blue of
copper, green of nickel, yellow and brown of iron.
Industrial dyes used in the textile, paper and leather
industries produce very intense colors which linger even

12
INTRODUCTION

after repeated dilution. The coloring of wastewater can also

be caused by suspensions of colloidal and oily substances,
fats and lubricants. In other cases, the color may develop in
the water because of the effect of mixing different types of
wastes (Metcalf & Eddy, 1987 and Murugesan et al.,
2007).
Release of these pollutants without proper treatment
poses a significant threat to both environment and public
health. Thus their treatment becomes inevitable (Sandrin
& Maier, 2003).
4. Treatment Process:-
It is only natural for industry to presume that its
wastewater can best be disposed of in the domestic sewer
system. However, city authorities should not accept any
wastewater discharges into the domestic sewer system
without first learning the facts about the characteristics of
the wastewater, the sewage systems ability to handle them
and the effects of the wastewater upon all components of
the city disposal system. Institution of a sewer ordinance
restricting the types or concentrations of wastewater
admitted in the sewer leading to a treatment plant; is one
means of protecting the system (Crites & Tchobanoglous,
1998). Many conventional processes were carried out to
treat wastewater:-

13
INTRODUCTION

4.1. Adsorption Process:-

Adsorption is the ability of the adsorbate to adhere
or attach to the adsorbent. It is a well-established separation
technique to remove dilute pollutants as well as to recover
valuable products from aqueous streams (Chia-Chang &
Hwai-Shen, 2000).
Adsorption was first observed by Lowitz, 1785 and
was soon applied as a process for removal of color from
sugar during refining. In the latter half of the nineteenth
century, American water treatment plants used inactivated
charcoal filters for water purification. In (1929) the first
granular activated carbon (GAC) units for treatment of
water supplies were constructed in Germany and in (1930)
at Bay City, Michigan (Montgomery, 1985).
Adsorption is divided in two types; one is due to
forces of physical nature called Vander-Waal's force
(relatively weak). They are not sufficiently strong to
influence the reactivity of the molecule adsorbed. The
second type is considerably stronger. The adsorbed
molecules are held to the surface by valence force of the
same type as those occurring between bond atoms in
molecules. This is known as chemisorption and the heat
evolved is of the order 10 to 100 kilo calories (k Cal) per
mole, compared to physisorption which has less than 5 kilo

14
INTRODUCTION

calories (k Cal) per mole (Motoyuki, 1990).

Adsorptive removal of heavy metals from aqueous
effluents which have received much attention in recent
years is usually achieved by using activated carbon or
activated alumina (Igwe & Abia, 2005). The conventional
adsorbents were employed in many processes for the
removal of heavy metals from wastewater such as chemical
precipitation, electrochemical treatment (chemical
oxidation or reduction), evaporative recovery, filtration,
reverse osmosis, ion exchange, membrane technologies and
adsorption on activated carbon (Padma et al., 2003). These
techniques have significant disadvantages including
incomplete metal removal, the need for expensive
equipments, monitoring systems and high reagent or energy
requirements or generation of toxic sludge or other waste
products that require disposal (Aksu et al., 1999). They
also are ineffective or expensive especially when the heavy
metal ions are in solutions containing in the order of 1-100
ppm levels (Volesky, 1990 a,b and Volesky & Holan,
1995). A major drawback with precipitation was sludge
production. Ion exchange was considered a better
alternative technique, but it was not economically
appealing because of high operational cost (Pagnanelli et
al., 2000).

15
INTRODUCTION

A search for a low-cost and easily available

adsorbent had led to the investigation of materials of
agricultural and biological origin as potential metal
sorbents (Hammaini et al., 1999).
4.2. Biological Wastewater Treatment :-
Biological treatment of wastewater is evaluated as a
good treatment method for industrial effluents. There are
various techniques that have been developed to treat
wastewater by biological means. They accomplish what is
generally called secondary treatment (Farabegoli et al.,
2004).
Conventional wastewater treatment goes through
three stages. In the first stage which is primary treatment,
solids settle out of the wastewater. The solids are called
sludge and are usually taken to the landfill. The next stage
is called secondary treatment, where dissolved or
suspended materials are converted to a microbial biomass
so that they can be separated from the water. The third step
called tertiary treatment is where particles and nutrients are
removed. Finally, the water is disinfected, usually with
chlorine and sometimes ozone or ultraviolet (UV) radiation
(Reed, 1990).
An innovative heavy metal removal process called
bioremediation or biosorption which has gained enormous

16
INTRODUCTION

impetus. Biosorption is such a technology for treatment of

wastewater by interaction with live or dead biological
matter and is, at present, the most practical and widely
used approach for bioremediation of toxic metals
(Volesky, 1990 a). Biosorption is also defined as a process
that utilizes inexpensive dead biomass to sequester toxic
heavy metals and to remove contaminants from effluents
(Ting et al., 1991 and Singh et al., 1998).
The natural affinity of biological compounds for
metallic elements could contribute to economically
purifying heavily metal-loaded wastewater (Volesky, 1987;
Ferraz & Teixera, 1999 and Padma et al., 2003).
Biosorption technology employs various types of biomass
as source to trap heavy metals in contaminated water. The
biosorbent is prepared by subjecting biomass to various
processes like pretreatment, granulation and
immobilization, finally resulting in metal entrapped in bead
like structures. These beads are stripped of metal ions by
desorption which can be recycled and reused for
subsequent cycles (Gadd & White, 1989; Luef et al.,
1991; Mclean et al., 1994; Tobin et al.,1994; Volesky &
Holan, 1995; Volesky & May-Philips, 1995; Gupta &
Mukerji, 2001 and Alluri et al., 2007).
Heavy-metal ions can be entrapped in the cellular

17
INTRODUCTION

structure and subsequently biosorbed onto the binding sites

present in the cellular structure. This method of uptake is
independent of the biological metabolic cycle and is
known as biosorption or passive uptake. The heavy metal
can also pass into the cell across the cell membrane
through the cell metabolic cycle. This mode of metal
uptake is referred to as active uptake. The metal uptake by
both active and passive modes can be termed as
bioaccumulation (Fourest & Roux, 1992).
Bioaccumulation of metals and organic matter by
various micro- and macro-organisms was reported under
various conditions (Muraleedharan et al., 1994 and
Philip et al., 1999). The disadvantages include taking long
time for removal of metals. The idea behind all biological
methods of wastewater treatment is to introduce contact
with biological cells, which feed on the organic materials
(the chemical and biological oxygen demands (COD and
BOD), which are also significant problems in wastewater
(Wong & Yuen, 1996).
The use of biological materials including bacteria,
fungi, yeasts, algae, molds and etc. (Viladi, 2001 and
Madrid & Carmen, 2003) for heavy metal removal and
recovery technologies had gained important credibility
during recent years,

18
INTRODUCTION

1. Due to their natural occurrence, it uses relatively low-

cost, low-technology techniques, hence requires moderate
capital investment.
2. It's a natural process and environmental safe and it can
be used along side other technologies.
3. Because of the good performance, regeneration of
biosorbent, possibility of metal recovery, low cost of this
complexing material and could be considered as an eco-
friendly complementary device to the existing high cost
technologies.
Although the methodologies employed are not
technically complex, considerable experience and expertise
may be required to design and implement a successful
bioremediation program, due to the need to thoroughly
assess a site for suitability and to optimize conditions to
achieve satisfactory results (Hu, 2001).
5. Wastewater Microorganisms :-
Although most organisms in biological wastewater
treatment plants are microscopic in size, there are some
organisms are macroscopic. All living cells can be
classified as prokaryotic or eukaryotic as shown in Fig. (2).
Prokaryotic cells lack a nucleus and other membrane-bound
structures, while eukaryotic cells possess these structures.
The nucleus is the primary membrane-bound structure in

19
INTRODUCTION

eukaryotic cells. It regulates cellular activity and contains

the genetic information. Examples of membrane-bound
structures or organelles found in eukaryotic cells include
the Golgi apparatus (which regulates cellular metabolism)
and Lysomes (which contain hydrolytic enzymes)
(Gerardi, 2006).

Fig. (2) prokaryotic cell (a) and eukaryotic cell (b)

5.1. Fungal Bioremediation:-
Fungi are a Kingdom of eukaryotic microorganisms
which includes yeasts and moulds. Fungi have a vegetative
structure known as mycelium. The mycelium consists of a
rigid, branching system of tubes, through which flows a
multinucleate mass of cytoplasm. A mycelium arises by the
germination and outgrowth of a single reproductive cell, or
spore. Yeasts are exceptional fungi that cannot form a
mycelium, so are unicellular. Fungi decompose organic
matter and have significant roles in nutrient cycles. Most

20
INTRODUCTION

yeast and fungal species thrive in warm, sugary, acidic and

aerobic conditions; however they can grow under a wide
range of environmental conditions (Cochrane, 1958).
Wastewater has particular physicochemical and
microbiological characteristics. It can be expected,
therefore, that fungi occur abundantly in this environment
and the influence of environmental factors on their
qualitative and quantitative composition can be observed
more easily (Revankar & Lele, 2007).
Wastewater contains fungal spores, primarily from
the soil. Fungi have been reported to be capable of trapping
metals by cell wall components, altering metal uptake,
absorbing into the cells by forming metal complex,
producing metabolites extracellularly to chelate and
precipitate these metals, or storing metals in the cytosol in
association with various metal-binding proteins (Gadd,
1993 and Gadd et al., 1999).
Fungal biomass would have to be easily available in
substantial quantities. Fungi are used in a variety of
industrial fermentation or enzymatic production processes
such as P. chrysogenum in penicillin production or A. niger
in citric acid production which could serve as an
economical and constant supply source of biomass for the
removal of metal ions. Fungi can also be easily grown in

21
INTRODUCTION

substantial amounts using unsophisticated fermentation

techniques and inexpensive growth media. Therefore, a
fungal biomass could serve as an economical means for
removal/recovery of metal ions from aqueous solutions
(Singh et al., 2007).
Some compounds are poisonous to prokaryotic
organisms like bacteria but may be quite harmless to
eukaryotic organisms like fungi. In cases where a
wastewater are found to be toxic to bacteria, the use of
micro fungi can represent an efficient biological treatment
alternative (Clark, 1962; Bumpus et al., 1985; Archer et
al., 1994; Borchert & Libra, 2001; Maxima & Costa-
Ferreira, 2004; Khanongnuch et al., 2004; Mechichi et
al., 2006; Potentini & Rodriguez-Malaver, 2006; Susla et
al., 2007 and Demir et al., 2007).
Presence of a large and diverse population of fungi is
desired for the treatment of some industrial wastewater and
composting of organic wastes. Fungi have the ability to
degrade cellulose, tolerate low nutrient levels and ability to
excrete metabolites (e.g., organic acids) to form complexes
with metal ion, thus reducing its toxicity to the biomass
(Burgstaller & Schinner, 1993; Akthar & Mohan, 1995;
Castro et al., 2000; McGrath, 2001; Coulibaly, 2002 and
Baldrian, 2003). They are a versatile group, as they can

22
INTRODUCTION

adapt and grow under various extreme conditions of pH,

temperature and nutrient availability, as well as high metal
concentrations (Anand et al., 2006 and Baldrian &
Snajdr, 2006).
They offer the advantage of having cell wall material
which shows excellent metal-binding properties (Gupta &
Mukerji, 2001). The cell wall of A. niger, for instance,
consists chiefly of neutral carbohydrate (73% to 83%) and
hexosamine (9% to 13%), with smaller amounts of lipid
(2% to 7%) and protein (0.15% to 2.5%) (Cabuk et al.,
2005).
6. Biosorbent Pretreatment:-
Tuzun et al. (2005) had shown that heat inactivated
biomass could produce additional binding sites via
denaturation of proteins on cell wall structures. Alkali
treatment could cause hydrolysis of protein. It had been
reported that the performance of a microbial biomass
depended on its surface properties.
Gadd & White (1989) and Kapoor et al. (1999)
reported that the bioadsorptive capacity of the heat-
inactivated cells might be greater, equivalent or less than
that of living cell. However, the use of heat inactivated
biomass in industrial application might offer some
advantages over living cells, such as less sensitive to heavy

23
INTRODUCTION

metal ions concentration and adverse operating conditions

(i.e., pH and temperature). This is also due to the argument
that dead cells do not have toxicity limitations, no
requirement of growth and nutrient media and easy
desorption of adsorbed metal ions (Huang et al., 1988).
Kapoor & Viraraghavan (1998) examined A. niger
for biosorption of heavy metals and demonstrated that, the
pretreatment by NaOH resulted in a significant
improvement in metal ions removal in comparison with un-
pretreated biomass.
Both living and dead biomass could be used to
remove metals, but maintaining a viable biomass during
metal adsorption was difficult, because it required a
continuous supply of nutrients and avoidance of metal
toxicity to the microorganisms (Spinti et al., 1995). Using
of dead biomass could avoid these problems. Dead
biomass could be regenerated and reused for many cycles.
However, the use of dead biomass in powdered form had
some problems, such as; difficulty in the separation of
biomass after adsorption, mass loss after regeneration
(Kapoor & Viraraghavan, 1995,1998 and Yan &
Viraraghavan, 2000), low strength and small particle size,
which made it difficult to use in column applications
(Tsezos, 1990).

24
INTRODUCTION

Volesky (1989) solved the problem; dead biomass

could be immobilized in a granular or polymeric matrix to
improve the mechanical strength of the biosorbent. The
immobilized biomass was ideal for use in a conventional
ion-exchange column or adsorption column.
7. Immobilization of Biosorbent:-
An important part of the studies on biosorption is
based on the immobilization of the organisms on the natural
or synthetic polymeric materials (Baytak et al., 2004 and
Godlewska-Zykiewicz & Microchim, 2004).
In view of potential applications either industrial or
technical operations, in remediation of heavy metals from
aqueous solutions, the immobilization of the microbial cell
systems is generally necessary and provide additional
advantages over freely suspended cells. These included
easier handling, requiring less complex separation systems,
allowing a high biomass density to be maintained and
provide a greater opportunity for reuse and recovery (Ting
& Sun, 2000 and Tsekova et al., 2010).
Groboilot et al. (1994) and Mofidi et al. (2000)
indicated that, alginic acid is a heteropolysaccharide of -
L-glucuronic acid and -D-mannurinic acids and is found
in many algal species especially inside the brown algae.
This carboxylic polyelectrolyte is soluble in aqueous

25
INTRODUCTION

solutions and precipitates in the form of a co-acervate in

the presence of multivalent metal ions like Ca 2+, Co2+,
Fe2+, Fe3+ and Al3+.
Valdman et al. (2001) and Baldrian (2003)
reported that natural polymers such as cellulose
derivatives, alginate, chitosan and chitin had been mostly
used as the matrix for the immobilization of microbial cells
via entrapment. Immobilization of fungal cells in these
polymer supports could also enhance fungal cell
performance and adsorptive capacity of the biosorbent
system for the heavy metal ions. Scott (1987) cleared that
the choice of immobilization matrix is a key factor in
environmental application of immobilized biomass. The
polymeric matrix determines the mechanical strength and
chemical resistance of the final biosorbent particle, which
would be utilized for successive biosorptiondesorption
process.
The most extensively investigated biopolymer in
bioremediation studies is CMC (carboxy-methyl cellulose).
CMC is a natural polymer which is not toxic to microbial
cells, biodegradable and hydrophobic. The immobilization
methods using CMC could be performed under very mild
conditions without damage to living microbial cells (Arica
& Bayramoglu, 2005).

26
INTRODUCTION

The use of poly vinyl alcohol (PVA) as an

immobilization matrix started about 10 years ago. The PVA
immobilized biomass showed superior mechanical strength
and chemical stability (Galli et al., 2003). Also, PVA
offered several advantages like low cost, high durability, no
toxicity to viable cells and no loss in mass over a wide
range of pH (Khoo &Ting, 2001).
The fungus entrapped alginate beads were very
stable over the experimental pH range of 3.08.0. The
operational stability of the support under specified
experimental conditions is a very important parameter in
the cell entrapment. One of the most important
disadvantages of cell entrapment is the increase in the mass
transfer resistance due to the polymeric matrix. From this
point of view, entrapped fungus containing alginate could
also get rid of this disadvantage when compared with other
support materials, such as agar, carrageenan, poly vinyl
alcohol and 2-hydroxy ethyl methacrylate, because the
presence of carboxylic groups in the alginate structure
enhanced the heavy metal ion adsorption capacity of the
system with a combination of microbial cells (Arica et al.,
2001).

27
INTRODUCTION

8. Desorption and Reuse :-

Regeneration of biomass for heavy metal recovery
and biomass reuses have been suggested by utilizing
various acids and chelating agents such as HNO3, HCl,
H2SO4, Na2CO3 and EDTA and therefore, the metal ions
bound on the surface can be eluted (Yan & Viraraghavan,
2003; Lu et al., 2006 and Wang & Chen, 2006).
9. Treatment Mechanisms:-
The biosorption process involves a solid phase
(sorbent or biosorbent; biological material) and a liquid
phase (solvent, normally water) containing a dissolved
species to be sorbed (sorbate, metal ions). Due to higher
affinity of the sorbent for the sorbate species, the latter is
attracted and bound there by different mechanisms. The
process continues till equilibrium is established between
the amount of solid-bound sorbate species and its portion
remaining in the solution. The degree of sorbent affinity
for the sorbate determines its distribution between the solid
and liquid phases. Mechanisms of metal biosorption are
essential to be understood first in order to successfully
exploit the phenomenon and for regeneration of biosorbent
materials in multiple reuse cycles. The complex nature of
biosorbent materials makes this task particularly
challenging (Volesky, 1986 and Fourest & Roux, 1992).

28
INTRODUCTION

A number of mechanisms by which microorganisms

tolerated and remove heavy metals had been proposed.
Microorganisms modulated metal toxicity by maintaining a
low intracellular concentration of toxic metals via three
categories: biosorption of metal ions onto the surface of
microorganisms (Ozdemir et al., 2003 and Liu et al.,
2004), chemical transformation of metal ions and
intracellular uptake (accumulation in the pericellular or
endocellular regions of the cell) of metal ions by
microorganisms. The latter two processes needed viable
microorganisms and were called bioaccumulation.
Transport of metal ions across the cell membrane yields
intracellular accumulation, which is dependent on the cell's
metabolism. It is often associated with an active defense
system of the microorganism, which reacts in the presence
of toxic metal. So, the biosorption mechanisms may be
classified according to various criteria. According to the
dependence on the cell's metabolism, biosorption
mechanisms can be divided into: metabolism dependent
and non-metabolism dependent. According to the location
where the metal removed from solution is found,
biosorption can be classified as extra-cellular accumulation
and cell surface sorption and intracellular accumulation
(Aksu et al., 1991; Muraleedharan et al., 1991; Gadd &

29
INTRODUCTION

White, 1993; Bridge et al., 1999; Dursun et al., 2003 and

Uslu et al., 2003).
During non-metabolism dependent biosorption,
metal uptake is by physico-chemical interaction between
the metal cations and the cell surfaces of microorganisms
which are negatively charged (Cell walls of microbial
biomass, mainly composed of polysaccharides, proteins
and lipids have abundant of various anionic structures such
as ketones, aldehydes, carboxyl, hydroxyl, sulphate,
phosphate and amino groups for binding metal ions (Chen
& Hao, 1998; Kuyucak & Volesky, 1998; Bayramoglu et
al., 2003 and Zhou et al., 2004). This type of biosorption,
i.e., non-metabolism dependent is relatively rapid and can
be reversible. This is based on physical adsorption, ion
exchange and chemical sorption. It is often followed by a
slower metal binding process in which additional metal ion
is bound, often irreversibly. This slow phase of metal
uptake can be due to a number of mechanisms, including
covalent bonding, surface precipitation, redox reactions,
crystallization on the cell surface or, most often, diffusion
into the cell interior and binding to proteins and other
intracellular sites (Volesky, 1990 a; Crist et al., 1994 and
Ozer et al., 1999).
Several varied mechanisms exist by which fungi are

30
INTRODUCTION

able to deal with metals in such environments, e.g.

extracellular precipitation of secondary minerals, metal
binding to fungal cell walls, intracellular sequestration and
complexation, compartmentation or volatilization. The
extracellular precipitation of secondary minerals in
particular by fungi, often occurring as a result of
oxalate/oxalic acid production which immobilizes metals in
the process, is of environmental significance (Yakubu &
Dudeney, 1986; Malik, 2004 and Adeyemi, 2009).
Considering the above mechanisms of metal
resistance in fungi, it is expected that screening of metal
tolerant fungi may provide strains with improved metal
accumulation (Volesky & Holan, 1995 and Huang &
Huang, 1996).
Metal removal or stabilization is likely to be the first
step to detoxificate co-contaminated sites, as inorganic
pollutants in the ionic forms inhibit remediation through
interaction with enzymes directly involved in
biodegradation (e.g. specific oxygenases) or in general
metabolism, by binding to the enzymes sulfhydryl-groups
(Gadd, 1990 and Romero et al., 2006).
10. Factors affecting bioaccumulation:-
Simple cells eat the organic material present in the
wastewater. Through their metabolism, the organic material

31
INTRODUCTION

is transformed into cellular mass, which is no longer in

solution but can be precipitated at the bottom of a settling
tank or retained as slime on solid surfaces or vegetation in
the system. The water exiting the system is then much
clearer than it entered it. A key factor is the operation of
any biological system is an adequate supply of oxygen.
Indeed, cells need not only organic material as food but
also oxygen to breathe, just like humans. Without an
adequate supply of oxygen, the biological degradation of
the waste is slowed down (Gharieb et al., 1998).
The physiological state of the organism, the age of
cells, the availability of micronutrients during their growth,
metal ions concentration, the presence of certain co-ions
and the environmental conditions during the
bioaccumulation process were important parameters that
affect the performance of a living biosorbent (Volesky,
1990 a).
Biotransformation and biosorption are emerging
technologies, which utilize the potential of microorganisms
to transform or to adsorb metal ions (Chen & Hao, 1998).
Microbial viability was essential for biotransformation as
these reactions are enzyme mediated. Generally metal ions
were converted into insoluble form by specific enzyme
mediated reactions and were removed from the aqueous

32
INTRODUCTION

phase (Brierley et al., 1986). Endocellular accumulation

(bioaccumulation) of metals involved metabolism-
dependent uptake and storage in intracellular organelles
such as vacuoles, or binding to cysteine-rich small proteins
such as metallothioneins (Gadd & Rehm, 1988).
11. Factors affecting Biosorption:-
The investigation of the efficacy of the metal uptake
by the microbial biomass is essential for the industrial
application of biosorption, as it gives information about the
equilibrium of the process which is necessary for the design
of the equipment.
The following factors affect the biosorption
process:
1. Temperature seems not to influence the biosorption
performances in the range of 20-35C.
2. pH seems to be the most important parameter in the
biosorptive process: it affects the solution chemistry of the
metals, the activity of the functional groups in the biomass
and the competition of metallic ions.
3. Biomass concentration in solution seems to influence the
specific uptake: for lower values of biomass concentrations
there is an increase in the specific uptake.
4. Biosorption is mainly used to treat wastewater where
more than one type of metal ions would be present; the

33
INTRODUCTION

removal of one metal ion may be influenced by the

presence of other metal ions.
12. Biosorption equilibrium models - Assessment of
sorption performance:-
Examination and preliminary testing of solid-liquid
sorption system are based on two types of investigations:
equilibrium batch sorption tests and dynamic continuous
flow sorption studies (Gadd & Rehm, 1988). The
equilibrium of the biosorption process is often described by
fitting the experimental points with models usually used for
the representation of isotherm adsorption equilibrium. The
two widely accepted and linearized equilibrium adsorption
isotherm models for single solute system are Langmuir's
and Freundlich's model.
13. Biosorption Applications:-
Modern industry and human activities were, to a
large degree, responsible for contamination of the
environment through a high release of metals in the
environment at rates and concentrations sufficient to make
them pollutants. The notorious heavy metal pollution
requires an urgent solution (javaid et al., 2010). Although
the biosorption application is facing the great challenge,
there are two trends for the development of the biosorption
process for metal removal. One trend is to use hybrid
technology for pollutants removal, especially using living

34
INTRODUCTION

cells. Another trend is to develop the commercial

biosorbents using immobilization technology, and to
improve the biosorption process including
regeneration/reuse, making the biosorbents just like a kind
of ion exchange resin, as well as to exploit the market with
great endeavor (Wang & Chen, 2009).
Recent studies showed that strains isolated from
contaminated sites have an excellent ability of removing
significant quantities of metals both from aqueous solutions
and electroplating effluents (Malik, 2004). Also, metal-
sequestering properties of non-viable biomass provided a
basis for a new approach to remove heavy metals when
they occurred at low concentrations (Fujii et al., 1990;
Ohtake et al., 1990; Volesky, 1990 a; Volesky, 1990 b;
DeLeo & Ehrlich, 1994; Lovely & Phillips, 1994;
Ohtake & Silver, 1994; Shen & Wang, 1995 and Smith
& Gadd, 2000).
Parameswari et al. (2010) studied the role of
microorganisms in bioremediation of heavy metal in
contaminated soil and water ecosystem. Metal-resistant
fungi were isolated from municipal sewage contaminated
soil viz., Aspergillus niger, Phanerochaete chrysosporium
and Trichoderma viride. The laboratory study was
conducted to determine the Minimum Inhibitory
Concentrations (MICs) of Ni2+ and Cr6+ for isolated fungal

35
INTRODUCTION

cultures. The MICs ranged from 75 to 100 mg L-1 for Cr6+,

followed by Ni2+ (50 to 100 mg L-1), depending on the
isolate. These isolates were tested for their metal
biosorption potential for Cr6+ and Ni2+ in vitro condition.
Biosorption experiments were conducted with initial metal
concentrations of 25, 50, 75 and 100 mg L-1 with a contact
time of 48 h and wet fungal biomass (6 mm diameter) at
25C. Maximum biosorption of Cr6+ and Ni2+ ions was
found at 100 mg L-1. Among the fungi P. chrysosporium
accumulated 64.25 % of Cr6+ and 57 % of Ni2+ than other
fungi.
Ezzouhri et al. (2009) studied thirty-six micro-
organisms, represented by fungi and yeasts strains, were
isolated from heavy metal contaminated sites in Tangier,
Morocco. Filamentous fungi isolated belonged to the
genera Aspergillus, Penicillium, Fusarium, Alternaria and
Geotrichum. They were screened for their resistance to
heavy metals. The results revealed that the majority of the
isolates were resistant to Pb2+, Cr6+, Cu2+ and Zn2+, whereas
to Cd2+, only the fungus Penicillium sp. was able to grow.
The level of resistance depended on the isolate tested, as
well as the site of its isolation. Minimum inhibitory
concentrations (MICs) for Pb2+, Cr6+, Cu2+ and Zn2+ were
also determined. Aspergillus and Penicillium isolates were
the most tolerant to the heavy metals. Their MIC ranged

36
INTRODUCTION

from 20 - 25 mM for Pb2+, followed by 15 - 20 mM both

for Cu2+ and Zn2+ and 10 - 15 mM for Cr6+. These fungi
have shown a high level of resistance to all metals tested,
which makes them attractive potential candidates for
further investigations regarding their ability to remove
metals from contaminated wastewater.
Bai & Abraham (2001) investigated the Cr6+
sorption ability of powdered biomass of Rhizopus
nigricans at the best operating conditions. They studied the
influence of solution pH, agitation, Cr6+ concentration,
biomass dosage, contact time, biomass particle size and
temperature. The results cleared that, the optimum pH for
biosorption of Cr6+ was found to be 2.0. Higher adsorption
percentage was noted at lower initial concentrations of Cr
ions, while the adsorption capacity of the biomass
increased with increasing concentration of ions. Optimum
biomass dosage was observed as 0.5% (w/v). More than
75% of the ions were removed within 30 min of contact
and maximum removal was obtained after 8h. Biomass
particles of smaller size (90 m) gave maximum
adsorption (99.2%) at 100 ppm concentration. The
adsorption capacity increased with increase in temperature
and agitation speed and the optimum were determined as
45C at 120 rpm.

37
INTRODUCTION

Srivastava & Thakur (2006) evaluated the

potential of Aspergillus sp. for the removal of chromium in
shake flask culture at different pH, temperature, inoculum's
size, carbon and nitrogen source. The maximum chromium
was removed at pH-6; temperature-30C, sodium acetate-
0.2% and yeast extract- 0.1%. Aspergillus sp. was applied
in 2 L bioreactor for removal of chromium and it was
observed that 70% chromium was removed after 3 days.
Awofolu et al. (2006) studied biomasses of five
fungi species (Aspergillus niger, Penicillium austurianum,
Saccharomyces cerevisiae, Mucor arcindloides and
Trichoderma reesi) and evaluated for their uptake of lead
ion from aqueous solution using batch systems. Both dead
and live fungal biomasses were comparatively studied for
their adsorption efficiencies. The effect of pH as one of the
primary factors that influences sorption efficiency of metal
ions in solution was also studied. The percentage uptake of
lead ion by fungi species ranged from: Aspergillus niger:
6.71- 64.95% and 66.91-95.27%; Penicillium austurianum:
44.47-98.85% and 75.57-94.21%; Saccharomyces
cerevisiae: 52.61-88.68% and 61.20-89.95%; Mucor
arcindloides: 83.78-93.13% and 62.91-97.65% and
Trichoderma reesi: 52.52-80.70% and 35.31-88.13% for
dead and live biomass, respectively.

38
INTRODUCTION

Cabuk et al. (2005) studied the effect of

pretreatment on the Pb2+ biosorption capacity of fungal
biomasses, Aspergillus versicolor, Metarrhizium anisopliae
var. anisopliae, and Penicillium verrucosum, was
investigated. For this purpose, the biomasses were
subjected to physical treatments such as heat and
autoclaving, and chemical treatments such as sodium
hydroxide, formaldehyde, gluteraldehyde, acetic acid,
hydrogen peroxide, commercial laundry detergent,
orthophosphoric acid and dimethyl sulfoxide. Dimethyl
sulfoxide, hydrogen peroxide and gluteraldehyde increased
biosorption of Pb2+ in comparison with the A. versicolor
live biomass. M. anisopliae var. anisopliae biomass
pretreated with hydrogen peroxide, gluteraldehyde,
commercial laundry detergent, dimethyl sulfoxide and
formaldehyde significantly improved biosorption of Pb2+ in
comparison with live biomass. Pretreatment with all
methods of P. verrucosum increased biosorption of Pb2+ in
comparison with live biomass. The maximum biosorption
capacity of A. versicolor biomass subjected to dimethyl
sulfoxide was 30.6 mg g-1.
Yazdani et al. (2010) studied a strain of Trichoderma
atroviride, isolated from a river passing through the metal
polluted Serdang industrial area, was studied for its uptake
and tolerance to Cu2+. This study found that the uptake

39
INTRODUCTION

capacity of T. atroviride for Cu2+ ranged from 0.77 to 11.20

mg/g in Potato Dextrose Broth in liquid media over the
Cu2+ concentration range of 25 to 300 mg/L.
Kacar et al. (2002) used the alginate beads and both
entrapped live and heat inactivated fungal mycelia of
Phanerochaete chryosporium for the removal of Hg2+ and
Cd2+ ions from aqueous solution in the concentrations
range of 30500 ppm. The biosorption of Hg2+ and Cd2+
ions by the biosorbents increased as the initial
concentration of Hg2+ and Cd2+ ions increased in the
medium. Biosorption equilibrium was occurred after one
hour (1h) and the adsorbed heavy metal ions didn't change
further with time. The effect of pH was also investigated
and the maximum biosorption of Hg2+ and Cd2+ ions on all
the tested biosorbents were obtained between pH 5.0 and
6.0. Temperature over the range 1545C had no
significant effect on the biosorption capacity. The
equilibrium was well described by Langmuir and
Freundlich biosorption isotherms. The alginate-fungus
beads could be regenerated using 10 mM HCl, up to 97%
recovery. The biosorbents were reused in three
biosorption-desorption cycles with negligible decrease in
biosorption capacity.
El-Morsy (2004) studied thirty-two fungal species
were isolated from a polluted watercourse near the Talkha

40
INTRODUCTION

fertilizer plant, Mansoura Province, Egypt. Aspergillus

niger, A. flavus, Cunninghamella echinulata and
Trichoderma koningii were isolated frequently. On the
basis of its frequency, Cunninghamella echinulata was
chosen for biosorption studies. Free and immobilized
biomass of C. echinulata sequestered ions in this
decreasing sequence is: Pb2+ > Cu2+ > Zn2+. The effects of
biomass concentration, pH and time of contact were
investigated. The level of ion uptake rose with increasing
biomass. The maximum uptake for lead (45 mg/g), copper
(20 mg/g) and zinc (18.8 mg/g) occurred at 200 mg/L
biomass. The uptake rose with increasing pH up to 4 in the
case of Pb2+ and 5 in the case of Cu2+ and Zn2+. Maximum
uptake for all metals was achieved after 15 min. Ion uptake
followed the Langmuir adsorption model, permitting the
calculation of maximum uptake and affinity coefficients.
Treatment of C. echinulata biomass with NaOH improved
biosorbent capacity, as did immobilization with alginate.
Immobilized biomass could be regenerated readily by
treatment with dilute HCl. The biomass-alginate complex
efficiently removed Pb2+, Zn2+ and Cu2+ from polluted
water samples. Therefore, Cunninghamella echinulata
could be employed either in free or immobilized form as a
biosorbent of metal ions in waste water.

41
INTRODUCTION

Yan & Viraraghavan (2001) studied immobilized

Mucor rouxii biomass in a polysulfone matrix. The
spherical immobilized biomass beads were packed in a
column. The biosorption column was able to remove metal
ions such as Pb2+, Cd2+, Ni2+ and Zn2+ not only from
single-component metal solutions but also from multi-
component metal solutions. Column kinetics for metal
removal was described by the Thomas model. For single-
component metal solutions, the metal removal capacities of
the beads for Pb2+, Cd2+, Ni2+ and Zn2+ were more than that
removed by multi-component metal solution containing
Cd2+, Ni2+ and Zn2+. The adsorbed metal ions were easily
desorbed from the beads with 0.05N HNO3 solution. After
acid desorption and regeneration with deionized water, the
beads could be reused to adsorb metal ions at a comparable
capacity.
Patel et al. (2005) studied the biological agents
viz. fungi and bacteria (Pseudomonas sp.) to develop
a suitable technology for purification of industrial
effluent. Pure culture of Pseudomonas sp. and locally
isolated fungal sp. from mix industrial effluents were
utilized in the study. The laboratory study was carried
out to know the tolerance of Pseudomonas sp. against
different heavy metals concentration. The
Pseudomonas sp. had shown capacity to grow in

42
INTRODUCTION

mixture of heavy metals (Cd2+, Co2+, Ni2+, Cr6+, Pb2+)

of 5 ppm concentration of each element after 24 hrs
of incubation. The Pseudomonas sp. and isolated
fungi were subjected to different working parameters
in order to find out the suitable conditions for the
bioremediation process. The study indicated that
bioremediation using Pseudomonas sp. could be
effectively carried out under neutral pH value with 48
hrs. to 72 hrs. of contact time. The fungal sp. was also
effective in removing heavy metals from mix
industrial effluent having pH nearly neutral. The
enhancement in the removal of heavy metals was
quite remarkable with 72 hrs. contact time compared
to 48 hrs. The Pseudomonas sp. and locally isolated
fungal sp. from the effluent were found suitable for
treatment purpose to purify the industrial wastewater.
Abdulla et al. (2010) studied the recovery of
chromium was carried out by using precipitation process.
To this purpose, two common precipitating agents lime and
cement dust were used. The effects of precipitating agent
concentration, settling rate and supernatant volume were
studied in batch experiments. Results showed that the
characteristics of tannery wastewater samples collected
from the chromium tanning stage showed COD (4538
ppm), Cr3+ (2350 ppm) and Cr6+ (900 ppm). The maximum

43
INTRODUCTION

chromium recovery (98%) was achieved using 2g/100ml of

lime and 2hr settling rate. For biological removal of Cr6+,
thirty four actinomycete isolates were found to tolerate up
to 100 ppm Cr6+, five of these isolates could tolerate Cr6+ at
concentration up to 2500 ppm. The supernatant was further
analyzed for bioremoval of soluble chromium using
chromium resistant actinomycetes. The results showed that
isolate S65 is the most efficient isolate where nearly 97%
of chromium was removed over 4hrs period. These results
surveys the development of technologies that have rendered
the tanning process eco-sustainable, where the combination
between the chemical precipitation and the biological
removal of chromium from tanning wastewater make it
meet the environment safely.
Tsekova et al. (2010) investigate the immobilization
technology. The principal techniques that are available for
the application of biosorption are based on adsorption on
inert supports, on entrapment in polymeric matrix, on
covalent bonds in vector compounds, or on cell cross-
linking. the immobilization was carried out to Aspergillus
niger, strain B 77, was by inclusion in two different
polymers: poly vinyl alcohol hydrogel (PVA) and Ca-
alginate. The biomass/polymer matrices were formed into
equal size unites of the cubes and spheres, and the resulting
biomass/polymer matrices were used to remove heavy

44
INTRODUCTION

metals (Cu2+, Mn2+, Zn2+, Ni2+, Fe3+, Pb2+ and Cd2+) from
wastewater in shake flask experiments. Total biosorption
capacities of the biosorbents were in the following order:
free cells (33.3 mg/g) < PVA biomass (39.8 mg/g) < Ca-
alginate-biomass (44.6 mg/g). The metal removal
efficiencies of the beads Ca-alginate biomass were 96.2%
for Cd2+; 90.0% for Pb2+; 80.0% for Fe3+; 72. 8% for Cu2+;
55.4% for Zn2+; 54.4% for Ni2+ and 52.3% for Mn2+, while
the removal efficiencies of cubes PVA- biomass for the
same heavy metals ions were: 95.0%; 88.0%; 80.0%;
67.1%; 58.5%; 48.9% and 44.6%, respectively. The results
obtained from these experiments, were compared with
those using dispersed biomass as a sorbent. Various
applications are available for biomass immobilization.

45
SCOPE, OBJECTIVE AND AIM OF THE WORK

Scope of the work

The scope of this work is to regulate the industrial
wastewater pollution to become environmentally and safely
added to the sewer systems.

Objective and Aim of the work

The objective of this study is to explore the
possibilities of biological materials especially fungal
species in treating the polluting load from tannery
wastewater (chromium stage). This treatment includes
decrease in heavy metal concentrations especially
chromium and other constituents like the organic load from
tannery wastewater (chromium stage). This study therefore
aimed at elucidating this technology with emphasis on their
processes and applications.

The focus of this study therefore concerns with:-

1- Assessing the physiological adaptation of fungi
through characterization of metal resistant fungi
isolated from tannery wastewater constituents
(chromium stage), quantify the inhibitory effects of
the studied metals (Cr6+, Pb2+, Cu2+, Cd2+) on the
fungal isolates (The heavy metal MICs for each
fungus), the effect of the studied metal ions on the

46
SCOPE, OBJECTIVE AND AIM OF THE WORK

fungal growth rate and the effect of training on such

metals on resistance and metals removal potentials.
2- Studying the biosorption process and all factors
affecting on it (pH, contact time, temperature,
stirring rate, biomass weight and metal ions
concentrations).
3- Studying the effect of physical and chemical
pretreatment of fungal biomass on the biosorption
process.
4- Studying the treatment of tannery wastewater
(Chromium stage), by using free fungal biomass.
5- Studying the effect of immobilization technology on
the treatment process and its availability to be
recycled.

47
MATERIALS & METHODS

Materials and Methods

I-Materials:-
I.1.Chemicals:-
All chemicals used in the present work including
potassium dichromate K2Cr2O7, lead nitrate Pb(NO3)2,
copper sulphate CuSO4.5H2O, cadmium sulphate
3CdSO4.8H2O, calcium chloride CaCl2, sodium alginate
C6H7O6Na, glutraldehyde CH2(CH2CHO)2, sodium hydroxide
NaOH and hydrochloric acid HCl were of analytical purity
grade. Solutions of Cr6+, Pb2+, Cu2+ and Cd2+ were prepared
as stock solutions by dissolving an accurate weighted
quantity from each metal salt in DDW to obtain a
concentrated solution and other concentrations were
obtained by dilution. 0.1M from HCl and NaOH were used
to adjust the pH to the desired values.
I.2. Media:-
For experimental studies, maintenance of stock
cultures and identification, the following range of media
were used (Onions et al., 1981; Smith & Onions, 1983;
Tortora et al., 1995 and Sharma & Pandey, 2010) :-
1.2.A-Potato Dextrose Agar Medium (PDA): is one of
the most commonly used culture media
Ingredients: Potato (peeled) 200 g; Dextrose 20 g; Agar 20
g and DDW 1.0 L.

48
MATERIALS & METHODS

1.2.B-Czapek's-Dox Agar Medium (Cz-DA):

Ingredients: Sucrose 30.0 g, (NaNo3) 3.0 g, (K2HPO4) 1.0
g, (KCl) 0.5 g, (MgSO4.7H2O) 0.5 g, (FeSO4.7H2O) 0.01 g,
agar 15.0 g and DDW 1.0 L.
1.2.C-Czapek Yeast Autolysate Agar Medium (CYA):
Ingredients: Yeast extract 5.0 g, (NaNO3) 3.0 g, (K2HPO4)
1.0 g, (KCl) 0.5 g, (MgSO4.7H2O) 0.5 g, (FeSO4.7H2O)
0.01 g, Sucrose Solution 30 g / 100 ml, Agar 15.0 g and
DDW 1.0 L. pH 7.3 0.2 at 25C.
Method of preparation :- Component except sucrose
solution were reached to 900 ml DDW then Autoclave for
15 min at 1.5 psi and 121C. Aseptically add sterile sucrose
solution then pour into sterile Petri dishes.
1.2.D-Sabouraud's Dextrose Agar Medium (SDA):
Ingredients: Peptone 10.0 g, Glucose 40.0 g, Agar 15.0 g
and DDW 1.0 L.
1.2.E-Malt Extract Agar Medium (MEA):
Ingredients: Malt extract 20.0 g, peptone 1.0 g, glucose
20.0 g, agar 20.0 g and DDW 1.0 L.
Each medium was prepared by adding the solid
ingredients to one liter DDW then dissolved with heating to
70C and stirring. Media were sterilized by autoclaving at
121C for 15 min and when it became cool poured in 9 mm
diameter Petri-dishes. The liquid form of a medium (broth)
was obtained by eliminating the agar from its ingredients.
49
MATERIALS & METHODS

I.3. Leather process description:-

Beam House Processes Tanning Processes Finishing Processes
1. Soaking 7. Pickling 11. Shaving
2. Liming Unhairing 8. Chrome Tanning 12. Dyeing & Fat
3. Washing 1,2 9. Retanning Liquoring
4. Trimming, Fleshing & 10. Sammying 13. Drying, Trimming
Splitting & Sorting
5. Bating "Deliming" 14. Finishing &
6. Washing Leather Product

Leather Process Description

I.4. Instrumentations:-
I.4.A. Millipore (Elix) UV- Milli-Q Advantage A 10
System:- Doubled Distilled Water (DDW) from Millipore
Instrument was used throughout the study.
I.4.B. pH Meter:-
BOECO PT-370 pH/ mv meter Germany was used for pH
measurements.
I.4.C. Atomic Absorption Spectrophotometer (AAS):-
The quantitative determinations of metal ions were
carried out using atomic absorption spectrophotometer
(iCE 3000 Series AA Spectrometer Thermo Scientific)
as shown in photo (1).

Photo (1): Atomic Absorption Spectrophotometer

50
MATERIALS & METHODS

I.4.D. UV/VIS Spectrophotometer:-

T70+ UV/VIS Spectrometer PG instruments
Ltd was used for spectrophotometer measurements of the
COD concentrations (using Closed Reflux method at 600
nm), Ammonia concentrations (using Phenate method at
425 nm), Nitrate concentrations (using Closed Brucin
Sulphate method at 410 nm) and Phosphorous
concentrations (using Molybdate Blue method at 690 nm)
according to Standard Methods for the Examination of
Water and Wastewater, 2005. It was also used for
measuring color absorbance of samples at different
wavelengths through Scan Spectrum Curves as shown in
photo (2).

Photo (2): UV/VIS Spectrophotometer

I.4.E. Multi-parameter analyzer:-
Multi parameter analyzer - HANNA HI 9828 -
Italy - pH / ORP / EC / DO was used for measuring of
pH, temperature, EC, DO, TDS and salinity as shown in
photo (3).

Photo (3): Multi-parameter analyzer

51
MATERIALS & METHODS

I.4.F. Scanning Electron Microscope (SEM):-

The examinations of fungal beads were done using
Jeol JSM-5500LV SEM at the Regional Center for
Mycology and Biotechnology, Al-Azhar University. The
beads were also examined using ESEM (FEI INSPECT
S Holland) at Housing and Building National Research
Center and EDAX for beads were done by using the same
instrument.
I.4.G. Fourier Transform Infrared Spectrometry (FT-IR) :-
Shimadzu S 201 PC spectrophotometer Japan
was used for infrared spectroscopy. Sample was prepared
as a disc (2 mg sample + 200 mg spectroscopy KBr).
. II-Experimental Methods:-
II.1. Samples collection site :-
Samples of wastewater from contaminated sites with
tannery wastewater - chromium stage ELMONTAZA
TANNERY, Ain El Sira, Old Cairo, Egypt, were collected
in sterilized glass bottles of 1.5L capacity, transported on
icebox as shown in photo 4 and Fig. 3. The collected
samples were then mixed to become one sample, analyzed
within 8 h and stored in a refrigerator at 4C. The
physicochemical characteristics of tannery wastewater
sample are listed in Table (2).

52
MATERIALS & METHODS

Photo (4):(A, B and C) Samples collection site (D) Tannery

wastewater before and after filtration

Fig.(3): UV Scan Spectrum Curve for tannery wastewater

sample.

53
MATERIALS & METHODS

Table (2): Physico-chemical analysis for tannery wastewater

(chromium stage) sample.
Constituents Values Units
Color Dark Green
580nm 0.296
Absorbance Absorbance
418 nm 0.396

Temperature 28.5 (C)

pH 4.01
TDS 57.87 ppt
EC 63.6 ms/cm
Salinity 73.2
COD 550 mg O2/L
BOD 120 mg O2/L
ORP 173
ammonia nitrogen 200.11 mgNH4+ /L
Nitrate 25 mg(NO3-)/L
Nitrite 30 mg(N02-)/L
Phosphorous 15 mgPO4+ /L
Oil and Grease 28.3 mg/L
Chromium 500 mg/L
Cadmium 15.5 mg/L
Copper 2.7 mg/L
Lead 15.4 mg/L
Nickel 5.3 mg/L
Iron 10.8 mg/L
Manganese 2.1 mg/L

54
MATERIALS & METHODS

II.2. Isolation of fungal strains :-

Wastewater samples were collected from El-Montaza
Tannery site (chromium stage) in sterile bottles on icebox.
Serial dilution and plate count technique were employed
(Jenson, 1968 and Benson, 1985). Aliquots of one ml of
diluted sample (1:1) were plated both onto PDA and MEA
plates (three replicates) to ensure the growth of fungi
present in samples. After at least three days of incubation at
30 1 C, developed up colonies were isolated, counted
and differentiated to the genus level. Purified isolates were
obtained by streaking colonies repeatedly on PDA medium
and observed under light microscopy. The relative
frequency of occurrence was calculated as the number of
species isolated divided by the total number of isolates.
II.3. Screening and identification of isolated fungal
strains :-
This is a preliminary study in which purified isolates
were screened on the basis of their tolerance to the studied
metal ions (chromium, lead, copper and cadmium). The
choice of the studied metal ions is based on their frequently
encountered together in tannery wastewater sample and on
their known toxicity as environmental pollutants. A disc of
mycelium approximately 5 mm in diameter was inoculated
on PDA plates supplemented with 5 ppm from each metal
ion of the studied metal ions in mixture. The inoculated

55
MATERIALS & METHODS

plates were incubated at 30C for at least 7 days. Only

species which exhibit tolerance were isolated and identified
using the keys of Pitt (1979). The purified fungal isolates
were identified using the most documented keys in fungal
identification [Barnett et al., 1991; Domsch et al., 1993;
Kurtzman & Fell, 2000 for yeast species; Booth, 1971
for the genus Fusarium; Barron, 1977; Ellis, 1971&1976
for hyphomycetes; Pitt, 1979 for Penicillia; Raper &
Fennell, 1965 and Samson, 1992 for Aspergilli]. The
fungal isolates were subjected for certain morphological
studies by an image analysis system using Soft-Imaging
GmbH software (analysis Pro ver. 3.0), as well as using
the newly introduced RCMB database management system
for Aspergilli and Penicillia identification at the RCMB,
Al-Azhar University. The identified fungal strains are;
Aspergillus tamarii, Aspergillus niger, Penicillium
chrysogenum and Rhizopus stolonifer.
II.4. Growth and culture conditions :-
Different incubation temperatures (15, 20, 25, 30, 35
and 40C) and different growth media (CzDA, MEA,
PDA and SDA agar medium) were used to determine the
optimal growth condition for isolates to be used in metal
tolerance studies. Agar plugs (5 mm in diameter) were
prepared and inoculated on the intersection point of two
lines drawn on the bottom of the test medium. Colony
56
MATERIALS & METHODS

diameter of the fungi was measured along the perpendicular

lines after seven days of incubation. Colony diameter is
used to reflect the growth of the fungi.
(M) = (L + W) / 2
Where, Mean diameter (M), Length (L) and Width (W).
Also, Relative percentage of change (RP) was measured
are related to those at 30C as considered 100%
(Zapotoczny et al., 2006).
II.5. Tolerance Study:-
II.5.A: Isolation on solid medium:-
This is a preliminary study on the effects of the
studied metal ions on fungal growth. PDA was used as a
solid medium for fungal isolation despite the fact that agar
may affect metal speciation and solubility, because a solid
medium was more appropriate for growth observations of
all fungi. Stock solutions of the metal salt were prepared in
DDW and were added separately to PDA medium at
concentrations of (10, 25, 50, 75 and 100 ppm) and in
mixed concentrations ranged from 40 to 400 ppm with
equal ratio for each metal ion. The control was obtained on
PDA medium without any metal ions. The plates were
inoculated with 5 mm agar discs from young fungal
colonies, pre-grown on PDA. Three replicates of each
concentration. The inoculated plates were incubated at
30C for at least 7 to 15 days and the growth was observed
57
MATERIALS & METHODS

by naked eye. Colony diameter is used to reflect the growth

of the fungi (Zapotoczny et al., 2006).
Also, the resistance of the selected isolates to studied
metal ions was determined by measuring MIC. The
minimum inhibitory concentration (MIC) is defined as the
lowest concentration of metal that completely prevented
visible growth of the isolate. The isolates exhibiting growth
after incubation were considered as tolerant to the metals.
II.5.B:- Toxicity testing on liquid medium:-
This is a toxicity testing for the most tolerant fungal
isolates using PDB as a liquid medium. An appropriate
amount of a stock solution of metal ions was added to the
liquid medium (vol/vol) of each conical flask (250 ml) to
reach the desired concentration and volume of 100 ml. The
medium was inoculated with three disks of young
mycelium from the edge of the solid stock PDA of selected
culture which was cut using a sterile corkborer, each 0.5 cm
in diameter (Zapotoczny et al., 2006).
The cultures were raised for seven days both in static
and shaking incubation conditions (100 ml) containing
different concentrations of metal ions (Treated) or without
any metal ions (Control). After the desired incubation
period (seven days), The biomass was washed thoroughly
with DDW and dried overnight on a pre-dried and pre
weighted filter paper at 80C till a constant weight was
58
MATERIALS & METHODS

achieved and the total growth was determined as dry

weight (mg/L of the medium). The growth rate value
(mg/h) was obtained by divided the total growth value over
the incubation period per hour (Anand et al., 2006).
II.6. Heavy-metals training:-
A. niger, an efficient strain to tolerate the studied
metal ions, was grew on PDB and trained by serial transfer
every three days to medium containing increasing
concentrations of metal ions. The media were adjusted to
pH 5.0 0.2, as metals occur as divalent cations at this pH.
Not-exposed strains grew on the same medium without
metal ions (control strain). Then, the differences in metal
removal between control and training biotypes were
studied. Metal tolerance was assessed by the mycelial dry
weight (mg/L of the medium). The broth was digested with
aqua regia (HCl: HNO3 at 3:1 ratio). The digested samples
were then measured by AAS.
II.7.Biosorption isotherm models:-
The Adsorption isotherms are equilibrium equations
that applied to conditions resulting after the adsorbate
containing phase has been in contact with the adsorbent for
sufficient time to reach equilibrium, the equilibrium time
from the kinetic studies was kept as contact time. Generally
both Langmuir's and Freundlich's isotherms are used for
explaining the adsorption of metal ions.
59
MATERIALS & METHODS

II.7.A. Langmuir isotherm model:-

The Langmuir isotherm can be derived by assuming
that, the adsorbent forms only a monolayer of atoms on the
adsorbate, that there is a finite adsorption area that the
process is reversible and that equilibrium exists. Although
the final result is rather simple, the derivation is rather
complex and lengthy. The math will be omitted here, but
the result is
Qe = x/m = ACe / 1+BCe (1)
Where:
Qe = mass of solute adsorbed per mass of adsorbent used
i.e. capacity of adsorbent for adsorbate, (mg adsorbed/ gm
adsorbent); x = mass of solute adsorbed, (mg); m = mass of
adsorbent, (g); Ce = equilibrium concentration of solute,
(mg/l); A= the slope of the isotherm at zero coverage,
(l/mg) and B= the concentration at which half coverage is
attained, (i.e. is a constant related to the energy or net
enthalpy of adsorption), (l/mg)
Values of qe were obtained by simple mass balance
calculations according to the following equation:
Qe=V/m (C0Ce) (2)
Where:
V = volume of metal solution, (lit); m = mass of adsorbent,
(g) and C0 = initial concentration of metal solution, (mg/l)

60
MATERIALS & METHODS

To linearize the Langmuir isotherm, we invert the

equation (1) and then multiplying both sides by Ce,
obtaining:
Ce/Qe = 1/A + B/A Ce (3)
If Ce/qe is plotted versus Ce the data should fit a
straight line, if it does not, the Langmuir isotherm is not
applicable! That is, one or more of the assumptions is not
valid. From the slope and the intercept of the straight line,
the values of A and B have been calculated (Gadd &
Rehm, 1988).
II.7.B.Freundlich isotherm model:-
This model, which depicts an empirical relationship,
is expressed as
Qe = x/m = K( Ce)1/n (1)
Where:
K = experimental constant (Freundlich's constant)
indicative of the adsorption capacity of the adsorbent
(l/mg); n = experimental constant indicative of the
adsorption intensity of the adsorbent.
The Freundlich's equation can be linearized by taking
the logarithm of both sides of the equation yields
log (x/m) = log K+ 1/n log (Ce) (2)
If log (x/m) is plotted versus log (Ce) the data should
fit a straight line. Otherwise, the Freundlich's isotherm is
not applicable. If the data generates a straight line, the y-
61
MATERIALS & METHODS

intercept is log K, and the slope of the line is 1/n (Gadd &
Rehm, 1988).
II.8. Preparation of biosorbents:-
II.8.A.Fungal medium and cultivation:-
A. niger was grown on PDA media (plates or slants)
for approximately 4 days or until the plates were fully
grown. Inoculum of spores was transferred to 500- ml
Erlenmeyer flasks containing 75 ml of the cultivation
medium (PDB). After inoculation, the organisms were
grown on a rotary shaker at 30C and 150 rpm for three
days. After the cultivation was completed, the mycelium
was harvested by filtration through filter paper and washed
with DDW. The cultures were transferred to a dryer
overnight at 80C. Biomasses were then pulverized in clean
mortar and immediately frozen at -80C to become ready
for use as free fungal biomass (FC) (Awofolu et al., 2006).
II.8.B. Biomass pretreatment:-
Fresh fungal biomass was subjected to chemical
and physical pretreatment as following:
1- Washing with NaOH at different concentrations (0.1 and
0.2 N) for 15 min.
2- Washing with glutraldehyde at different concentration
(0.1 and 0.2% V/V) for 15 min.
3- Washing with CaCl2 at different concentrations (0.1 and
0.2% W/V) for 15 min.
62
MATERIALS & METHODS

4- Autoclaved for 15 min at 121C & 1.5 psi.

5- Boiled for 15 min in bi-distilled water.
Following the desired pretreatment, the fungal
biomass was washed with DDW until the pH of the
washing solution was close to neutral range (pH 6.8-7.2).
The biomass was then filtrated through filter paper and
washed with DDW. The cultures were transferred to a dryer
overnight at 80C. Biomasses were pulverized in clean
mortar and immediately frozen at -80C to become ready
for use as a pretreated biosorbent (Awofolu et al., 2006).
II.8.C.Immobilizing material and technique:-
Method described by El-Naggar et al., (2006) was
used to prepare Ca-alginate beads as shown in photo 5.
alkali pretreated A. niger. biomasses were immobilized by
inclusion in Ca-alginate polymer. The biomass/polymer
matrices were formed into equal size unites of spheres and
the resulting biomass/polymer matrices were used to
remove heavy metals from wastewater in shake flask
experiments.
The fungal biomass was immobilized using 3%
aqueous solution of sodium alginate. A desired weight of
fine biomass 0.1 g was taken and then mixed with 10 ml
alginate gel thoroughly. The biomass-alginate mixture were
kept in refrigerator for half an hour then injected into 2%

63
MATERIALS & METHODS

CaCl2 solution. The formed beads were left in CaCl2

solution for one hour and then washed twice with DDW.
The beads formed had a diameter ranged from 2.5
to 3.0 mm and the 10 ml alginate gel and biomass-alginate
gel gave about 500 beads. The obtained beads were kept in
refrigerator for use.

Photo (5): Immobilizing material and technique

II.9. Batch biosorption experiments:-
II.9.A. For synthetic metal ion solutions:-
When fungal biomass is placed in a solution
containing metal ions and agitated for an adequate time,
biosorption of metal ions occurs. Aqueous metal ions
solutions (100 ml) were used and the biosorption capacity
was determined at different environmental conditions. A
withdrawn sample from the solution was taken for
centrifugation and the supernatant was analyzed for

64
MATERIALS & METHODS

dissolved metal concentrations by AAS. Then the

biosorption capacity and isothermal studies were
conducted.
II.9.A.1. Effect of pH :-
0.1g of A. niger biomass was added to 100ml of
(Cr6+) 20 ppm at different pH values namely, 1.0 0.2, 3.0
0.2, 5.0 0.2 and 7.0 0.2 for different contact times 0,
30, 60, 90 and 120 min at temperature 25 3C and the
stirring of the solution was fixed at 250 rpm.
II.9.A.2. Effect of contact time :-
0.1 g of A. niger biomass was added to 100ml of
(Cr6+) 20 ppm at different contact times namely, 0, 60, 120,
300, 1440 min at temperature 25 3C, at pH 5.0 0.2 and
the stirring of the solution was fixed at 250 rpm.
II.9.A.3. Effect of temperature:-
0.1 g of A. niger biomass was added to 100ml of
(Cr6+) 20 ppm at different temperatures values namely, 10.0
3.0, 15.0 3.0, 25.0 3.0 and 50.0 3.0C at pH 5.0
0.2 and 250 rpm.
II.9.A.4. Effect of stirring rate:-
0.1 g of A. niger biomass was added to 100ml of
(Cr6+) 20 ppm at different stirring rates namely, 0, 100, 250
and 500 rpm, at pH 5.0 0.2 and at temperature 25 3C.

65
MATERIALS & METHODS

II.9.A.5. Effect of biomass weight:-

Different biomass weights namely; 0.05, 0.1, 0.5 and
1.0 gram was added to 100ml of (Cr6+) 20 ppm at pH 5.0
0.2, at temperature 25 3C and 250 rpm.
II.9.A.6. Effect of Cr6+ ion concentration:-
0.1 g of A. niger biomass was added to 100ml of
(Cr6+) at different conc. of Cr6+ namely; 20, 50 and 100
ppm at pH 5.0 0.2, at temperature 25 3C and 250 rpm.
II.9.B. For real tannery wastewater:-
For tannery wastewater biosorption studies, the
collected sample of tannery wastewater from tanning
process chromium stage was used. Batch biosorption
experiments were carried out in 500 ml Erlenmeyer flasks,
as follows: pre-weighed biosorbent samples (FC (0.1g) or
immobilized biomass {10 ml of control beads (alginate
only) or 10 ml of biomass-alginate beads} were immersed
in 100 ml of the real tannery wastewater. The pH of real
wastewater was firstly adjusted at 5.0 0.2, the stirring at
250 rpm on a Jar test at temperature 25 3C. Since the
process of tanning is batch, after finishing the process (2
hr.), the last stage, after settling time, a sample was taken
from the supernatant. Supernatant solution was then filtered
on filter paper and all most important parameters include
metal ions concentrations (chromium, cadmium, copper,

66
MATERIALS & METHODS

lead, nickel, iron and manganese) were measured by AAS.

TDS, Conductivity, DO, pH and salinity were measured by
Multi-parameter analyzer. Color absorbance, COD,
ammonia nitrogen and phosphorous concentrations were
measured by T70+ UV/VIS. These parameters were
determined as quick as possible (Tsekova et al., 2010).
II.10. Color absorbance measurement :-
The color absorbance of WW samples either in
presence or absence of fungal biomass was determined
using T70+ UV/visible spectrophotometer as follows;
sample of 2 ml of WW was measured directly at wide
range of wave lengths at 400 to 600 nm and the scan
spectrum curve was obtained (Wong and Yuen, 1996).
II.11. Determination of metal ions:-
Atomic absorption spectrophotometer (AAS) was
used to measure the concentrations of metal ions in the
filtrates with an air/ acetylene flame for all studied metal
ions except for chromium ions with an acetylene/Nitrous
Oxide flame. The measured wave lengths for chromium,
lead, copper and cadmium, iron and manganese ions were
at 357.9, 217, 324.8, 228.8, 248.3 and 279.5 nm,
respectively. Standard curves were done using standard
solutions and the concentration was given directly by the
instrument in ppm.
II.12. Calculation of biosorption capacity :-
67
MATERIALS & METHODS

The efficiency of heavy metal removal was

determined by calculation of the uptake percent (Uptake
%) and the amount of metal ions adsorbed per gram of the
biosorbent (Q), as the following equations:
The uptake percent was measured as:-
Uptake % = (C0 Ce)/ C0 * 100
The metal uptake per gram was calculated from a metal
mass balance yielding:-
Q = V (C0 - Ce)/m
Where, (Q) is mg metal ions per g dry biosorbent;
(V) is the reaction volume (L), (C0) and (Ce) are the initial
and residual metal concentrations (mg/l), respectively, and
(m) is the amount of dry biosorbent (g) (Gadd & Rehm,
1988).
II.13. Biosorption / desorption cycles:-
The reusability of fungal biomass was studied using
immobilized form only. The reuse of accumulated beads
either control (Ca-alginate bead) or immobilized cells
beads (Ca-alginate-biomass bead) were done for three
consecutive cycles. The accumulated beads after each cycle
were eluted using 0.05N HNO3 for one hour on a rotary
shaker (150 rpm). Then the beads were washed with bi-
distilled water three times until the pH of washing bi-
distilled water reach 6 6.5 and recycling of beads were
occurred(Tsekova et al., 2010).

68
MATERIALS & METHODS

II.14.SEM Examination:-
Fungal beads were prepared for Jeol JSM-5500LV
SEM examination as follows: 1) immobilized beads were
rinsed with bi-distilled water. 2) Then fixed in 2.5% (V/V)
glutraldehyde in 0.1 M. sodium cacodylate buffer, pH: 7.2
at 4C for 2 hr. 3) The beads were then washed three times
over 30 minutes in 0.1 M sodium cacodylate buffer. 4) The
beads were fixed with 1% osmium tetra-oxide in 0.1 M
cacodylate buffer for 2 hr.) Samples were dehydrated
through ethanol serious (30, 50, 70, 90, 100% V/V) then
transfer to critical point dryer for 2 hours then go to gold
coating and exam with SEM.
II.15. Data analysis:-
However the experimental conditions were changed
throughout the present study, all experiments were run
separately in triplicates with appropriate controls run
simultaneously. The data collected were subjected to mean
(average) value and drawing graph using Microcal Origin
5.0 software. Also, Statistical analyses were done by using
Minitab 15 English software.
II.16. safety precautions:-
The laboratory safety precautions were completely
considered during fungal isolation, cultivation and biomass
formation. It also extended to take care when acidic or
alkaline materials were used.
69
RESULTS

RESULTS
I. Fungal communities in tannery wastewater:-
The purpose of this investigation is to obtain
filamentous fungi from tannery wastewater (Chromium
stage) for their possible exploitation in biosorption studies.
Collected samples are mixed to become one sample in
sterilized bottle and physicochemical analyses are done as
shown in Table (2). Samples are then diluted with
sterilized DDW in 1:1 ratio. Aliquots of 1ml of diluted
sample are plated both onto PDA and MEA plates (three
replicates) to ensure the growth of fungi present in sample.
Results obtained both on PDA and MEA media are similar;
therefore, only PDA medium is used for colony count.
Fifteen fungal colonies represents five genera are obtained
on PDA medium at this dilution. Genus Aspergillus is
found to occur in maximum percentage (40 %) followed by
Penicillium (26.7 %). Genera of Rhizopus and Fusarium
are of similar percentage (13.3 %). Genus Alternaria has
the minimum percentage (6.7 %) as shown in Table (3)
and Fig. (4). Basic Statistical analysis for fungal colonies
count showed significance 0.336 as shown in Fig. (5).

70
RESULTS

Table (3): Fungal genera isolated from tannery wastewater

At dilution 1:1 Total Count
Colonies Count Percent (%)
Fungal genera
Aspergillus 6 40 %
Penicillium 4 26.7 %
Rhizopus 2 13.3 % 15
Fusarium 2 13.3 %
Alternaria 1 6.7 %

Rhizopus 13.3%
Fusarium 13.3%

Penicillium 26.7%
Alternaria 6.7%

Aspergillus 40%

Fig. (4): Prevalence of isolated fungi from tannery wastewater

Probability Plot of Fungal Colonies Count

Normal
99
Mean 3
StDev 2
95 N 5
AD 0.334
90
P-Value 0.336
80
70
Percent

60
50
40
30
20

10

1
0.0 2.5 5.0 7.5
Fungal Colonies Count

Fig. (5): Basic Statistics for fungal colonies count

71
RESULTS

II. Screening and identification of fungal strains:-

Pure cultures of isolated fungal colonies are initially
screened for heavy metal ions tolerance by streaking
colonies on PDA medium containing 5 ppm from each of
selected metal ions (Cr6+, Pb2+, Cu2+ and Cd2+) in mixture
and checked for growth. Based on their growth they are
ranked and identified using image analysis instrument at
RCMB, Al-Azhar University.
II.1. Isolate No. (1):-
Greenish brown colonies reaching 45mm in diameter
on Czapek's-Dox Agar media after seven days at 28C.
Conidia and vesicle are globose to sub-globose in shape
and have diameter 3.4m and 25m, respectively.
Conidiophore is 10m in diameter. The dimensions of first
and second sterigmata are 11.76.1m and 9.45.1m,
respectively. Therefore, it's identified as Aspergillus
tamarii (plate 1).

Plate (1) Aspergillus tamarii ( Mag. 400X)

72
RESULTS

II.2. Isolate No. (2):-

Luxuriant, velvety, white, yellow to black colonies
reaching 50 mm in diameter on Czapek's-Dox Agar media
after seven days at 28C and Pale yellowish colony on
reverse side. Conidia well-arranged globose with diameter
4.0 m and radiate black conidial heads. Globose to sub-
globose vesicle, 70.0 m. Conidiophore is 19.0 m. The
dimensions of first and second Sterigmata are 15.74.5m
and 7.03.8m, respectively. Therefore, it's identified as
Aspergillus niger (plate 2).

Plate(2) Aspergillus niger (Mag. 400X)

II.3. Isolate No. (3):-
Colonies are flat on CYA attending 35-50 mm
diameter during 10 days at 25C. Produce green yellow
pigment at the center with age. Conidia are yellow with
pale green blue globose, 3.2 m. Penicillus type
Biverticillate Metulae 13.02.7 m Phialides 8.02.8 m.

73
RESULTS

Therefore, it's identified as Penicillium chrysoginum (plate

3).

Plate (3) Penicillium chrysogenum (Mag. 400X)

II.4. Isolate No. (4) :-
Colonies are whitish to grayish-brown reaching 40-
60 mm at 25C. Sporangiophores are dark-brown either
solitary or in groups of 2-7. Smooth or slightly rough-
walled stolons opposite the branched rhizoids, 14.04.5m.
Sporangia and columella are Globose to sub-globose with
diameter 80.0m and 60.0m, respectively.
Sporangiophores are irregular in shape often polygonal, 5.9
m. Therefore, it's identified as Rhizopus stolonifer (plate
4).

Plate (4) Rhizopus stolonifer (Mag. 400X)

74
RESULTS

III. Optimization of fungal growth :-

This study determines the optimal growth conditions
include temperature and suitable media for growth of
fungal isolates to be used in metal - tolerance study through
estimation of RG (mm) and RP (%). Where, RG: Radial
growth of the colony (mm), RP: Relative percentage of
change (%) and (*): The values of percentage are related to
those at high value as considered 100%.
III.1. Effect of medium type on fungal growth:-
Different media types are used for fungal cultivation
to determine suitable medium for growth. The isolated
fungal strains are grown on different media types (MEA,
PDA, SDA and Czapek's-Dox) and incubated at 30C.
Results showed that the optimum medium for all isolates is
PDA medium using radial growth as a criterion as shown in
Table (4) and Fig. (6). Statistical analysis for optimization
of fungal growth at different media types by One-way
ANOVA is showed significance 0.004 as shown in Table
(6).
Table (4): Effect of medium type on fungal growth

Fungal R. P.
A. niger A. tamarii
isolates
stolonifer chrysoginum
Growth RG RP* RG RP* RG RP* RG RP*
medium (mm) (%) (mm) (%) (mm) (%) (mm) (%)
Cz-A 60 85.7 58 90.6 50 71.4 45 64.3
PDA 70 100 64 100 70 100 70 100
MEA 64 91.4 62 96.9 66 94.3 70 100
SDA 68 97.1 58 90.6 62 88.6 60 85.7

75
RESULTS

R. stolonifer
100 P. chrysogenum
A. niger
A. Tamarii
80

60
RP %

40

20

0
Cz-A PDA MEA SDA
Growth Media

Fig. (6): Fungal isolates grown on different media types

III.2. Effect of incubated temperature on fungal

growth:-

The growth of identified fungal strains is

dramatically affected by incubated temperatures. Changing
the incubated temperatures is studied at 15, 20, 25, 30, 35
and 40C. Only A. niger can grow at a wide range from 15
40C. A. tamarii and P. chrysoginum can grow from 15-
35C. R. stolonifer can grow from 20-35C. The optimum
incubated temperature's for all is the same at 30C as shown
in Table (5) and Fig. (7). Statistical analysis for
optimization of fungal growth at different incubated
temperatures by One-way ANOVA is showed significance
0.000 as shown in Table (6).

76
RESULTS

Table (5): Effect of incubated temperature on fungal growth

Fungal R. P.
A. niger A. Tamarii
isolates stolonifer chrysoginum
RG RP* RG RP* RG RP* RG RP*
Incubated
(mm) (%) (mm) (%) (mm) (%) (mm) (%)
Temp.
15C 0.0 0 15 23.4 25 35.7 20 28.6
20C 23 32.9 35 54.7 54 77.1 52 74.3
25C 68 97.1 58 90.6 62 88.6 60 85.7
30C 70 100 64 100 70 100 70 100
35C 21 30 23 35.9 40 57.1 35 50
40C 0.0 0 0.0 0 10 14.3 0.0 0

100 R. stolonifer
P. chrysogenum
A. niger
80 A. Tamarii

60
RP %

40

20

0
15C 20C 25C 30C 35C 40C
Incubated temperature (C)

Fig. (7): Fungal isolates grown at different incubated

temperatures

Table (6): Statistical analysis for optimization of fungal growth

by One-way ANOVA

At different Growth media Incubated Temp.

P(Sig.) 0.004 0.000

77
RESULTS

IV. Metal tolerance potential of fungal isolates:-

This study aims to show the metal-resistant fungi and

the effect of the studied metal ions on the fungal growth.
This study is processed firstly on solid PDA media, so
MIC (Minimum Inhibitory Concentration) and RP
(Relative Percent of radial growth in comparison to control)
are determined. Then the most tolerant isolates are grown
on liquid media (PDB), so the total growth (G) and the
growth rate () are calculated.
IV.A. Isolation of the most tolerant fungus on solid
(PDA) media:-

In this preliminary test, the resistance of the fungal

isolates to the studied metal ions is determined by the
dilution method. PDA is used as a solid medium for fungal
isolation despite the fact that agar may affect metal
speciation and solubility, because a solid medium is more
appropriate for growth observations of all fungi. The MIC
is defined as the lowest concentration of metal ions that
inhibit visible growth of the isolate. The effect of the heavy
metal on the growth of the tested isolates is estimated by
measuring the radial growth RG (mm). Statistical analysis
for metal tolerance potential of fungal isolates by One-way
ANOVA is showed in Table (12).

78
RESULTS

IV.A.1. Fungal growth at different Cr6+ ions conc.:-

The growth of fungal isolates at different chromium
(Cr6+) ions conc. ranged from 10 to 200 ppm is observed
until fifteen days of incubation period at 30C. The
tolerance of the studied fungal species for Cr6+ ions is
A. niger = P. chrysoginum (100 ppm) < R. stolonifer = A.
tamarii (75 ppm) as shown in Table (7) and Fig. (8).

Table (7): Fungal isolates grown at different Cr6+ ions conc.

P.
Fungal. R. stolonifer A. niger A. tamarii
chrysoginum
sp.
RG(mm)
RG(mm)

RG(mm)

RG(mm)
IP(days)

IP(days)

IP(days)

IP(days)
RP*(%)

RP*(%)

RP*(%)

RP*(%)
Cr6+
ppm
Control 70 100 3 64 100 7 70 100 3 70 100 3
10 55 78.6 7 60 92.3 7 64 91.4 7 60 85.7 7
25 38 54.3 7 55 84.6 7 57 81.4 7 45 64.3 7
50 26 37.1 7 43 66.2 7 48 68.6 7 33 47.1 7
75 17 24.3 10 30 46.2 7 35 50 7 12 17.1 15
100 0 0 15 15 23.1 15 25 35.7 10 0 0 15
200 0 0 15 0 0 15 0 0 15 0 0 15

110
100 R. stolonifer
P. chrysogenum
90
A. niger
80 A. Tamarii
RP %

70
60
50
40
30
20
10
Control 10 25 50 75 100
6+
Cr Concs ( ppm )

Fig. (8): Fungal isolates grown at different Cr6+ ions conc.

79
 RESULTS

IV.A.2. Fungal growth at different Pb2+ ions conc.:-

The growth of fungal isolates at different lead (Pb2+)
ions conc. ranged from 10 to 200 ppm is observed until
fifteen days of incubation period at 30C. The tolerance of
the studied fungal species for Pb2+ ions is
A. niger = P. chrysoginum = A. tamarii (100 ppm) < R.
stolonifer (75 ppm) as shown in Table (8) and Fig. (9).

Table (8): Fungal isolates grown at different lead ions conc.

P.
Fungal. R. stolonifer A. niger A. tamarii
chrysoginum
sp.
IP (days)

IP (days)

IP (days)

IP (days)
RG(mm)
RG(mm)

RG(mm)

RG(mm)
RP*(%)

RP*(%)

RP*(%)

RP*(%)
Pb2+
ppm
Control 70 100 3 64 100 7 70 100 3 70 100 3
10 58 82.9 7 50 76.9 7 65 92.9 7 50 71.4 7
25 45 64.3 15 45 69.2 7 60 85.7 7 45 64.3 7
50 30 42.9 15 36 55.4 7 50 71.4 7 35 50 15
75 15 21.4 15 32 49.2 15 45 64.3 10 25 35.7 15
100 0 0 15 15 23.4 15 40 57.1 15 12 17.1 15
200 0 0 15 0 0 15 0 0 15 0 0 15

110
R. stolonifer
100
P. chrysogenum
90 A. niger
80 A. Tamarii

70
RP %

60
50
40
30
20
10
Control 10 25 50 75 100
2+
Pb Concs ( ppm )

Fig. (9): Fungal isolates grown at different lead ions conc.

80
 RESULTS

IV.A.3. Fungal growth at different Cu2+ ions conc.:-

The growth of fungal isolates at different copper
(Cu2+) ions conc. ranged from 10 to 100 ppm is observed
until fifteen days of incubation period at 30C. With P.
chrysoginum . strain, pale blue mycelium is observed at
high copper conc. The tolerance of the studied fungal
species for Cu2+ ions is
A. niger (75 ppm) < P. chrysoginum = A. tamarii (50
ppm) < R. stolonifer (25 ppm) as shown in Table (9) and Fig.
(10).
Table (9): Fungal isolates grown at different copper ions conc.
P.
Fungal. R. stolonifer. A. niger A. tamarii
chrysoginum
sp.
IP (days)

IP (days)

IP (days)

IP (days)
RG(mm)

RG(mm)

RG(mm)

RG(mm)
RP*(%)

RP*(%)

RP*(%)

RP*(%)
Cu2+
ppm
Control 70 100 3 64 100 7 70 100 3 70 100 3
10 22 31.4 15 50 76.9 7 62 88.6 7 35 50 10
25 15 21.4 15 40 61.5 15 55 78.6 7 20 28.6 15
50 0 0 15 30 46.2 15 40 57.1 15 15 21.4 15
75 0 0 15 0 0 15 20 28.6 15 0 0 15
100 0 0 15 0 0 15 0 0 15 0 0 15

110
R. stolonifer
100
P. chrysogenum
90 A. niger
80 A. Tamarii
RP %

70
60
50
40
30
20
10
Control 10 25 50 75
2+
Cu Concs ( ppm )

Fig. (10): Fungal isolates grown at different copper ions conc.

81
 RESULTS

IV.A.4. Fungal growth at different Cd2+ ions conc.:-

The growth of fungal isolates at different cadmium
(Cd2+) ions conc. ranged from 10 to 100 ppm is observed
until fifteen days of incubation period at 30C. The whitish
color of the PDA media is decreased around fungal growth
at high cadmium conc. The tolerance of the studied fungal
species for cadmium (Cd2+) ions is
A. niger (75 ppm) < P. chrysoginum (50 ppm) = A. tamarii (50 ppm) < R.
stolonifer (25 ppm) as shown in Table (10) and Fig. (11).
Table (10): Fungal isolates grown at different cadmium ions conc.
P.
Fungal. R. stolonifer A. niger A. Tamarii
chrysoginum
sp.
IP (days)

IP (days)

IP (days)

IP (days)
RG(mm)

RG(mm)

RG(mm)

RG(mm)
RP*(%)

RP*(%)

RP*(%)

RP*(%)
Cd2+
ppm
Control 70 100 3 64 100 7 70 100 3 70 100 3
10 25 35.7 15 46 70.8 15 54 77.1 7 35 50 15
25 12 17.1 15 27 41.5 15 32 45.7 15 20 28.6 15
50 0 0 15 13 20 15 23 32.9 15 10 14.3 15
75 0 0 15 0 0 15 12 17.1 15 0 0 15
100 0 0 15 0 0 15 0 0 15 0 0 15

110
R. stolonifer
100 P. chrysogenum
90 A. niger
A. Tamarii
80
70
RP %

60
50
40
30
20
10
Control 10 25 50 75
2+
Cd Concs ( ppm )

Fig. (11): Fungal isolates grown at different cadmium ions conc.

82
 RESULTS

IV.A.5. Fungal growth at different conc. of the studied

ions in mixture:-
The growth of fungal isolates at different conc. of the
studied ions in mixture ranged from 40 to 500 ppm with
equal ratio from each metal ion is observed until fifteen
days of incubation period at 30C. The tolerance of the
studied fungal species for studied ions in mixture is
A. niger (400 ppm 100 from each metal ion) < P.
chrysoginum = A. tamarii (300 ppm 75 from each metal
ion) < R. stolonifer (200 ppm 50 from each metal ion) as
shown in Table (11) and Fig. (12).

Table (11): Fungal isolates grown at different conc. of the studied

ions in mixture
Fungal. P.
R. stolonifer A. niger A. tamarii
sp. chrysoginum
IP (days)

IP (days)

IP (days)

IP (days)
RG(mm)
RG(mm)

RG(mm)

RG(mm)
RP*(%)

RP*(%)

RP*(%)

RP*(%)
Mixed
ions
Conc.
Control 70 100 3 64 100 7 70 100 3 70 100 3
40 42 60 15 50 78.1 7 55 78.6 7 48 68.6 10
100 30 42.9 15 41 64.1 10 42 60 7 30 42.9 15
200 10 14.3 15 32 50 15 36 51.4 10 22 31.4 15
300 0 0 15 25 39.1 15 26 37.1 15 11 15.7 15
400 0 0 15 0 0 15 14 20 15 0 0 15
500 0 0 15 0 0 15 0 0 15 0 0 15

Table (12): Statistical analysis for metal tolerance potential of

fungal isolates by One-way ANOVA
At
different Cr6+ Pb2+ Cu2+ Cd2+ Mixture
conc.

P(Sig.) 0.0 0.0 0.0 0.0 0.0

83
RESULTS

110
R. stolonifer
100
P. chrysogenum
90 A. niger
80 A. Tamarii
RP %
70
60
50
40
30
20
10
Control 40 100 200 300 400
Studied ions concs in mixture ( ppm )

Fig. (12): Fungal isolates grown at different conc. of the studied

ions in mixture

Results showed that all fungal isolates grew faster in

the absence of heavy metals and showed concentration
dependent reduction in their growth response especially for
both copper and cadmium, while recording some tolerance
to mixture, chromium and lead, respectively as shown in
Tables (13, 14) and Fig. (13).
Table (13): Metal toxicity toward fungal isolates
Fungal isolates Metal Toxicity
A. niger Cd2+ = Cu2+ > Cr6+ = Pb2+ > Mixture
P. chrysoginum Cd2+ = Cu2+ > Mixture > Pb2+ = Cr6+
A. tamarii Cd2+ = Cu2+ > Mixture = Cr6+ > Pb2+
R. stolonifer Cd2+ = Cu2+ > Mixture = Cr6+ = Pb2+

Table (14): MIC values of the studied fungal isolates toward

metal ions
MIC Cr Pb Cu Cd
Mixture
Fungal isolates (VI) (II) (II) (II)
R. stolonifer >75 > 75 >25 >25 > 200
P. chrysoginum >100 >100 >50 >50 >300
A. niger >100 >100 >75 >75 >400
A. tamarii >75 >100 >50 >50 >300
84
RESULTS

400
R. stolonifer
350 P. chrysogenum
A. niger
300 A. Tamarii

250
MIC(ppm)

200

150

100

50

0
Cr(VI) Pb(II) Cu(II) Cd(II) Mix
Studied Metal Ions

Fig. (13): MIC value of the studied metal ions toward fungal
isolates

From this experiment we can conclude that, the most

resistant fungal isolates toward studied metal ions are A.
niger then P. chrysoginum. The most toxic metals for all
fungal isolates are cadmium and copper, respectively and
the mixture of the studied ions will decrease their toxicity.
Statistical analysis for MIC difference with consideration
to fungal isolates showed significance 0.937. While with
consideration to metal ions, it showed significance 0.000
as shown in Table (15).
Table (15): Statistical analysis for MIC values of the studied
fungal isolates toward metal ions by One-way ANOVA
MIC With With
difference consideration to consideration
fungal isolates to metal ions

P(Sig.) 0.937 0.000

85
RESULTS

IV.B. Toxicity testing of selected species on liquid (PDB)

media:-

The most tolerant fungal isolates (A. niger and P.

chrysoginum) are studied on PDB media to determine the
total growth (G) and the growth rate () after seven days of
incubation at 30C. A. niger and P. chrysoginum showed
significant decrease in the total growth (G) and the growth
rate () both in static (forms mat) and shaking incubation
(forms pellet) along with increasing concentration of the
studied metal ions concentration ranged from 10 to 100
ppm when compared to control. Results observed that
shaking increased the growth rate () and total growth (G)
and the isolated A. niger strain had the maximum growth
rate than P. chrysoginum either in control or in presence of
metal ions. Studied fungal strains grew faster in the absence
of heavy metals and their cultures showed concentration
dependent reduction in their growth response to both
copper and cadmium, while recording some tolerance to
chromium and lead when compared with control medium.
With shaking incubation, the pellet formed had significant
advantages, decrease of the viscosity and the easier
separation of the biomass from the cultivation broth.
Results also showed that Pellets size decreased and become
more compact with more or less circular morphology as
shown in Tables (16 -19) and Figures (14 -17).

86
 RESULTS

IV.B.1. Effect of Cr6+ ions on fungal growth rate:-

Table (16): G and of P. chrysoginum and A. niger at different
Cr6+ ions conc. in static and shaking conditions

Fungal. P. chrysoginum A. niger

sp.
Static Shaking Static Shaking
Total Growth Total Growth Total Growth Total Growth
Cr6+ Growth Rate Growth Rate Growth Rate Growth Rate
(G) () (G) () (G) () (G) ()
Conc. (mg/L) (mg/h) (mg/L) (mg/h) (mg/L) (mg/h) (mg/L) (mg/h)
Control 3200 19.0 4000 23.8 5000 29.8 5600 33.3
10 2800 16.7 3000 17.9 4200 25 5000 29.8
25 1200 7.2 1500 8.9 3750 22.3 3950 23.5
50 850 5.1 1000 5.9 2840 16.9 3000 17.9
75 400 2.4 750 4.5 1250 7.4 1970 11.7
100 150 0.9 350 2.1 500 2.9 780 4.6

35 P. static
P. shaking
30
Growth rate (mg/h)

A. static
25 A. shaking

20

15

10

0
Control 10 25 50 75 100
6+
Cr Concs ( ppm )

Fig. (14): P. chrysoginum and A. niger tolerance and growth

response to Cr6+ ions in static and shaking conditions

87
 RESULTS

IV.B.2. Effect of Pb2+ ions on fungal growth rate:-

Table (17): G and of P. chrysoginum and A. niger at different
lead ions conc. in static and shaking conditions

Fungal. P. chrysoginum . A. niger .

sp.
Static Shaking Static Shaking
Total Growth Total Growth Total Growth Total Growth
Pb2+ Growth Rate Growth Rate Growth Rate Growth Rate
(G) () (G) () (G) () (G) ()
Conc. (mg/L) (mg/h) (mg/L) (mg/h) (mg/L) (mg/h) (mg/L) (mg/h)
Control 3200 19.0 4000 23.8 5000 29.8 5600 33.3
10 3000 17.9 3620 21.5 3980 23.7 4630 27.6
25 1050 6.3 1520 9.0 2540 15.1 3790 22.6
50 400 2.4 810 4.8 1850 11.0 3600 21.4
75 105 0.6 250 1.5 980 5.8 1970 11.7
100 50 0.3 105 0.6 150 0.9 300 1.8

35 P. static
P. shaking
30
A. static
Growth rate (mg/h)

A. shaking
25

20

15

10

0
Control 10 25 50 75 100
2+
Pb Concs ( ppm )

Fig. (15): P. chrysoginum and A. niger tolerance and growth

response to lead ions in static and shaking conditions

88
 RESULTS

IV.B.3. Effect of Cu2+ ions on fungal growth rate :-

Table (18): G and of P. chrysoginum and A. niger at different
copper ions conc. in static and shaking conditions

Fungal. P. chrysoginum A. niger

sp.
Static Shaking Static Shaking
Total Growth Total Growth Total Growth Total Growth
Cu2+ Growth Rate Growth Rate Growth Rate Growth Rate
(G) () (G) () (G) () (G) ()
Conc. (mg/L) (mg/h) (mg/L) (mg/h) (mg/L) (mg/h) (mg/L) (mg/h)
Control 3200 19.0 4000 23.8 5000 29.8 5600 33.3
10 1500 8.9 2000 11.9 2980 17.7 3100 18.5
25 850 5.1 1200 7.1 1540 9.2 2020 12.0
50 200 1.2 810 4.8 850 5.1 1550 9.2
75 0 0 0 0 180 1.1 220 1.3

35 P. static
P. shaking
30 A. static
Growth rate (mg/h)

A. shaking
25

20

15

10

0
Control 10 25 50 75
2+
Cu Concs ( ppm )

Fig. (16): P. chrysoginum and A. niger tolerance and growth

response to copper ions in static and shaking conditions

89
 RESULTS

IV.B.4. Effect of Cd2+ ions on fungal growth rate:-

Table (19): G and of P. chrysoginum and A. niger at different
cadmium ions conc. in static and shaking conditions

Fungal. P. chrysoginum A. niger

sp.
Static Shaking Static Shaking
Total Growth Total Growth Total Growth Total Growth
Cd2+ Growth Rate Growth Rate Growth Rate Growth Rate
(G) () (G) () (G) () (G) ()
Conc. (mg/L) (mg/h) (mg/L) (mg/h) (mg/L) (mg/h) (mg/L) (mg/h)
Control 3200 19.0 4000 23.8 5000 29.8 5600 33.3
10 180 1.1 520 3.1 620 3.7 1620 9.6
25 80 0.5 200 1.2 250 1.5 1000 5.9
50 0 0 110 0.7 150 0.9 750 4.5
75 0 0 0 0 50 0.3 150 0.9

35 P. static
P. shaking
30
A. static
Growth rate (mg/h)

25 A. shaking

20

15

10

Control 10 25 50 75
2+
Cd Concs ( ppm )

Fig. (17): P. chrysoginum and A. niger tolerance and growth

response to cadmium ions in static and shaking conditions

90
RESULTS

V. Heavy-metals training
The relative toxicity of heavy metals for each fungal
strain had become obvious at the higher conc. This test
indicated the possible adaptation of the fungal strain to heavy
metal stress conditions and their potential for use in
extracting metal ions from solution. As the isolated A. niger
strain had the most tolerance effect among the rest of fungal
isolates. A. niger is trained with high studied metal ions levels
ranged from 10 to 100 ppm according to tolerance study
previously mentioned. The data analysis for biosorption of
the studied metal ions by control and trained A. niger had
been done in light of two isotherm models (Langmuir's and
Freundlich's). Results showed that the A. niger trained strain
had the maximum removal for all studied metal ions than
control strain. It also showed that the total growth (G) growth
rate () of control and trained strain were initially inhibited,
being the control strain was more sensitive. It is found that
the sorption isotherms of fungi for the studied metal ions
appeared to fit both or either Freundlich's or Langmuirs
models. According to correlation coefficient (R2) the best fit
model is determined. The best correlation coefficient (R2) is
equal one (R2 = 1). Low correlation coefficients (R2 < 0.7)
were not statistically significant while high correlation
coefficients (R2 > 0.7) were statistically significant at 95%
confidence level.

91
 RESULTS

V.1. Control and trained A. niger at different Cr6+ concs:-

In Cr6+ assays, the control A. niger biotypes grew at
100 ppm with 4.6 mg/h and removal 82.0 %; while the
trained strain were grew at 100 ppm with 7.3 mg/h and
94.0 %. According to correlation coefficient (R2), the best
fit to the Langmuirs models is obtained by trained stain
while the best fit to Freundlich's model is by control strain
as shown in Tables (20, 21) and Figures (18-23).
Table (20): Cr6+ removed by control and trained strain at
different concs.
Trained strain Control strain
Fungal sp.
Cr6+Conc.

Total Growth Total Growth

Removed

Removed
removed

removed
Cr6+

Cr6+

Cr6+

Cr6+
Growth Rate Growth Rate
%

%
(G) () (G) ()
(mg/L) (mg/h) (mg/L) (mg/h)

Control 5600 33.3 ---- ---- 5600 33.3 ---- ---

10 5420 32.3 6.9 69 5000 29.8 6.03 60.3
25 5030 29.9 20.25 81 3950 23.5 18.775 75.1
50 4100 24.4 43 86 3000 17.9 41 82
75 3200 19.0 67.5 90 1970 11.7 63 84
100 1220 7.3 94 94 780 4.6 82 82
Table (21): Functional isotherm parameters for control and
trained strain at different Cr6+ concs.
A. niger Log Log Lang. Freund
C0 Ce Qe Ce/Qe
. Ce Qe R2 R2
10 3.1 0.13 0.5 -0.9 24.4
25 4.75 0.40 0.7 -0.4 11.8
50 7 1.05 0.9 0.02 6.7 0.7633* 0.6003
Trained 75 7.5 2.11 0.9 0.3 3.6
100 6 7.70 0.8 0.9 0.8
10 3.97 0.1 0.6 -0.9 32.9
25 6.23 0.5 0.8 -0.3 13.1
Control 50 9 1.4 0.95 0.1 6.6 0.6548 0.9999*
75 12 3.2 1.1 0.5 3.8
100 18 10.5 1.3 1.02 1.7
Model parameters estimated are statistically significant at
* 95% confidence level.
92
RESULTS

95
90
85
Removal % 80
75
70
65
Trained
60 Control

0 20 40 60 80 100
6+
Cr Concs ( ppm )

Fig. (18): Cr6+ removal by both control and trained strain

35 Trained
30 Control
Growth rate (mg/h)

25
20

15
10

5
0
Control 10 25 50 75 100
6+
Cr Concs ( ppm )

Fig. (19): Growth response to Cr6+ ions in trained and control

strain

Fig. (20): Langmuir isotherm for Cr6+ removal by trained strain

93
RESULTS

Fig. (21): Langmuir isotherm for Cr6+ removal by control strain

Fig. (22): Freundlich isotherm for Cr6+ removal by trained strain

Fig. (23): Freundlich isotherm for Cr6+ removal by control strain

94
 RESULTS

V.2. Control and trained A. niger at different Pb2+ concs:-

In the lead assays, the control A. niger biotypes grew
at 100 ppm with 1.8 mg/h and removal 69.1 %; while the
trained strain were grew at 100 ppm with 2.5 mg/h and
74.2 %. According to correlation coefficient (R2), the best
fit for Langmuirs models is obtained by control and
trained strain as shown in Tables (22, 23) and Figures (24-
29).
Table (22): Lead removed by control and trained strain at
different concs.
Trained strain Control strain
Pb2+ Conc

Fungal.

Total Growth
Removed

Total Growth

Removed
removed

removed
sp.

Pb2+

Pb2+

Pb2+

Pb2+
Growth Rate Growth Rate
%

%
(G) () (G) ()
(mg/L) (mg/h) (mg/L) (mg/h)

Control 5600 33.3 ---- ---- 5600 33.3 ---- ---

10 4368 26 1.89 18.9 3192 19 1.53 15.3
25 4200 25 6.775 27.1 3790 22.6 5.625 22.5
50 3800 22.6 19.1 38.2 3600 21.4 15.4 30.8
75 2000 11.9 37.125 49.5 1970 11.7 30.075 40.1
100 420 2.5 74.2 74.2 300 1.8 69.1 69.1
Table (23): Functional isotherm parameters for control and
trained strain at different lead concs.
A. niger Log Log Lang. Freund
C0 Ce Qe Ce/Qe
. Ce Qe R2 R2
10 8.11 0.04 0.9 -1.4 187.4
25 18.23 0.16 1.3 -0.8 113.1
50 30.9 0.50 1.5 -0.3 61.5
Trained 75 37.88 1.86 1.6 0.3 20.4 0.7468* 0.5056
100 25.8 17.67 1.4 1.2 1.5
10 8.47 0.05 0.9 -1.3 176.7
25 19.38 0.15 1.3 -0.8 130.7
Control 50 34.6 0.43 1.5 -0.4 80.9 0.7062* 0.4784
75 44.93 1.5 1.7 0.2 29.4
100 30.9 23.0 1.5 1.4 1.3
Model parameters estimated are statistically significant at
* 95% confidence level.
95
RESULTS

80

70

60
Removal %
50

40

30

20 Trained
Control
10
0 20 40 60 80 100
2+
Pb Concs ( ppm )

Fig. (24): Percent of lead removal by both control and trained

strain

35 Trained
Control
Growth rate (mg/h)

30
25
20

15
10

5
0
Control 10 25 50 75 100
2+
Pb Concs ( ppm )

Fig. (25): Growth response to lead ions in trained and control

strain

Fig. (26): Langmuir isotherm for lead removal by trained strain

96
RESULTS

Fig. (27): Langmuir isotherm for lead removal by control strain

Fig. (28): Freundlich isotherm for lead removal by trained strain

Fig. (29): Freundlich isotherm for lead removal by control strain

97
 RESULTS

V.3. Control and trained A. niger at different Cu2+

concs:-
In the copper assays, the control A. niger biotypes
grew at 75 ppm with 2.1 mg/h and removal 33.1 %; while
the trained strain were grew at 75 ppm with 5.9 mg/h and
39.5 %. According to correlation coefficient (R2), the best
fit for control and trained strain is Freundlich's model as
shown in Tables (24, 25) and Figures (30-35).
Table (24): Copper removed by control and trained strain at
different concs.
Fungal. Trained strain Control strain
sp.
Removed%

Removed%
Total Total
removed

removed
Growth Growth
Cu2+

Cu2+

Cu2+

Cu2+
Growth Growth
2+ Rate () Rate ()
Cu (G) (G)
(mg/h) (mg/h)
(mg/L) (mg/L)
Conc
Control 5600 33.3 ---- ---- 5600 33.3 ---- ---
10 4830 28.8 0.89 8.9 3100 18.5 0.58 5.8
25 3150 18.8 4.475 17.9 2020 12.0 3.175 12.7
50 2200 13.1 14 28 1550 9.2 10 20
75 1000 5.9 29.625 39.5 220 1.3 24.825 33.1

Table (25): Functional isotherm parameters for control and

trained strain at different copper concs.

A. niger Log Log Lang. Freund

C0 Ce Qe Ce/Qe
. Ce Qe R2 R2
10 9.11 0.02 0.96 -1.7 494.4
25 20.53 0.14 1.3 -0.8 144.6
Trained 0.8185* 0.9691*
50 36 0.64 1.6 -0.2 56.6
75 45.38 2.96 1.7 0.5 15.3
10 9.42 0.02 0.97 -1.7 503.5
25 21.83 0.16 1.3 -0.8 139.1 0.8096* 0.8866*
Control
50 40 0.65 1.6 -0.2 62
75 50.18 11.28 1.7 1.1 4.4
Model parameters estimated are statistically significant at
* 95% confidence level.

98
RESULTS

40
35
30
Removal %
25
20
15
10
Trained
5 Control

0 10 20 30 40 50 60 70 80
2+
Cu Concs ( ppm )

Fig. (30): Percent of copper removal by both control and trained

strain

35 Trained
Control
Growth rate (mg/h)

30

25
20

15

10

5
0
Control 10 25 50 75
2+
Cu Concs ( ppm )

Fig. (31): Growth response to copper ions in trained and control

strain

Fig. (32): Langmuir isotherm for copper removal by trained

strain
99
RESULTS

Fig. (33): Langmuir isotherm for copper removal by control

strain

Fig. (34): Freundlich isotherm for copper removal by trained

strain

Fig. (35): Freundlich isotherm for copper removal by control

strain

100
 RESULTS

V.4. Control and trained A. niger at different Cd2+

concs.:-
In the cadmium assays, the control A. niger biotypes
grew at 75 ppm with 0.9 mg/h and removal 8.5 %; while
the trained strain were grew at 75 ppm with 5.4 mg/h and
10.2 %. According to correlation coefficient (R2), the best
fit for control and trained strain is Freundlich's model as
shown in Tables (26, 27) and Figures (36-41).
Table (26): Cadmium removed by control and trained strain at
different Concs.

Fungal. Trained strain Control strain

sp.
Removed%

Removed%
Total Total
removed

removed
Growth Growth
Cd2+

Cd2+

Cd2+

Cd2+
Growth Growth
2+ Rate () Rate ()
Cd (G) (G)
(mg/h) (mg/h)
(mg/L) (mg/L)
Conc
Control 5600 33.3 ---- ---- 5600 33.3 ---- ---
10 3210 19.1 0.64 6.4 1620 9.6 0.47 4.7
25 2820 16.8 3.25 13 1000 5.9 2.35 9.4
50 1050 6.3 6.4 12.8 750 4.5 4.9 9.8
75 900 5.4 7.65 10.2 150 0.9 6.375 8.5

Table (27): Functional isotherm parameters for control and

trained strain at different cadmium Concs.
A. niger Log Log Lang. Freund
C0 Ce Qe Ce/Qe
. Ce Qe R2 R2
10 9.36 0.02 0.97 -1.7 469.5
25 21.75 0.12 1.3 -0.9 188.7
50 43.6 0.61 1.6 -0.2 71.5 0.6842 0.9835*
Trained
75 67.35 0.85 1.8 -0.1 79.2
10 9.53 0.03 0.98 -1.5 328.5
25 22.65 0.24 1.4 -0.6 96.4
Control 0.7103* 0.9685*
50 45.1 0.65 1.7 -0.2 69.03
75 68.625 4.25 1.8 0.6 16.1
Model parameters estimated are statistically significant at
* 95% confidence level.

101
RESULTS

15

12
Removal %

6
Trained
Control

0 10 20 30 40 50 60 70 80
2+
Cd Concs ( ppm )

Fig. (36): Percent of cadmium removal by both control and

trained strain

35 Trained
Growth rate (mg/h)

30 Control
25
20

15
10

5
0
Control 10 25 50 75
2+
Cd Concs ( ppm )

Fig. (37): Growth response to cadmium ions in trained and

control strain

Fig. (38): Langmuir isotherm for cadmium removal by trained

strain
102
RESULTS

Fig. (39): Langmuir isotherm for cadmium removal by control

strain

Fig. (40): Freundlich isotherm for cadmium removal by trained

strain

Fig. (41): Freundlich isotherm for cadmium removal by control

strain

103
RESULTS

VI. BIOSORPTION CAPACITY

Biosorption capacity of metal ions is expressed by

the uptake percentage or the removal percentage and the
specific uptake (Q). In this study, batch experiments are
used in the optimization of metal sorption by fungal
biomasses.
VI.A. Biosorption capacity of chromium ions Cr6+ :-
The affinity and the capacity of the most tolerant
fungal species (A. niger) towards chromium ions (Cr6+)
were investigated using different parameters which affects
the biosorption study. These parameters include pH,
contact time, temperature, stirring rate, biomass weight and
metal ions conc. which called environmental conditions.
This study is done in batch experiments to determine the
optimum conditions for maximum uptake.
VI.A.1. Effect of pH :-
Obtained results indicated that the pH of the
solution is an important factor for the biosorption study.
Results showed that the best uptake capacity of Cr6+ (86%,
17.2 mg/g) were obtained at pH 5.0 0.2 while the
minimum uptake percent were obtained at pH 1.0 0.2, 3.0
0.2 and 7.0 0.2. So, the uptake percent is increased as
pH of the solution increased and the maximum uptake
occurred at 3 p H 7.0 0.2 as shown in Table (28) and
Fig. (42).
104
 RESULTS

Table (28): The biosorption capacity of Cr6+ ions at different pH

values

PH 1.0 0.2 3.0 0.2 5.0 0.2 7.0 0.2

Conc. ppm

Conc. ppm

Conc. ppm

Conc. ppm
Uptake %
Uptake %

Uptake %
Uptake %
Q (mg /g)

Q (mg /g)

Q (mg /g)

Q (mg /g)
Time
(min)

0 20 0 0 20 0 0 20 0 0 20 0 0
30 15.8 21 4.2 13.1 34.5 6.9 11.9 40.5 8.1 17.4 13 2.6
60 13.7 31.5 6.3 10.8 46 9.2 7.8 61 12.2 14.7 26.5 5.3
90 10.4 48 9.6 6.6 67 13.4 4.6 77 15.4 11.0 45 9
120 7.2 64 12.8 4.2 79 15.8 2.8 86 17.2 9.6 52 10.4

Uptake % Q (mg of ion/ g dry biomass)

100 30

specific uptake (Q)

25
80
20
Uptake %

60
15
40
10
20
5

0 0
0 1 2 3 4 5 6 7 8
pH values

Fig.(42): Effect of pH on the biosorption capacity of Cr6+ ions

after two hour of contact

VI.A.2. Effect of contact time :-

Results showed that the hexavalent chromium
concentration decreases from an initial value to an
equilibrium value provided the contact time is sufficient.
The time needed to reach equilibrium is defined as
"equilibrium time"'. Results showed that the uptake

105
RESULTS

capacity of Cr6+ ions increased with contact time till two

hour and then the uptake capacity doesn't show any
significant results as shown in Table (29) and Fig. (43).
Table (29): The biosorption capacity of Cr6+ ions at different
contact times
Time (min) Cr6+ Conc. Uptake % Q (mg /g)
0 20 0 0
60 7.8 61 12.2
120 2.8 86 17.2
300 2.4 88 17.6
1440 3.2 84 16.8

Uptake % Q (mg of ion/ g dry biomass)

100 100

specific uptake (Q)

80 80
Uptake %

60 60

40 40

20 20

0 0
0 200 400 600 800 1000 1200 1400

Time (min)

Fig. (43): Effect of contact times on the biosorption capacity of

Cr6+ ions

VI.A.3. Effect of temperature:-

Results showed that the maximum uptake capacity
of Cr6+ is observed at temperature range between 25 and
50C where about 86.0 %, 17.2 mg/g and 89.5 %, 17.9
mg/g were accumulated after two hours, respectively. The
minimum uptake capacity was showed at 15C and 10C,

106
RESULTS

respectively as shown in Table (30) and Fig. (44). Results

indicating that the maximum uptake could occur at wide
range of temperature.
Table (30): Effect of temperature on the biosorption capacity of
Cr6+ at different contact times

Temp 10 C 15 C 25 C 50 C
Conc. ppm

Conc. ppm

Conc. ppm

Conc. ppm
Uptake %
Uptake %

Uptake %
Uptake %
Q (mg /g)

Q (mg /g)

Q (mg /g)

Q (mg /g)
Time
(min)

0 20.0 0 0 20.0 0 0 20.0 0 0 20.0 0 0

30 16.9 15.5 3.1 14.8 26 5.2 11.9 40.5 8.1 11.1 44.5 8.9
60 14.3 28.5 5.7 12.2 39 7.8 7.8 61 12.2 6.9 65.5 13.1
90 13.4 33 6.6 11.5 42.5 8.5 4.6 77 15.4 3.8 81 16.2
120 12.9 35.5 7.1 11.0 45 9 2.8 86 17.2 2.1 89.5 17.9

Uptake % Q (mg of ion/ g dry biomass)

30
100
25
specific uptake (Q)
80
Uptake %

20
60
15

40 10

20 5

0 0
0 5 10 15 20 25 30 35 40 45 50 55
o
Temperature values ( C )

Fig. (44): The biosorption capacity of Cr6+ at different

temperature values after two hours of contact

VI.A.4. Effect of stirring rate:-

Results showed that the maximum uptake capacity
88.5 %, 17.7 mg/g and 86.0 %, 17.2 mg/g were obtained at
stirring rates 500 and 250, respectively. The minimum
107
RESULTS

uptake capacity were showed at low stirring rates (0 rpm

and 100 rpm), respectively as shown in Table (31) and
Fig. (45). Results indicating that, the uptake of metal ions
increased with the increase of stirring rate.
Table (31): Effect of stirring rate on the biosorption capacity of
Cr6+ at different contact times

0 rpm 100 rpm 250 rpm 500 rpm

Stir
Conc. ppm

Conc. ppm

Conc. ppm

Conc. ppm
r
Uptake %

Uptake %

Uptake %

Uptake %
Q (mg /g)

Q (mg /g)

Q (mg /g)

Q (mg /g)
Time
(min)

0 20.0 0 0 20.0 0 0 20.0 0 0 20.0 0 0

30 15.5 22.5 4.5 14.6 27 5.4 11.9 40.5 8.1 11.3 43.5 8.7

60 14.9 25.5 5.1 10.8 46 9.2 7.8 61 12.2 6.0 70 14

90 13.9 30.5 6.1 8.5 57.5 11.5 4.6 77 15.4 3.4 83 16.6

120 13.7 31.5 6.3 7.6 62 12.4 2.8 86 17.2 2.3 88.5 17.7

Uptake % Q (mg of ion/ g dry biomass)

100 30
specific uptake (Q)

25
80

20
Uptake %

60
15
40
10

20
5

0 0
0 100 200 300 400 500
Stirring rate (rpm)

Fig. (45): The biosorption capacity of Cr6+ at different stirring

rates after 2 h of contact

108
RESULTS

VI.A.5. Effect of biomass weight:-

Results showed that there an increase in the uptake
percentage of Cr6+ with the increase in biomass weight
from 0.05 to 0.5 g than from 0.5 g to 1.0 g. While, the mg
uptake/g dry weight decreased with the increase in the
biomass weight. The best uptake capacity were showed at
biomass weight 0.1g, it achieves 86.0 % and 17.2 mg/g as
shown in Table (32) and Fig. (46).
Table (32): Effect of biomass weight on the biosorption capacity
of Cr6+ at different contact times
Biomass 0.05 0.1 0.5 1.0
weight
Uptake %

Uptake %

Uptake %

Uptake %
Q (mg /g)

Q (mg /g)

Q (mg /g)

Q (mg /g)
(g)
Conc.

Conc.

Conc.

Conc.
Ppm
ppm

ppm

ppm
Time
(min)

0 20.0 0 0 20.0 0 0 20.0 0 0 20 0 0

30 15.6 22 8..8 11.9 40.5 8.1 10.2 49 1.96 9.5 52.5 1.05

60 10.5 47.5 19 7.8 61 12.2 5.9 70.5 2.82 4.1 79.5 1.59

90 8.0 60 24 4.6 77 15.4 3.5 82.5 3.3 2.9 85.5 1.71

120 5.5 72.5 29 2.8 86 17.2 1.1 94.5 3.78 0.9 95.5 1.91

Uptake % Q (mg of ion/ g dry biomass)

120
30
specific uptake (Q)

110 25
Uptake %

20
100
15
90
10

80 5

0
70
0.0 0.2 0.4 0.6 0.8 1.0
Biomass Weight (g)

Fig. (46): The biosorption capacity of Cr6+ at different biomass

weights after 2 h of contact
109
RESULTS

VI.A.6. Effect of Cr6+ ion concentration:-

It is found that, 0.1 gram of A. niger . could fix
86.0, 64.8 and 58.9 % from 20, 50 and 100 ppm Cr6+
solutions, respectively. Results also showed that one gram
dry weight of A. niger . could accumulate 17.2, 32.4 and
58.9 mg of Cr6+ from 20, 50 and 100 ppm solutions,
respectively as shown in Tables (33, 34) and Figures (47-
49).
Results obtained showed that the amount of Cr6+
accumulated increased with the increase in Cr6+ conc.
According to correlation coefficient (R2), the Freundlich's
model is best fit for Cr6+ biosorption by A. niger than
Langmuirs model. Statistical analyses for biosorption of
Cr6+ ions by A. niger strain at different environmental
conditions by One-way ANOVA are shown in Table (35).

Table (33): Effect of Cr6+ ion concentration on biosorption

capacity at different contact times

Cr6+ 20 50 100
(ppm)
Conc. ppm

Conc. ppm
Uptake %

Uptake %

Uptake %
Q (mg /g)

Q (mg /g)

Q (mg /g)
Conc.
Ppm

Time

(min)
0 20.0 0 0 50.0 0 0 100 0 0
30 11.9 40.5 8.1 46.0 8 4 95.0 5 5
60 7.8 61 12.2 39.2 21.6 10.8 70.1 29.9 29.9
90 4.6 77 15.4 28.5 43 21.5 53.5 46.5 46.5
120 2.8 86 17.2 17.6 64.8 32.4 41.1 58.9 58.9

110
RESULTS

Table (34): Functional isotherm parameters of control A. niger .

at different Cr6+ Concs.

Freundlich's
Langmuirs
Functional
parameter

log Qe
log Ce

Ce/Qe
Qe
C0

R2

R2
Ce
20 2.8 17.2 0.45 1.24 0.16
A. 50 17.6 32.4 1.25 1.51 0.54 0.8696* 0.9632*
niger .
100 41.1 58.9 1.61 1.77 0.69
Model parameters estimated are statistically
*
significant at 95% confidence level.

Uptake % Q (mg of ion/ g dry biomass)

100
90 60

80
50

specific uptake (Q)

70
Uptake %

60 40

50
30
40
30 20
20
10
10
0 0
0 10 20 30 40 50 60 70 80 90 100 110
6+
Cr conc (ppm)
L ang muirs is otherm model for c hromium
6+
Fig. (47): The biosorption
removal capacity
by c ontrol of. Cr
A . nig s trainfrom
afterdifferent
2h of conc. of
Cr6+ after 2 h of contact c ontac t

0.8 y = 0.0133x + 0.1954

0.6 R 2 = 0.8696
C e / Qe

0.4
0.2
0
0 10 20 30 40 50
Ce

c e/qe L inear (c e/qe)

111
 RESULTSF reundlic h's is otherm model for c hromium
removal by c ontrol A . nig . s train after 2h of
Fig. (48): Langmuir isothermcfor Cr6+
ontac t removal by control strain
y = 0.4399x + 1.0205
2 R 2 = 0.9632
1.5
log Qe

1
0.5
0
0 0.5 1 1.5 2
log C e

log qe L inear (log qe)

Fig. (49): Freundlich isotherm for Cr6+ removal by control strain

Table (35): Statistical analysis for biosorption of Cr6+ ions by A.

niger strain at different environmental conditions by One-way
ANOVA

At ion
Contact Stirr. Biomass
diff. pH Temp. conc.
time rate wt.
P(Sig.) 0.000 0.036 0.002 0.001 0.000 0.033

VI.B. Biosorption capacity of lead, copper and cadmium

ions :-
The affinity and the capacity of the most tolerant
fungal species (A. niger) towards lead, copper and
cadmium ions were investigated. This single state study is
done in batch experiments for initial metal ions
concentration 20 ppm at pH 5.0 0.2, temperature 25 3C
and at stirring rate 250 rpm as shown in Table (36) and
Fig. (50). Results showed that the uptake capacity had the
following order: - Cr6+ (86%, 17.2 mg/g) > Pb2+ (70.5%, 14.1
mg/g) > Cu2+ (66.5%, 13.3 mg/g) > Cd2+ (55.5%, 11.1 mg/g).
Also, Statistical analysis for biosorption of the studied

112
RESULTS

metal ions by A. niger strain by One-way ANOVA is

shown in Table (37).
Table (36): The biosorption capacity of lead, copper and
cadmium at different contact times

ions Pb2+ Cu2+ Cd2+

Uptake %
(mg /g)

(mg /g)

(mg /g)
Uptake

Uptake
Conc.

Conc.

Conc.
Ppm

ppm

ppm
Time
%

%
Q

Q
(min)

0 20 0 0 20 0 0 20 0 0
30 15.4 23 4.6 16.9 15.5 3.1 15.5 22.5 4.5
60 10.2 49 6.8 11.8 41 8.2 13.2 34 6.8
90 8.5 57.5 9.5 9.5 52.5 10.5 10.1 49.5 9.9
120 5.9 70.5 14.1 6.7 66.5 13.3 8.9 55.5 11.1

Uptake % Q (mg of ion/ g dry biomass)

100

15
80

specific uptake (Q)

Uptake %

60
10

control A. nig
40 6+
Cr 20 ppm
2+ 5
Pb 20 ppm
20
2+
Cu 20 ppm
2+
Cd 20 ppm
0 0
Chromium Lead Copper Cadmium
Different metal ions

Fig. (50): The biosorption capacity of the studied metal ions after
2 h of contact

Table (37): Statistical analysis for biosorption of the studied

metal ions by A. niger strain by One-way ANOVA

metal ions Cr6+,Pb2+, Cu2+ and Cd2+

0.000
P(Sig.)

113
RESULTS

VII. PRETREATMENT STUDY

Several experiments were done to investigate the
effect of physical and chemical pretreatments of fungal
biomass on the biosorption capacity of Cr6+ ions (50 ppm).
Also, all studies were done in batch experiments at
temperature 25 3C, pH 5.0 0.2 and 250 rpm.
VII.A. Chemical pretreatments:
VII.A.1. Sodium hydroxide as alkali- pretreatment:-
Isolated A. niger strain is pretreated with different
NaOH conc. (0.1 and 0.2 N) to study the biosorption
capacity for Cr6+ ion (50 ppm). Results showed that the
maximum uptake (81.6%) and the highest mg/g dry weight
(40.8) were achieved by with 0.1N then 0.2N NaOH
pretreatment, while the minimum uptake (64.8%) and the
lowest mg/g dry weight (32.4) were obtained by control
biomass as shown in Table (38) and Fig. (51).
Table (38): Effect of pretreatment of A. niger . with NaOH on the
biosorption capacity of Cr6+ ion (50 ppm)

NaOH
Conc. Control 0.1 0.2
(N)
Uptake

Uptake

Uptake
(mg/g)

(mg/g)

(mg/g)
Conc.

Conc.

Conc.
ppm

ppm

ppm
%

%
Q

Time
(min)
0 50.0 0 0 50.0 0 0 50.0 0 0
30 46.0 8 4 37.9 24.2 12.1 40.6 18.8 9.4
60 39.2 21.6 10.8 22.4 55.2 27.6 28.2 43.6 21.8
90 28.5 43 21.5 18.6 62.8 31.4 22.3 55.4 27.7
120 17.6 64.8 32.4 9.2 81.6 40.8 12.5 75 37.5

114
RESULTS

Uptake % Q (mg of ion/ g dry biomass)

100
6+ 40
Cr 50 ppm

specific uptake (Q)

80
30
Uptake %

60

20
40

10
20

0 0
Control 0.1 0.2

treatment of A. nig by NaOH

Fig.(51): The biosorption capacity of Cr6+ by A. niger . pretreated

with different NaOH conc. after 2 h of contact

VII.A.2. Glutraldehyde pretreatment:

Isolated A. niger strain is pretreated with different
glutraldehyde conc. (0.1% and 0.2%) to study the
biosorption capacity for Cr6+ ion (50 ppm). Results showed
that the maximum uptake (69%) and the highest mg/g dry
weight (34.5) were achieved by A. niger pretreated with
glutraldehyde of conc. 0.1% then control biomass, while
the minimum uptake (64.0%) and the lowest mg/g dry
weight (32.0) were obtained with glutraldehyde 0.2% as
shown in Table (39) and Fig. (52).

115
RESULTS

Table (39): Effect of pretreatment of A. niger . with glutraldehyde

on the biosorption capacity of Cr6+ ion (50 ppm)

Glutraldehyde
Conc. (%) Control 0.1 0.2

Uptake

Uptake

Uptake
(mg/g)

(mg/g)

(mg/g)
Conc.

Conc.

Conc.
ppm

ppm

ppm
%

%
Q

Q
Time (min)
0 50.0 0 0 50.0 0 0 50.0 0 0
30 46.0 8 4 40.9 18.2 9.1 43.4 13.2 6.6
60 39.2 21.6 10.8 33.8 32.4 16.2 34.1 31.8 15.9
90 28.5 43 21.5 22.4 55.2 27.6 29.6 40.8 20.4
120 17.6 64.8 32.4 15.5 69 34.5 18.0 64 32

Uptake % Q (mg of ion/ g dry biomass)

100
6+
Cr 50 ppm 40

80

specific uptake (Q)

30
Uptake %

60

20
40

10
20

0 0
Control 0.1 0.2

treatment of A. nig by Glutraldehyde

Fig.(52): The biosorption capacity of Cr6+ by A. niger . pretreated

with different glutraldehyde conc. after 2 h of contact

VII.A.3. CaCl2 pretreatment:

Isolated A. niger strain is pretreated with different
CaCl2 conc. (0.1% and 0.2%) to study the biosorption
capacity for Cr6+ ion (50 ppm). Results showed that the
maximum uptake (73.2%) and the highest mg/g dry weight
(36.6) were achieved by A. niger pretreated with 0.1% then
0.2% CaCl2, while the minimum uptake (64.8%) and the
116
RESULTS

lowest mg/g dry weight (32.4) were obtained by control

biomass as shown in Table (40) and Fig. (53).

Table (40): Effect of pretreatment of A. niger . with CaCl2 on the

biosorption capacity of Cr6+ ion (50 ppm)

CaCl2
Conc. Control 0.1 0.2
(N)
Time
Uptake

Uptake

Uptake
(mg/g)

(mg/g)

(mg/g)
Conc.

Conc.

Conc.
ppm

ppm

ppm
%

%
Q

Q
(min)

0 50.0 0 0 50.0 0 0 50.0 0 0

30 46.0 8 4 30.8 38.4 19.2 39.8 20.4 10.2
60 39.2 21.6 10.8 25.7 48.6 24.3 28.7 42.6 21.3
90 28.5 43 21.5 17.3 65.4 32.7 22.3 55.4 27.7
120 17.6 64.8 32.4 13.4 73.2 36.6 15.9 68.2 34.1

Uptake % Q (mg of ion/ g dry biomass)

100
6+
Cr 50 ppm 40

80

specific uptake (Q)

30
Uptake %

60

20
40

10
20

0 0
Control 0.1 0.2

treatment of A. nig by CaCl2

Fig. (53): The biosorption capacity of Cr6+ by A. niger .

pretreated with different CaCl2 conc. after 2 h of contact

VII.B. Physical pretreatments:

VII.B.1. Boiling and autoclaving effects:
Isolated A. niger strain is pretreated with boiling and
autoclaving. It showed that the maximum uptake (75.2%)
and the highest mg/g dry weight (37.6) were achieved by A.
117
RESULTS

niger pretreated with autoclaving then control biomass,

while the minimum uptake (52.6%) and the lowest mg/g
dry weight (26.3) were obtained by A. niger pretreated with
boiling as shown in Table (41) and Fig. (54).
Table (41): Effect of physical pretreatment (boiling and
autoclaving) of A. niger . on the biosorption capacity of Cr6+ 50
ppm

Treatment
type Control Boiling Autoclaving
Uptake

Uptake

Uptake
(mg/g)

(mg/g)

(mg/g)
Conc.

Conc.

Conc.
Ppm
ppm

ppm
%

%
Time (min)
Q

Q
0 50.0 0 0 50.0 0 0 50.0 0 0
30 46.0 8 4 48.9 2.2 1.1 42.1 15.8 7.9
60 39.2 21.6 10.8 42.6 14.8 7.4 35.8 28.4 14.2
90 28.5 43 21.5 32.4 35.2 17.6 23.2 53.6 26.8
120 17.6 64.8 32.4 23.7 52.6 26.3 12.4 75.2 37.6

The previous data were collected in Fig. (55), which

showed the uptake percentage of Cr6+ 50 ppm after two
hours of contact with A. niger subjected to different
pretreatment processes. Results showed that the rate of Cr6+
uptake has the following orders; pretreated with 0.1N
NaOH > autoclaving > 0.1% CaCl2 > 0.1% Glutraldehyde
> control biomass > Boiling. Statistical analysis for
biosorption of Cr6+ ions by A. niger strain after different
treatment methods by One-way ANOVA is showed in
Table (42).

118
RESULTS

Table (42): Statistical analysis for biosorption of Cr6+ ions by A.

niger . strain at different treatment methods by One-way
ANOVA
At different treatment Chemical and Physical
methods treatment
P(Sig.) 0.000

Uptake % Q (mg of ion/ g dry biomass)

100
Cr
6+
50 ppm 40

80

specific uptake (Q)

30
Uptake %

60

20
40

10
20

0 0
Control Boiling Autoclaving

Physical treatment of A. nig

Fig. (54): The biosorption capacity of Cr6+ by control, Autoclaved

and Boiled A. niger . after 2 h of contact

0.1 N 6+
Cr 50 ppm
80
0.1 %
70 0.1 %
60
Uptake %

50

40

30

20

10

0
NaOH Autoclaved Cal Chlor Glutr. Control Boiled

Types of Pretreatment

Fig. (55): The order of uptake percentage of Cr6+ at conc. 50 ppm

by physical and chemical pretreated A. niger . after 2 h of contact

119
RESULTS

VIII. Treatment of tannery wastewater

The purpose of this study is to determine the ability
of alkali-treated A. niger (free and immobilized biomass) to
remove toxic constituents from tannery wastewater. Batch
biosorption experiments were carried out at temperature 25
3C, pH 5.0 0.2 and 250 rpm for two hours of contact.
VIII.A. Treatment by alkali-treated biomass (FC):-
Tannery wastewater sample had high mixed heavy
metals pollutants and high organic loads. The BOD/COD
ratio is nearly 0.22, which is very low in comparison to
domestic wastewater (i.e., 0.5). Therefore, the
biodegradability of the influent is found to be low. The
COD/ ammonia nitrogen as (NH4+)/ phosphorous as (PO4-3)
ratio averaged 550/200/15 indicated that this liquor
contained high amounts of nitrogen but lesser amounts of
phosphorus. Hyper saline wastewater means wastewater
containing more than 35 g L-I TDS.
Alkali-treated A. niger biomass showed decrease of
all metal ions present in tannery wastewater. The
biosorption capacity are as follow; chromium (31%, 155
mg/g), cadmium (40%, 6.2 mg/g), copper (57.4%, 1.55
mg/g), lead (46.1%, 7.1 mg/g), nickel (52.8%, 2.8 mg/g),
iron (43.1%, 4.66 mg/g) and manganese ions (61.9%, 1.3
mg/g). The removal percentages order at equilibrium is:
Mn > Cu > Ni > Pb > Fe > Cd > Cr. Also, alkali-treated A.
120
 RESULTS

niger biomass were effective in decreasing the organic

loads from tannery wastewater. It decreases TDS, EC and
Salinity up to 32.1, 32.2 and 27.4 %, respectively. It also
decreases COD up to 51.8 %, BOD up to 48.6 %, ammonia
nitrogen up to 66.1 %, nitrate up to 63.0 % and
phosphorous up to 43.3 %, respectively as shown in Tables
(43, 44) and Figures (56-58).
Table (43): Chemical constituents values before and after
treatment of tannery WW by FC after 2 h of contact

Before After Removal

Constituents Units
treatment treatment %
580nm 0.296 0.178 39.9
Absorbance Absorbance
418nm 0.396 0.215 45.7
TDS 57.87 39.3 32.1 PPt
EC 116 78.6 32.2 ms/cm
Salinity 73.2 53.2 27.4
DO 45.0 32.0 28.9 %
COD 550 265.0 51.8 mg O2/L
BOD 120 61.7 48.6 mg O2/L
Ammonia nitrogen 200.11 67.9 66.1 mgNH4+ /L
Nitrate 25 9.3 63.0 mg(NO3-)/L
Phosphorous 15 8.5 43.3 mgPO4+ /L

Fig.(56): Scan spectrum curve shows the decrease in absorbance

of tannery wastewater sample after treatment with free biomass
after 2 h of contact
121
RESULTS

70 alkali-treated A. niger 66.1

63.0
60
51.8
48.6
50
43.3
Removal %

40
32.1 32.2
27.4 28.9
30

20

10

0
TDS EC Salinity DO COD BOD Amonia Nitrate Phosphorous

Chemical constituents of tannery WW

Fig. (57): Removal percentage of chemical constituents in tannery

WW after treatment by free biomass.

Table (44): Heavy metals values before and after treatment of

tannery WW by free biomass

Before After
Constituents Q(mg/g) Removal %
treatment treatment
Chromium 500 345 155 31
Cadmium 15.5 9.3 6.2 40
Copper 2.7 1.15 1.55 57.4
Lead 15.4 8.3 7.1 46.1
Nickel 5.3 2.5 2.8 52.8
Iron 10.8 6.14 4.66 43.1
Manganese 2.1 0.8 1.3 61.9

122
RESULTS

Q (mg of ion/ g dry biomass) Removal %

Chromium Cadmium Copper Lead Nickel Iron Manganese

155 (A. niger treated with NaOH) 100

150
specific uptake (Q)

80
61.9

Removal %
100 57.4 52.8 60
46.1 43.1
40
50 31 40

6.2 7.1 2.8 4.66 1.3 20

1.55
0
0
Chromium Cadmium Copper Lead Nickel Iron Manganese

Tannery WW metals

Fig. (58): Heavy metals biosorption capacity from tannery

wastewater by free biomass.

VIII.B. Treatment by immobilization:-

The proposal of this study is to examine the use of
alginate cross-linked beads in treating tannery wastewater
streams.
VIII.B.1. Effect of immobilization by different alginate
forms:-
The effect of immobilization of alkali-treated A. niger
by alginate particularly, Ca-alginate, Al-alginate and Fe-
alginate on the treatment is investigated. Results showed
that the decrease in absorbance at : 580 nm and 418 nm is
in the following order: Immobilization by calcium ions
(72.3 72.7 %) > Free biomass (39.9 - 45.7 %) >
Immobilization by aluminum ions (37.8 - 39.9 %) >
Immobilization by ferric ions (24.7 - 34.8 %).
Immobilization by Ca-alginate is also effective in treating
123
 RESULTS

chromium and iron ions with removal percentages 51 %

and 58.3 %, respectively, while by free biomass is 31 %
and 43.1 % as shown in Table (45) and Figures (59, 60).
Table (45): Effect of different immobilized forms of alkali-treated
biomass on tannery wastewater treatment

Ca2+ Al3+ Fe3+

Before
Constituents alginate alginate- alginate-
treatment
Biomass Biomass Biomass
580 nm 0.082 0.184 0.223
0.296
Removal % 72.3 37.8 24.7
Abs.
418 nm 0.108 0.238 0.258
0.396
Removal % 72.7 39.9 34.8
Conc.(ppm) 245 302 328
Removal % 51 39.6 34.4
Chromium Q 500
(mg/10ml 25.5 19.8 17.2
alginate-biomass)
Conc.(ppm) 4.5 5.2 5.8
Removal % 58.3 51.9 46.3
Iron Q 10.8
(mg/10ml 0.63 0.56 0.5
alginate-biomass)

Fig. (59): Scan spectrum curve shows the decrease in absorbance

of tannery wastewater sample after treatment by different
immobilized forms of alkali-treated biomass after 2h of contact

124
RESULTS

Q (mg of ion/10ml alginate- biomass) (Removal %)

Chromium Ca Chromium Al Chromium Fe Iron Ca Iron Al Iron Fe
100
specific uptake (Q) 25
25.5 19.8
17.2 80
20

Removal %
58.3
15 51.9 60
51
46.3
10 39.6
34.4 40
5
0.63 0.56 0.5
20
0
Chromium Ca Chromium Al Chromium Fe Iron Ca Iron Al Iron Fe

Tannery WW metals

Fig. (60): Heavy metals biosorption capacity of treated tannery

WW by different immobilized forms of alkali-treated biomass

VIII.B.2. Treatment by Ca-alginate and Ca-alginate-

biomass:-
Alkali-treated biomass is immobilized by inclusion in
Ca-alginate. The biomass/polymer matrices were formed
into equal size unites of spheres (1.5 - 2.5mm), and the
resulting biomass/polymer matrices were used to treat
tannery wastewater. The biomass/polymer matrices showed
significant decrease of all metal ions present in tannery
wastewater than free biomass. The removal percentages
were as follow; chromium (51 %), cadmium (70.97 %),
copper (91.9 %), lead (79.9 %), Nickel (93.8 %), iron (58.3
%) and manganese ions (87.6 %). The removal percentages
order is: Ni > Cu > Mn > Pb > Cd > Fe > Cr. Also, it is
effective in decreasing the organic loads from tannery
wastewater. It decreases TDS, EC and salinity up to 52.1
%, 52.2 % and 47.4 %, respectively. It also decreases COD
125
RESULTS

up to 71.8 %, BOD up to 68.6 %, ammonia nitrogen up to

86.1 %, nitrate up to 83.0 % and phosphorous up to 63.3
%, respectively. While the removal efficiencies of Ca-
alginate for the same wastewater was for chromium (43.5
%), cadmium (51.6 %), copper (67.4%), lead (57.8%),
nickel (73.02%), iron (51.02%) and manganese ions (74.8
%). The removal percentages order is: Mn > Ni > Cu > Pb
> Cd > Fe > Cr. Also, it is effective in decreasing the
organic loads from tannery wastewater. It decreases TDS,
EC and salinity up to 44.6 %, 44.7 % and 39.9 %,
respectively. It also decreases COD up to 64.3 %, BOD up
to 61.1 %, ammonia nitrogen up to 78.6 %, nitrate up to
75.5 % and phosphorous up to 55.8 %, respectively.
Results showed that the immobilization of alkali-
treated biomass in Ca-alginate beads is effective in treating
tannery wastewater by decreasing the wastewater
absorbance at : 580 nm and 418 nm to 72.3 % and 72.8%,
respectively then by Ca-alginate alone to 71.6 % and 71.9
%, respectively. The results obtained from these
experiments, were compared with those using dispersed
biomass as a sorbent (39.9 % and 45.7 %). So, the total
biosorption capacities of the biosorbents were in the
following order: Free cells < Ca-alginate < Ca alginate-
biomass as shown in Tables (46-48) and Figures (61-64).

126
RESULTS

Table (46): Chemical constituents value after treatment of

tannery WW by Ca-alginate and Ca-alginate-biomass.

Ca-
Ca- Removal Removal
Constituents alginate- Units
alginate % %
biomass
580nm 0.084 71.6 0.082 72.3
Abs.
Abs. 418nm 0.111 71.9 0.108 72.8
TDS 32.1 44.6 27.7 52.1 PPt
EC 64.1 44.7 55.4 52.2 ms/cm
Salinity 44.01 39.9 38.5 47.4
DO 26.4 41.4 23 48.9 %
COD 196.3 64.3 155 71.8 mg/L
BOD 46.68 61.1 37.7 68.6 mg/L
Ammonia
42.9 78.6 27.9 86.1 mg/L
nitrogen
Nitrate 6.1 75.5 4.3 83 mg/L
Phosphorous 6.7 55.8 5.5 63.3 mg/L

Fig. (61): Scan spectrum curve shows the decrease in absorbance

of tannery wastewater sample after treatment with Ca-alginate
and Ca-alginate-biomass forms after 2 h of contact.

127
RESULTS

Ca-alginate 86.1 83
80 Ca-alginate + A.niger
71.8
68.6
63.3
60
Removal %
52.1 52.2
47.4 48.9

40

20

0
TDS EC Salinity DO COD BOD Amonia Nitrate Phosphorous

Chemical constituents

Fig. (62): The removal percentage of chemical constituents in

tannery WW after treatment by Ca-alginate and Ca-alginate
biomass

Table (47): Heavy metals values before and after treatment of

tannery WW by Ca-alginate

Before After
Constituents Q Removal %
treatment treatment
Chromium 500 282.5 21.75 43.5
Cadmium 15.5 7.5 0.8 51.6
Copper 2.7 0.88 0.182 67.4
Lead 15.4 6.5 0.89 57.8
Nickel 5.3 1.43 0.387 73.02
Iron 10.8 5.29 0.551 51.02
Manganese 2.1 0.53 0.157 74.8

128
RESULTS

Q (mg of ion/10ml alginate) Removal %

Chromium Cadmium Copper Lead Nickel Iron Manganese
25
Ca alginate 100
specific uptake (Q)
20
73.02 74.8 80
67.4
15

Removal %
57.8
51.6 51.02 60
10
40
5
43.5
20
0
0
Chromium Cadmium Copper Lead Nickel Iron Manganese

Tannery WW metals

Fig. (63): Heavy metals biosorption capacity from tannery

wastewater by Ca-alginate

Table (48): Heavy metals values before and after treatment of

tannery WW by Ca-alginate-biomass

Before After Removal

Constituents Q
treatment treatment %
Chromium 500 245 25.5 51
Cadmium 15.5 4.5 1.1 70.97
Copper 2.7 0.22 0.248 91.9
Lead 15.4 3.1 1.23 79.9
Nickel 5.3 0.33 0.497 93.8
Iron 10.8 4.5 0.63 58.3
Manganese 2.1 0.26 0.184 87.6

129
RESULTS

Q (mg of ion/10ml alginate- bimass) Removal %

Chromium Cadmium Copper Lead Nickel Iron Manganese
30
91.9 Ca alginate - Biomass 100
25
79.9
specific uptake (Q)

70.97 93.8 80
20 87.6

Removal %
15 51 60

58.3
10 40

5
20

0
0
Chromium Cadmium Copper Lead Nickel Iron Manganese

Tannery WW metals

Fig. (64): Heavy metals biosorption capacity from tannery

wastewater by Ca-alginate-biomass.

130
RESULTS

IX. Recycling or Reusability

In order to show the reusability of the biosorbents
(Ca-alginate and Ca-alginate-biomass) beads, they were
eluted using 0.05N HNO3 and then reused. This process is
carried out for three subsequent cycles. All studies were
done in batch experiments at temperature 25 3C, pH 5.0
0.2 and 250 rpm.
IX.A. Recycling of Ca alginate:-
Treatment of tannery wastewater by Ca-alginate beads
is investigated for three cycles. It is found that, the removal
efficiencies of Ca-alginate decreased through three cycles
for chromium (43.5 %, 36 % and 28.5%), cadmium (51.6
%, 41.9 % and 31.6 %), copper (67.4 %, 55.6 % and 33.3
%), lead (57.8 %, 51.3 % and 44.2%), nickel (73.02 %,
65.5 % and 52.8 %), iron (51.02 %, 40.6 % and 23.1%) and
manganese ions (74.8 %, 52.4 % and 28.1%) at 1st cycle,
2nd cycle and 3rd cycle, respectively. Also, the efficacy in
removal of organic loads from tannery wastewater through
three cycles is decreased as shown in Tables (49, 50) and
Figures (65 - 67).

131
RESULTS

Table (49): Treatment of tannery wastewater by Ca-alginate

beads in three repeated cycles

Constituents

Cycles of Ca
alginate
1St Removal 2nd Removal 3rd Removal
cycle % cycle % cycle %

580nm 0.084 71.6 0.169 42.9 0.176 40.5

Abs. 418nm 0.111 71.9 0.192 51.5 0.216 45.5
TDS 32.1 44.6 36.4 37.1 40.8 29.6
EC 64.1 44.7 72.8 37.2 81.5 29.7
Salinity 44.01 39.9 49.5 32.4 54.9 24.9
DO 26.4 41.4 29.8 33.9 33.1 26.4
COD 196.3 64.3 237.5 56.8 278.8 49.3
BOD 46.68 61.1 55.44 53.8 64.68 46.1
Ammonia
42.9 78.6 57.9 71.1 72.9 63.6
nitrogen
Nitrate 6.1 75.5 8 68 9.9 60.5
Phosphorous 6.7 55.8 7.8 48.3 8.9 40.8

Fig. (65): Scan spectrum curve shows the decrease in absorbance

of tannery wastewater sample after treatment with Ca-alginate in
three repeated cycles, after two hours of contact

132
RESULTS

80 First cycle
70 Second cycle
60 Third cycle

Removal % 50
40
30
20
10
0
TDS EC Salinity DO COD BOD Amonia Nitrate PO4
Chemical constituents

Fig. (66): The removal percentage of chemical constituents in tannery

WW after recycling of Ca-alginate in three repeated cycles
Table (50): Heavy metals values before and after treatment of tannery
WW by Ca-alginate in three repeated cycles
Cycles of Ca
Constituents

alginate

1St Removal 2nd Removal 3rd Removal

cycle % cycle % cycle %

Chromium 282.5 43.5 320 36 357.5 28.5

Cadmium 7.5 51.6 9.0 41.9 10.6 31.6
Copper 0.88 67.4 1.2 55.6 1.8 33.3
Lead 6.5 57.8 7.5 51.3 8.6 44.2
Nickel 1.43 73.02 1.83 65.5 2.5 52.8
Iron 5.29 51.02 6.42 40.6 8.31 23.1
Manganese 0.53 74.8 1.0 52.4 1.51 28.1

100
First Cycle Recycling by Ca alginate
90
Second Cycle
80
Third Cycle
70
Removal %

60
50
40
30
20
10
0
Chromium Cadmium Copper Lead Nickel Iron Manganese

Tannery WW metals

Fig. (67): Heavy metals biosorption capacity from tannery wastewater

by Ca-alginate in three repeated cycles.

133
RESULTS

IX.B. Recycling of Ca- alginate- biomass :-

Treatment of tannery wastewater by Ca-alginate-
biomass beads was investigated for three cycles. It is found
that, the removal efficiencies of Ca-alginate-biomass
decreased through three cycles for chromium (51 %, 41 %
and 34.5 %), cadmium (70.97 %, 54.8 % and 41.7 %),
copper (91.9 %, 66.7% and 59.3 %), lead (79.9 %, 69.6 %
and 56.04 %), nickel (93.8 %, 72.5 % and 63.4 %), iron
(58.3 %, 51.5 % and 39.8 %) and manganese ions (87.6 %,
66.7 % and 57.6 %) at 1st cycle, 2nd cycle and 3rd cycle,
respectively. Also, the efficacy in removal of organic loads
from tannery wastewater is decreased as shown in Tables
(51, 52) and Figures (68-70).
Table (51): Treatment of tannery wastewater by Ca-alginate-
biomass beads in three repeated cycles
Cycles of Ca
Constituents

alginate-
biomass

1St Removal 2nd Removal 3rd Removal

cycle % cycle % cycle %

580nm 0.082 72.3 0.151 48.9 0.169 42.9

Abs.
418nm 0.108 72.8 0.162 59.1 0.191 51.8
TDS 27.7 52.1 33.5 42.1 37.3 35.6
EC 55.4 52.2 67 42.2 74.5 35.7
Salinity 38.5 47.4 45.8 37.4 50.6 30.9
DO 23 48.9 27.5 38.9 30.4 32.4
COD 155 71.8 210 61.8 245.8 55.3
BOD 37.7 68.6 49.7 58.6 57.5 52.1
Ammonia
27.9 86.1 47.9 76.1 60.9 69.6
nitrogen
Nitrate 4.3 83 6.8 73 8.4 66.5
Phosphorous 5.5 63.3 7 53.3 7.9 46.8

134
RESULTS

Fig. (68): Scan spectrum curve shows the decrease in absorbance

of tannery wastewater sample after treatment with Ca-alginate-
biomass in three repeated cycles, after two hours of contact

100
First cycle
90
Second cycle
80
Third cycle
Removal %

70
60
50
40
30
20
10
0
TDS EC Salinity DO COD BOD Amonia Nitrate PO4
Chemical constituents

Fig. (69): The removal percentage of chemical constituents in

tannery WW after recycling of Ca-alginate-biomass in three
repeated cycles
Table (52): Heavy metals values before and after treatment of
tannery WW by Ca-alginate Biomass in three repeated cycles
Constituents

Cycles of Ca

1St Removal 2nd Removal 3rd Removal

alginate-
biomass

cycle % cycle % cycle %

Chromium 245 51 295 41 327.5 34.5

Cadmium 4.5 70.97 7.0 54.8 9.03 41.7
Copper 0.22 91.9 0.9 66.7 1.1 59.3
Lead 3.1 79.9 4.68 69.6 6.77 56.04
Nickel 0.33 93.8 1.46 72.5 1.94 63.4
Iron 4.5 58.3 5.24 51.5 6.5 39.8
Manganese 0.26 87.6 0.7 66.7 0.89 57.6
135
RESULTS

110
First Cycle Recycling by Ca alginate-A. niger
100 Second Cycle
Third Cycle
Removal % 90
80
70
60
50
40
30
ChromiumCadmium Copper Lead Nickel Iron Manganese
Tannery WW metals

Fig. (70): Heavy metals biosorption capacity from tannery

wastewater by Ca-alginate-biomass in three repeated cycles

Photo (6): Ca-alginate and Ca-alginate-biomass beads before and

after tannery wastewater treatment
136
RESULTS

X. Examination of biosorbents by SEM

SEM micrograph and EDAX analysis were done for
alkali-treated A. niger biomass, Ca-alginate beads and Ca-
alginate-biomass (alkali-treated or autoclaved) beads either
before and or after treatment of tannery wastewater. Semi-
transparent layer above the mycelial biomass is observed.
EDAX analysis showed the quantity of metals biosorbed in
the pointed area not all of biomass. Before treatment there
are C (59.4 %), O (40.32 %) and Na (0.28 %). While, after
treatment there are C (27.03 %), O (52.73 %), Na (3.73 %),
Mg (0.42 %), Si (0.26 %), S (2.58 %), Cl (4.16 %), Ca
(0.48 %) and Cr (8.62 %) as shown in Plate 5 (a,b,c) & 6
(a.b,c).
Also, Ca-alginate beads before and after treatment of
tannery wastewater were studied. The SEM micrograph of
control Ca-alginate beads is plain, smooth and clear. EDAX
of Ca-alginate beads after treatment showed C (60.64 %),
O (22.06 %), P (0.75 %), S (1.28 %), Cl (0.38 %) and Cr
(14.90 %) as shown in Plate 7 (a, b, c).
While beads of Ca-alginate with alkali-treated biomass
showed mycelial fragments all around the bead and also the
surface of beads showed unifier distributed mycelial
protrusion indicating that the entrapped mycelia isn't
localized. EDAX analysis of these beads after treatment
showed C (26.62 %), O (34.77 %), Na (6.26 %), As (1.49

137
RESULTS

%), Cl (3.83 %), Pd (0.53 %), Ca (1.63 %), Cr (20.40 %),

and Pb (4.46 %) as shown in Plate 8 (a, b, c).
Also, beads of Ca-alginate with autoclaved biomass
showed fungal mycelia all around the bead as in alkali-
treated biomass beads. EDAX analysis of these beads after
treatment showed C (23.36 %), O (52.21 %), Na (9.5 %),
As (1.5 %), Pd (1.75 %), Ca (1.16 %), Cr (8.81 %) and Pb
(1.71 %) as shown in Plate 9 (a, b, c).

138
RESULTS

A) Mag. 800 X B) Mag. 3000 X

C) EDAX analysis
Plate 5 (a, b and c) shows A. niger biomass treated with NaOH
before tannery wastewater treatment

139
RESULTS

A) Mag. 400 X B) Mag. 6000 X

C) EDAX analysis
Plate 6 (a, b and c) shows alkali-treated A. niger . biomass after
tannery wastewater treatment

140
RESULTS

A) Mag. 70 X and 500 X for control Ca-alginate bead

b) Mag. 142 X and 485 X for Ca-alginate bead after tannery WW

treatment

c) EDAX analysis for Ca-alginate bead after tannery WW

treatment
Plate 7 (a, b and c) shows Ca-alginate bead before and after
tannery wastewater treatment

141
RESULTS

A) Mag. 137 X B) Mag. 4000 X

C) EDAX analysis
Plate 8 (a, b and c) shows immobilized bead with
alkali-treated biomass after tannery wastewater
treatment

142
RESULTS

A) Mag. 70 X B) Mag. 500 X

C) EDAX analysis
Plate 9 (a, b and c) shows immobilized bead with autoclaved
biomass after tannery wastewater treatment
XI. Fourier Transform Infrared Spectrometry
(FT-IR)
Another investigation related to the fungal
biosorption phenomenon is FT-IR. It is used for
determination of functional groups in organic materials in
frequency range from 500 to 4000 Cm-1. FT-IR is carried
out to A. niger fungal strain, alkali-treated A. niger, Ca-
alginate beads. FT-IR is also carried out to Ca-alginate-
biomass beads (alkali-treated) before and after tannery
wastewater treatment. The fungal biomass and beads were
dried to almost nil moisture and send for FT-IR analysis.
143
RESULTS

Spectral region consists of a set of peaks. This analysis had

eventually confirmed the difference between functional
groups in relation to biosorption occurred and to change in
chemical composition. A change of absorption bands can
be seen when comparing the FT-IR spectra of pristine and
tannery-loaded Ca-alginate-biomass beads. An interesting
phenomenon is the shifting and decrease in the band
intensity at (1041.5 to 1130.2 Cm-1), shifting and decrease
in the band intensity at (1651 to 1639.4 Cm-1) and shifting
and sharp decrease in the band intensity at (3552.6 to
3375.8 Cm-1) after tannery wastewater binding. This
decrease in the bands intensity was considered as
adsorption bands as shown in Tables (53-57) and Figures
(71-75).
XI.A. FT-IR spectrum for A. niger strain:-

144
RESULTS

Fig. (71): FT-IR spectrum for A. niger strain

Table (53): FT-IR spectral characteristics of A. niger strain

IR peak
Frequency Assignment T%
(Cm -1)
783.0 Strong broad N-H amines ( RNH2 Or R2NH) 41
or medium =C-H out of plane
1080.1 Strong C-O stretch (Carboxylic acids or 28
Esters or Ethers)
1523.7 Strong N=O nitroso Or strong N-O 43
asymmetric stretch
1639.4 Strong NH2 in plane bend (RNH2) amines or 46
strong N-H out of plane (RCONH2) amides
or strong C=O stretch (RCONHR) amides
3614.4 Strong O-H free Hydroxyl 48

XI.B. FT-IR spectrum for alkali-treated A. niger

strain:-
FT-IR spectrum of Alkali-treated A. niger showed
increase of bands than FT-IR spectrum of control A. niger
strain.

145
RESULTS

Fig. (72): FT-IR spectrum for NaOH treated A. niger strain

Table (54): FT-IR spectral characteristics of alkali-treated A.
niger strain
IR peak
Frequency Assignment T%
-1
(Cm )
709.8 Strong broad N-H amines ( RNH2 Or 43.5
R2NH) or medium =C-H out of plane
1041.5 Strong C-O stretch (Carboxylic acids or 43.5
Esters)
1326.9 Strong N-O asymmetric stretch 46
1388.7 Strong S=O sulphate ester 44
1461.9 Strong CH2 and CH3 Alkanes 44.5
1527.5 Strong N=O nitroso or N-O asym. stretch 41
1643.2 Strong C=O stretch (RCONHR) amides 36
2137.0 Medium Alkynes 49
2927.7 Strong broad dimer OH(Carboxylic acids) 48.5
or strong C-H stretch
3417.6 Varies O-H stretch 33.5
XI.C. FT-IR spectrum for Ca-alginate beads:-

146
RESULTS

Fig. (73): FT-IR spectrum for Ca-alginate beads

Table (55): FT-IR spectral characteristics of Ca-alginate beads

IR peak
Frequency Assignment T%
(Cm -1)
Strong broad N-H amines (RNH2 Or 46.5
713.6
R2NH) or medium =C-H out of plane
Strong C-O stretch (Carboxylic acids or 44.5
1037.6
Esters)
1323.1 Strong C-X stretch Alkyl halides 46.5
Medium C-O stretch (RCO-O-H) 44.5
1400.2
(Carboxylic acids)
Strong N=O nitroso Or strong N-O 41.5
1527.5
asymmetric stretch
1662.5 C=C Stretch or C=N 41.5
Strong broad dimer O-H (Carboxylic 45.5
3220.9
acids)
3622.1 Strong O-H free Hydroxyl 43

XI.D. FT-IR spectrum for Ca-alginate-biomass beads

before tannery wastewater treatment:-
147
RESULTS

Fig. (74): FT-IR spectrum for Ca-alginate-biomass beads before

tannery wastewater treatment

Table (56): FT-IR spectral characteristics of Ca-alginate-biomass

beads before tannery wastewater treatment
IR peak
Frequency Assignment T%
-1
(Cm )
Strong broad N-H amines ( RNH2 Or
721.3 44
R2NH) Or medium =C-H out of plane
Strong C-O stretch (Carboxylic acids
1041.5 42
or Esters)
1342.4 Strong C-X stretch alkyl halides 45
Medium C-O stretch (RCO-O-
1407.9 43
H)(Carboxylic acids)
Strong N=O nitroso Or strong N-O
1531.4 38
asymmetric stretch
Strong C=O stretch (RCONHR) 37.5
1651.0
amides
Strong broad dimer OH (Carboxylic
3552.6 38.5
acids)

XI.E. FT-IR spectrum for Ca-alginate-biomass beads

after tannery wastewater treatment:-

148
RESULTS

Fig. (75): FT-IR spectrum for Ca-alginate-biomass beads after

tannery wastewater treatment

Table (57): FT-IR spectral characteristics of Ca-alginate-biomass

beads after tannery wastewater treatment
IR peak
Frequency Assignment T%
(Cm -1)
Strong C-O stretch (Carboxylic acids or
1130.2 34
Esters)
1326.9 Strong C-X stretch alkyl halides 48
Medium C-O stretch (RCO-O-
1404.1 42
H)(Carboxylic acids)
1554.5 Strong N=O nitroso 40
1639.4 Strong C=O stretch (RCONHR) amides 26
Strong broad dimer OH (Carboxylic
3375.8 10
acids)

XII. Schematic Diagram Of Treatment Using

Immobilization Technology
149
RESULTS

Many conventional processes were carried out to treat

wastewater from tanneries such as biological process,
oxidation process and chemical process. Biological
treatment of wastewater is evaluated as a good treatment
method for industrial effluents. The processes used most
frequently for biological treatment of tannery wastewater
are the Activated Sludge Process (ASP), the Up-flow
Anaerobic Sludge Blanket (UASB) process and A
Common Effluent Treatment Plant (CETP) based on
activated sludge process. Furthermore, there is growing
interest in the development of biological reactor
configurations (suspended biomass, fixed biomass, aerobic
granular biomass systems, membrane bioreactors, etc.) to
optimize the time consuming step in the treatment line to
maximize efficiency and design specific biological systems
for each target industrial wastewater. A study is conducted
to develop simple design criteria to be used as a part of any
common biological systems for wastewater treatment as it
realize part of treatment as shown in Fig. (76). The
incoming wastewater, called influent, passes through
settling tank or sedimentation tank allowing heavier solids
to settle to the bottom of the tank. The partially treated
wastewater from the settling tank then flows to the aerated
tank where wastewater and immobilized media are mixed.

150
RESULTS

The aerated wastewater then flows as effluent to the other

part in the treatment system or it can be re-circulated back
to the aerated tank. More studies on how system
characteristics enhance efficiencies, and eventually, the
overall treatment cost, must be performed to be cost-
competitive one.

Fig. (76): Schematic diagram shows operation for tannery

wastewater treatment using immobilization technique.

151
DISCUSSION

DISCUSSION
Nowadays, it is common to observe environments
with organic and inorganic pollutants, defined as co-
contamination. Most industrial and urban effluents release
both pollutant types, leading to complex environmental
problems (Romero et al., 2006).
Wastewater which are rich in organic matter, are
habitat for many groups of microorganisms, such as
viruses, bacteria, fungi, algae, protozoa and worms.
Microorganisms play a significant role in bioremediation of
heavy metal contaminated soils and water ecosystems (Yan
& Viraraghavan, 2000 and Massaccesi et al., 2002).
Several studies have shown that wastewater contain
different types of fungi, which can be transferred and
distributed from these areas (Parameswari et al., 2010).
Applications of fungal biomass to treatment domestic and
industrial wastewater are very common. Fungi were known
to tolerate and detoxify metals by several mechanisms
including valence transformation, extra- and intra-cellular
precipitation and metabolic active uptake (Gadd & White,
1993 and Zafar et al., 2007). Treatment process means to
remove heavy metals from industrial wastewater and or to
recover economically valuable metals (Arica et al., 2001).

152
DISCUSSION

The current study is mainly concerned with the

treatment of wastewater especially tannery wastewater by
using fungi. The heavy metals contents of tannery
wastewater sample are examined. The concentrations of
heavy metals were found to be above the maximum
permissible limits according to WHO standard for fresh
water (Olajire & Imeokpara, 2000).
Isolation of fungi from tannery wastewater was done.
The most frequently encountered isolates were from genus
Aspergillus followed by Penicillium followed by Rhizopus
and Fusarium. While genus Alternaria showed the
minimum percentages. The occurrence of various fungi
such as Aspergillus, Rhizopus, Penicillium, Fusarium,
Chaetomium, Geomyces, Paecilomyces species in water
polluted with heavy metals (Cu, Cd, Pb, As, and Zn) has
also been reported by other workers from different parts of
the world (Zafar et al., 2007, Ezzouhri et al., 2009 and
Al-Sohaibani, 2011).
Basic statistical analysis using Normality Test by
Minitab 15 English software showed that at confidence
level 95 %, the significance was more than 5 %, indicating
that the fungal isolates were withdrawn from community
that follows normal distribution.

153
DISCUSSION

The isolated fungal genera were then subjected to

concentration of 5 ppm from the studied metal ions in
mixture. Four fungal strains belong to Aspergillus;
Penicillium and Rhizopus genera were tolerant (exhibited
growth). While Fusarium and Alternaria genera couldn't
grow at this concentration despite their site of isolation.
These results were almost in agreement with Jones &
Hutchinson (1988); Howe et al. (1997); Rudawska &
Leski (1998) and Baldrian & Gabriel (2002). They
demonstrated that the resistance against individual metals
was much more dependent on the isolate than on the sites
of its isolation. Almost similar explanation was showed by
Verma et al. (2001); Say et al. (2003); Hildebrandt et al.
(2007) and Zafar et al. (2007).
In contrast, Gadd (1993); Gadd & Sayer (2000);
Massaccesi et al. (2002) and Malik (2004) demonstrated
that, metal ions whether an essential or non-essential will
tend to show toxicity at certain levels. Their toxicity may
be presented differently, depending on the isolate and its
site of isolation. Some isolates were tolerant, while others
reacted negatively even at low metal ion concentrations.
This could be explained by the heterogeneity of pollution in
the locality from which the tested isolates originated. So,
microbial diversity appears to be closely linked to the

154
DISCUSSION

degree of heavy metal pollution. This is due to the

extinction of species sensitive to the stress imposed, and
enhanced growth of other resistant species. It must also be
taken into account that the contamination at the polluted
sites is usually not caused by a single metal and that the
selection is probably drived either by the most toxic
element or by more different metals acting synergistically.
So, micro-biota isolated from co-contaminated
environments could exhibit resistance to more than one ion
and consequently, co-tolerance may be a common natural
response.
Results showed that the type of culture media and
incubated temperatures were significantly affected the
mycelial growth of fungal strain. Being PDA medium and
incubation at 30C the best condition for mycelial growth.
Statistical analysis using One-way ANOVA showed that at
confidence level 95%, the significant at different media
types and at different incubated temperatures are less than 5
%, so they are significantly different (at least two of fungal
isolates are different in radial growth with difference of
media type's or temperatures value). These results were in
agreement with Raper & Fennell (1965); Zhae & Simon
(2006) and Sharma & Pandey (2010).

155
DISCUSSION

Tolerance test revealed heterogeneity in the heavy

metal tolerance of our isolates. Results showed that the
hexavalent chromium ions were less toxic in comparison
with the others metals studied followed by lead, copper and
cadmium, respectively. Results also showed that the colony
diameters of A. niger, P. chrysogenum, A. tamarii and R.
stolonifer isolates were decreased with the increase of the
studied ions concentrations from 10 to 100 ppm. Almost
similar results were reported by Wang et al., (1989); Lilly
et al. (1992); Falih (1998); Bader (1999); Baldrian &
Gabriel (2002); Levinskaite (2002); Sanyal et al. (2005);
Srivastava & Thakur (2006) and Ezzouhri et al. (2009).
Srivastava & Thakur (2006) showed that the
detoxification of chromium may be mediated by an
enzymatic antioxidant system such as peroxidase, catalase
and ascorbate peroxide.
Mergeay et al. (2003) and Sun & Shao (2007)
demonstrated that both intracellular bioaccumulation and
extracellular biosorption may contributed to the high
resistance to lead. Also, the resistance to lead ions may be
mediated by a P-type ATP-ase, which can transport lead
out of the cell.
The growth of A. niger isolates on agar media
containing high lead concentrations was accompanied with

156
DISCUSSION

the removal of white coloration of PDA medium around the

colony. This is probably due to a period of adaptation
where cells of the Aspergillus isolate synthesized some
enzymes essential for the uptake of lead, as reported by
Pelmony (1993).
Copper is a co-factor in numerous enzymatic
processes and represents the third most abundant transition
metal found in living organisms (Brandolini et al., 2002).
The copper adaptation may be due to an active process
involving copper metallothionein synthesis (Kermasha et
al., 1993 and Romero et al., 2006).
The blue color of the isolate's mycelia on agar media
amended with copper may be due to binding of copper ions
to the fungal cell wall. Almost similar observation was also
made by Anand et al., (2006).
Gadd (2010) attributed the morphological changes
during development to; microbes interact with metals and
minerals, altering their physical and chemical state, affect
microbial growth, activity and survival.
Perfus-Barbeoch et al. (2002) have reported that
cadmium ions exerted severe inhibition of physiological
processes in micro-organisms, such as growth and
photosynthesis at concentration less than 2 ppm, and often
in the ppb range.

157
DISCUSSION

Experiments indicated that tolerance was increased

in case of mixed ions solution in comparison to single ions
solution using the same isolates. Interestingly, metals
combinations were less toxic than single ones, and co
tolerance development indicated that the cellular
mechanisms that conferred resistance were non-specific.
This also may be due to the presence of co-metal ions as
well as to the differences in the heavy metal ions
concentrations. These explanations were in agreement with
Gadd & Sayer (2000); Amor et al. (2001); Muhammad
et al. (2009); Chatterjee et al. (2010) and Tsekova et al.
(2010).
Statistical analysis using One-way ANOVA showed
that at confidence level 95%, the Significant at different
Cr6+, Pb2+, Cu2+, Cd2+ and mixture of these ions are less
than 5 %, so they are significantly different (at least the
growth of two fungal isolates are different with difference
of metal ions concentrations).
The differences in resistance levels were probably
due to the presence of different types of tolerance processes
or potential variation of resistance mechanisms exhibited
by different isolates such as detoxification mechanism of
each fungal species, physiological mechanisms, genetic
adaptation, the activated biochemical processes and

158
DISCUSSION

metabolism uptake mechanisms. It could be also explained

by low toxicity of such metal ions (Roane & Pepper,
2000; Baldrian & Gabriel, 2002 and Nazareth &
Marbaniang, 2008).
Results showed that isolates of the same genus could
present a marked difference in the levels of metal
resistance. Major differences in the studied metal ions
tolerance have been found among our isolates.
It was also found that the period needed for growth
increased with the increase of metal ions concentrations. At
lower metal ions concentrations, the tested fungal isolates
were very resistant and exhibited growth similar or weakly
after the normal incubation period. Higher metal ions
concentration caused a reduction in growth and increased
the incubation period needed for growth compared to the
control. It mainly depends on the metal used and its
concentration. Similarly, Jones & Hutchinson (1988)
reported an increase in lag time among different strains
cultivated on metal ions amended media. In contrast,
Darlington & Rauser (1988) did not find any dependence
of lag time on metal ions concentrations.
The isolated fungal species showed different
resistances and so showed different minimum inhibitory
concentration (MIC values) toward the studied metal ions.

159
DISCUSSION

The highest resistance and MIC values were observed for

mixture of studied ions, Cr6+ and Pb2+, respectively. While
the lowest resistance and MIC values were for Cu2+ and
Cd2+, respectively. Also, statistical analysis using One-way
ANOVA showed that at confidence level 95%, the
significance of MIC at different Cr6+, Pb2+, Cu2+, Cd2+ and
mixture of these ions are less than 5 %, so they are
significantly different (at least two of fungal isolates are
different with difference of any metal MIC value). While,
the significance of MIC at different fungal isolates are
more than 5 %, so they are non-significantly different (at
least two of fungal isolates are equal with metal MIC
value).
Our results emphasized the detoxification abilities of
A. niger and P. chrysogenum isolates and the adaptation to
all metals tested, which may make them promising
candidates for further investigations to heavy metal
resistance.
The total growth and the growth rate of these fungal
strains in the presence of heavy metal relative to the control
were studied. The growth pattern appears to suggest some
tolerance development or adaptation of these fungi to the
presence of the studied heavy metals. The reduction in the
growth rate is a typical response of fungi to toxicants. Our

160
DISCUSSION

results were comparable with those reported by Gadd

(1993); Badar et al. (2000); Verma et al. (2001); Bai &
Abraham (2003); Malik (2004); Zouboulis et al. (2004);
Yoshida et al. (2006) and Zafar et al. (2007).
Mycelial growth occurs in form of pellets in liquid
medium with shaking. Tucker & Thomas (1992)
explained that; it is usually results from aggregation of
spores before germination, entrapment of spores by germ
tubes or less commonly and aggregation of young mycelia.
So, it depends on the physico-chemical and physiological
characteristics of spores and hyphae. The biomass dry
weight and pellet size were used to follow growth
characteristics of filamentous fungi as it reported by Kelly
et al. (2006). Advantages of pellet cultivations are the
significant, decrease of the viscosity and the easier
separation of the biomass from the cultivation broth.
The obtained results affirmed that A. niger strain had
the most tolerance effect between the rest of isolated
isolate. So, some investigations were done on that isolate,
regarding their ability to remove metals from contaminated
environments.
A. niger, was trained with the studied metal ions. The
growth rates of control and trained isolate were initially
inhibited but the control isolate was more sensitive.

161
DISCUSSION

Furthermore, trained mycelia removed metal ions more

than that of the control one. Doelman et al. (1994) and
Vadkertiova & Slavikova (2006) explained that by; long-
time exposure to heavy metals can produce considerable
modification of microbial populations, induces
morphological and physiological changes in the microbial
communities.
Kuyucak & Volesky (1998) also explained that by,
shaking incubation cause an increase in growth rate and
amount of metal biosorbed because it make good
distribution for the absorbent in the metal ion solution and
hence, increased the chances for metal ion to be adsorbed
on the cell wall surface.
Roane et al. (2001) and Romero et al. (2006)
reported that; training of fungal strains is a valuable
environmental technology for the detoxification of co-
contaminated habitats, by bioaugmentation strategy. These
data suggesting that such tolerance and co-tolerance could
be acquired in natural environments. So, a simple
bioremediation strategy could enhance the decontamination
of polluted areas, as the adapted organisms could be
present.
The removal efficiency of heavy metals by trained A.
niger isolate was found to be in most cases fitted to linearly

162
DISCUSSION

transformed Langmuirs and Freundlich's adsorption

isotherm models than control isolate. So, this conspicuous
potential was considered to select A. niger trained strain for
further assays.
The adsorption phenomenon was called biosorption
as fungal species were used for the adsorption. The effect
of environmental conditions on the uptake process
indicated that; pH value of metal solution is the most
important factor in the biosorption process.
The medium pH affected the solution chemistry of
the metals (the solubility of metal ions and the competition
of metallic ions), the activity and the ionization state of the
functional groups (i.e. carboxylic, phosphate, and amino
groups) on the fungal cell wall. This explanation was in
agreement with Friis & Myers-Keith (1998); Kapoor et
al. (1999); Gadd (2000) and Ting & Sun (2000).
Results showed that metal uptake was low initially at
pH 1.0 0.2 before rising to a maximum value at pH 5.0
0.2 and then suddenly drops at pH 7.0 0.2. At optimum
pH (5.0 0.2), there is another advantage being optimal pH
value for growth and sporulation in a majority of fungi and
thus enhance the bioaccumulation tendency which is in
agreement with Awofolu et al. (2006).

163
DISCUSSION

At low pH (less than 4.0) heavy metal removal was

inhibited, possibly as a result of the increase of H+
concentration which competes with positively charged
metal ions for the active sites on fungal cell wall. At high
pH (4.0 7.0) of metal ion solution the H+ concentration
decreased and the negative charge density on the cell
surface increases, hence the fixation of ions on fungal cell
wall increased. This explanation was in agreement with
Cotton & Wilkinson (1962); Andres et al. (1993);
Kapoor & Viraraghavan (1997) and Saglam et al.
(1999).
After pH 7.5 the efficiency of the metal removal
process increases drastically due to the formation of metal
hydroxides with their respective metal ions. This is mostly
due to the metal precipitation as hydroxides which depend
on the pH and ion concentration, but not due to the
biosorption as reported by Zouboulis et al. (2003).
These explanations indicated that the biosorption of
metal ions on the fungal mycelium could take place in two
mechanisms. Metal ions can be attracted by the negative
charge of the cell wall component. This electrostatic
binding is non-specific and it is only influenced by the
valence of the ion. The second mechanism is specific
binding of metal to the reactive sites on the cell wall. This

164
DISCUSSION

explanation was in agreement with Pirszel et al. (1995);

He & Tebo (1998); Kefala et al. (1999) and Naja et al.
(2005).
Our results showed that the changes in residual metal
concentration with time at optimum pH value indicated that
the contact time is also an important factor in the
biosorption process. Biosorption was reached equilibrium
in 2 h at pH 5.0 0.2. The plot shows that kinetics of
biosorption of Cr6+ ions consisted of two phases; the first an
initial rapid phase where biosorption was fast due to fixed
number of active sorption sites. The second phase was
slower as the competition for decreasing availability of
active sites intensifies by the metal ions remaining in
solution (whose contribution to the total metal biosorption
was relatively small). This means that the higher sorption
rate at the initial period (2h) may be due to an increased
number of vacant sites available at the initial stage, which
results in an increased concentration gradient between
sorbate in the solution and sorbate in the adsorbent surface.
As time increases, this concentration gradient was reduced
due the sorption of metal ions onto vacant sites, leading to a
decrease in sorption rate at later stages. So, two hours were
sufficient to establish equilibrium between the solid and
liquid phases because a metal in sorption reaction occurs in

165
DISCUSSION

milliseconds or minutes. These explanations were in agree

with Low et al. (1993) and Awofolu et al. (2006),
In agreement to our results; Aksu et al. (1992);
Kefala et al. (1999) and Ting & Sun (2000) indicated that,
the temperature seemed not to influence the biosorption
performances in the range of 20-50C. At low temperature
the rate of biosorption decreased. The increase in
biosorption of metal ions with the increase of temperature
indicated that the metal ions removed by fungal cells are
mainly adsorption phenomena because the temperature of
the adsorption medium could be important for energy
dependent mechanisms in metal biosorption by
microorganisms. Energy independent mechanisms were
less likely to be affected by temperature since the processes
responsible for biosorption were largely physico-chemical
in nature.
Our results indicated that, there were increases in
biosorption rate of Cr6+ with the increase of stirring rate
from 0 to 500 rpm. Stirring made good distribution for the
absorbent in the metal ion solution and hence, increased the
chances for metal ion to be adsorbed on the cell wall
surface. A moderate stirring were selected, since high
stirring rates over long times may resulted in the release of
metal ions adsorbed on the cell wall and degradation of

166
DISCUSSION

fungal cells. These results go parallel with that obtained by

Kuyucak & Volesky (1998).
Our results also indicated that the choice of biomass
weight was dependent on both uptake percent and specific
uptake. The increase in fungal biomass weight resulted in
increase in Cr6+ uptake percent while decrease the amount
removed in mg/g dry weight which reflects the specific
uptake. Almost similar results were showed by Gadd &
Rehm (1988) and Tsekova et al. (2010). They suggested
that as the biosorbent mass increased; the increase in the
uptake percent may be due to increases of the number of
available binding sites or surface area for the heavy metal
ions while the decrease in specific uptake may be due to
interference between the binding sites.
Fourest & Roux (1992) invalidated this hypothesis
attributing the responsibility of the specific uptake decrease
to metal concentration shortage in solution. Hence this
factor needed to be taken into consideration in any
application of microbial biomass as biosorbent.
Our results also showed that the increase in Cr6+ ions
concentration from 20 to 100 ppm resulted in increase in
specific uptake and decrease in the uptake percent. The
increase in specific uptake could be explained by with a
fixed adsorbent weight the competition of metal ions on the

167
DISCUSSION

biosorbent surface was increased and so, increased in metal

ions accumulated until reach saturation of active sites. The
decrease in the uptake percent could be explained by; at
lower initial metal ion concentrations, sufficient adsorption
sites are available for adsorption. However, at higher
concentrations the numbers of ions are relatively higher
compared to availability of the adsorption sites. Almost
similar explanation was also reported by Ilhan et al.
(2004).
A. niger strain showed highest accumulation capacity
for Cr6+ ions rather than other ions. These differences in
accumulation capacities could be attributed to variation in
the chemical properties of metal ions. Almost similar
explanations were taken by Gadd (1990).
Obtained results also showed that biosorption of Cr6+
by pretreated A. niger either increased or decreased
depending on the pretreatment method. Maximum uptake
was found to have the following order according to the
pretreatment methods; pretreated with 0.1N NaOH >
autoclaving > 0.1% CaCl2 > 0.1 % Glutraldehyde > without
treatment (control strain) > Boiling. An increase in
biosorption as a result of pretreatment could be due to an
exposure of active metal binding sites embedded in the cell
wall or chemical modifications of the cell wall components.

168
DISCUSSION

Huang and Huang (1996) stated that the increase

in metal biosorption after treatment of the biomass could be
due to the removal of surface impurities and to the
exposure of available binding sites for metal biosorption.
Our results showed that pretreatment with sodium
hydroxide resulted in an improvement in biosorption. In
agreement, Fourest & Roux (1992); Ashkenazy et al.
(1997) and Yan & Viraraghavan (2000) showed that the
biosorption was more efficient after sodium hydroxide
treatment due to unmasking of some cellular groups (such
as protein and protein-carbohydrate complexes) which
couldn't participate in the sorption process without
treatment with alkali. The higher affinity might be also
attributed to the chitin and chitosan content of the fungus
cell wall, exposed after NaOH treatment. NaOH appears to
remove amorphous polysaccharides from the cell wall,
generating accessible space within the -glucan-chitin
skeleton and hence permitting metal ions to precipitate on
this surface. Thus it is reasonable to assume that improved
metal ion removal was due to the changes in sorptive
characteristics of the biomass as a result of sodium
hydroxide pretreatment.
In contrast to our results, Kapoor & Viraraghavan
(1998) reported that sodium hydroxide pretreated decrease

169
DISCUSSION

biosorption in comparison with live cells. As a result of

sodium hydroxide treatment, the number of protein amino
groups that can be engaged in metallic ion binding
markedly decreased.
Results also showed that the treatment with calcium
chloride and glutraldehyde increase the biosorption
capacity than without any treatment. This may be attributed
to the presence of multi-functional groups. These results
were in agreement with that reported by Yan &
Viraraghavan (2000). In contrast to our study, Jianlong
(2002) demonstrated that glutraldehyde pretreated biomass
reduced biosorption.
In parallel to our results, Kuyucak & Volesky
(1998) showed that pretreating by boiling caused a
significant decrease in the biosorption capacity of microbial
biomass due to the extraction or denaturation of some cell
components (such as oligo- & poly-saccharides, proteins
and low molecular weight substances) which are
concerning with metal binding as well as loss in the
biomass dry weight. The reduction of biosorption capacity
may be attributed to the loss of some active groups.
Results also showed that treatment by autoclaving
was significantly increased the biosorption of Cr6+. In
agreement to our results; Galun et al. (1987); Churchill et

170
DISCUSSION

al. (1995) and Tuzun et al. (2005) reported that autoclave

pretreatment increased the biosorption capacity of
microbial biomass due to the exposure of latent binding
sites after pretreatment. Heat treatment could also erode
microbial cell surface integrity causing the walls to become
leaky with a marked increase in the passive diffusion of
metal ions to the interior part of the cell wall.
In contrast, Kapoor & Viraraghavan (1998) and
Yan & Viraraghavan (2000) reported that biomass
pretreated with autoclave reduced the biosorption of heavy
metals.
Statistical analysis showed that at confidence level
95%, the Significant is less than 5 %, so the estimated
treatment values are statistically significantly different (at
least two types of treatment are different).
From our results we can decide that this conflict is
normal since the cell wall composition can be characteristic
of the fungal species.
Tannery wastewater is one of the most important
sources of environment pollutants as it highly complex and
characterized by high contents of organic, inorganic
pollutants such as nitrogenous compounds, chromium,
sulfides, suspended solids and dissolved solids. The
inhibition of biodegradation which represented by low

171
DISCUSSION

BOD value to less than 1/3 of COD value due to the

presence of high metal ions concentrations especially
chromium ions and high concentrations of salts. This is also
confirmed by many other studies Ro & Ganter (1998) and
Song et al. (2000).
Based on above results; it was decided to conduct
batch biosorption experiments using pretreated A. niger
biomass with 0.1 N sodium hydroxide for treating tannery
wastewater. Results showed that this treated biomass can
biosorb all metal ions present in tannery wastewater in
different uptake percent depends on metal ions
concentration and metal type. For high metal
concentrations, results showed low uptake percent and high
accumulation capacity (Q) and vice versa. The removal
percentages order at equilibrium was: Mn > Cu > Ni > Pb >
Fe > Cd > Cr. Also, the treated biomass showed higher
biosorption for organic loads presented in the effluent.
These results were almost similar to those reported by
Durai & Rajasimman (2011).
In industrial technical operation, immobilized
microbial cell systems could provide additional advantage
over freely suspended cells. These include; regeneration
and reuse of the biomass, easier solid liquid separation and

172
DISCUSSION

minimal clogging in continuous-flow systems (Arica et al.,

1993).
Results showed that the treatment of tannery
wastewater was affected by immobilization of alkali-treated
A. niger in comparison to the removal efficiency by free
treated biomass. Alginate carboxyl groups are known to
play an important role in metal binding (Kuyucak &
Volesky, 1998). However, there were considerable
differences in total biosorption capability. Three ions
{Ca2+, Al3+ and Fe3+} were examined for alginate
polymerization in tannery wastewater treatment. Results
showed that Ca-alginate-biomass beads had high
biosorption capacity for chromium and iron. Also, it
decreased the color absorbance of tannery wastewater
sample than Al-alginate-biomass and Fe-alginate-biomass
beads, respectively. This could be due to the difference in
valence type or the similar properties of the two elements
(Al and Fe) either in valence or in chemical behavior. Also,
the calcium ions increase the biosorption capacity of
alginate-biomass as in the pretreatment study previously
mentioned.
Tobin et al. (1994) reported that the use of entrapped
fungus in Ca-alginate beads may be advantageous over the
ion-exchange resins when Ca2+, Mg2+ and K+ ions were

173
DISCUSSION

present in adsorption medium or industrial wastewater at

high concentrations.
Ca-alginate-biomass showed highest total
biosorption capacity for all heavy metal ions and also for
organic loads presented in the effluent at higher initial
concentrations followed by Ca-alginate alone followed by
free treated biomass, respectively. The displayed high
removal potential for metal ions in comparison to other
heavy metal ions from tannery wastewater may be due to
their low initial concentration or its affinity to be biosorbed
by the biosorbent. This could be also explained by the
increase of active sites available for metal uptake. These
results and explanations were in agreement with
Muhammad et al. (2009) and Chatterjee et al. (2010).
Tsekova et al. (2010) indicated that the time needed
for the sorption equilibrium reached much faster in case of
industrial wastewater sample in comparison to single ions
solution using same biosorbent. These results are
important, as equilibrium time is one of the important
parameters for selecting a wastewater treatment system.
This may be due to the presence of co-metal ions in the
industrial effluents as well as to the differences in the heavy
metal ions concentrations.

174
DISCUSSION

Recycling or reuses of the immobilized system were

one of the most advantages over free biomass. Our results
showed that the reuses of control alginate and entrapped
biomass beads were recycled and showed decrease to some
extent in the absorption capacity. This could be explained
by the presence of free active adsorption sites available for
metal ions uptake on both alginate gel and entrapped
biomass. Further addition of raw wastewater resulted in
competition of metal cations for complexation with the
active biosorption binding sites leading to decrease in the
uptake process. This decrease may be also attributed to the
negative effect of both metals on each other for removal or
the difference in ionic radii of metal ions. These results and
observations were paralleled to that obtained by Zhou &
Kiff (1991); Mclean et al. (1994); Sag & Kutsal (1996)
and Sag et al. (2000).
SEM examinations for the studied biosorbent in this
work were investigated. Immobilized biomass (alkali-
treated and autoclaved) beads showed the same fungal
mycelial protrusion, indicated that there are unifier
distribution of the fungal mycelia, which is an important
extension for the proper biosorption of heavy metal ions on
the entire surface area of the fungal entrapped beads. This

175
DISCUSSION

explanation was almost similar to Tobin et al. (1994) and

Abdel-Hameed (2006).
The SEM micrograph and EDAX analysis of beads
after tannery wastewater treatment showed precipitation of
metal ions on the surface of the beads. On the other hand
the effect of biosorption on the immobilized biomass beads
appeared as white shades around the mycelial protrusion,
which indicated the precipitation of the metal ion on the
bead surface and around the mycelial protrusion distributed
around surface. Almost similar observation was obtained
by Arica et al. (2001) and Chatterjee et al. (2010).
FT-IR investigations related to A. niger showed
change of peak frequency bands when comparing to the
FT-IR spectra of alkali-treated A. niger. This change may
be due to change in chemical composition. FT-IR
investigations related to the fungal biosorption showed
change of peak frequency bands. It was reasonable to
assume that these peak values suggested the metal
chelating. The structure of the metal bound to carboxyl
band is likely to take place. Also, FT-IR spectrum showed
difference in peaks and little change in the fingerprint
region (The typical infra-red spectrum). This shifting of
functional group and the change in this region suggested
metal bonding during the adsorption. The results of FT-IR

176
DISCUSSION

analysis suggested that the binding sites were most likely

carboxyl groups where, carboxylic acid dimers display very
broad, intense OH stretching absorption. Almost similar
explanation was obtained by Silverstein & Webster
(1998) and Tsekova et al. (2010).
The cost of the adsorbent is an important parameter
for consideration in the choice of materials to be used for
metal removal from aqueous systems. A sorbent can be
assumed or referred to as low cost if minimal processing
of the material is required, is quite abundant in nature, or is
a byproduct or waste material from industrial processes. A.
niger fits into one of these conditions because it could
readily be obtained as industrial waste resulting from citric
acid production which makes its utilization both cost
effective and environmental friendly. In addition, the
proposed modification protocols could be said to be
economically viable and worthy given the significant
difference in sorption efficiency as the modifying agent; a
common, cheap, simple, fast and efficient.
The modified biomass of A. niger and the simplified
protocol demonstrate their potential industrial applications
for the removal of metals from contaminated water
systems.

177
CONCLUSION

CONCLUSION
Current interest in the state of environment has
resulted in increased research to evaluate the global impacts
of pollution on the biosphere. In order to fight damage to
the environment by organic and inorganic pollutants, there
are needs to develop treatment technologies.
Microorganisms have been shown to take up heavy metals
as well as organic loads from aqueous solutions. Our
findings revealed that the isolated fungal strains had a
potential to tolerate and remove chromium, lead, copper
and cadmium at a laboratory scale. The biosorption process
was mainly influenced by environmental conditions (such
as pH, contact time, temperature, stirring rate, biomass
weight and initial metal ion concentration). It also affected
by biomass pretreatment. The ability of A. niger biomass to
bind and remove heavy metals as well as organic loads
from real wastewater was investigated. To overcome the
separation problems of using freely suspended biomass
form, as well as, mass loss after regeneration of the
biosorbent, the biomass was immobilized in the polymer
matrixes Ca-alginate gels. Biosorption studies of Ca-
alginate-biomass beads have been found to be effective in
removing toxic constituents from wastewater. Also,
recycling of Ca-alginate and Ca-alginate-biomass beads
were studied for three subsequent cycles. So, it can be
concluded that the immobilization of fungal biomass could
enhance the biosorption of color, organic contents and
metal-polluted industrial wastewater. The obtained data are
useful in the design of treatment of wastewater containing
heavy metals.

178
RECOMMENDATION

RECOMMENDATION
This study examined the extent of pollution created
by tanneries and the different fungal processes available for
the treatment and disposal of tannery wastewater. Further
investigations are needed to optimize the conditions for
metal removal from multimetal aqueous solutions and
diluted wastewaters for large scale operation. At present, no
single technology for wastewater treatment (chemical
remediation, phytoremediation or microbial remediation) is
without some form of merits and demerits. Due to the
enormous benefits and drawbacks of each of the existing
remediation technologies/processes, there is a need for the
implementation of an integrated remediation
technology/multiple technology which can have great
potential. The application of combined process of physical
or chemical with biological process to treat tannery
wastewater would give satisfactory results compared to
individual treatment processes. This can be attained
through further wastewater remediation research, which
will help to enhance decisions that are science-based. Also,
to achieve a safe and economical remediation option, there
is need to review and assess the current costs and market
share of the established remediation processes. The
application of this may offer enormous environmental
public health and cost benefits.

179
SUMMARY

SUMMARY
The continuous technological development in all
fields of life led to a continuous and great increase in
pollution of environment. Rules organized for
environmental affairs have been set to limit/restrict the
effect of pollution on environment and man health.
Recently, microorganisms were appeared as alternative
promising method for treatment and removal of toxic
constituents in liquid waste especially heavy metals.
This work investigated the ability of fungal species
in treatment of wastewater. Tannery wastewater was
selected as example of wastewater containing heavy metals.
The study of treatment was mainly concerned with heavy
metal removal mainly Cr6+, Pb2+, Cu2+ and Cd2+. The fungal
isolates were isolated from chromium stage - tannery
wastewater (ELMONTAZA TANNERY), Ain El Sira, Old
Cairo, Egypt. Fifteen fungal colonies represent five genera
(Aspergillus (40 %), Penicillium (26.7 %), Rhizopus (13.3
%), Fusarium (13.3 %) and Alternaria (6.7%). These
colonies were screened firstly on PDA media containing 5
ppm from the studied metal ions in mixture. The tolerated
fungal isolates were purified and identified as Aspergillus
tamari, Aspergillus niger, Penicillium chrysogenum and
Rhizopus stolonifer.

180
SUMMARY

The physiological studies of these fungal isolates

were studied at different medial types (PDA was the best
media's type) and different temperatures values (30C was
the best temperature's value).
The ability of studied fungal species to grow in the
presence of different concentrations of the studied metal
ions was investigated on solid media to determine the most
tolerant fungal isolates and the maximum tolerance for each
fungal strain toward metal ions expressed as MIC value. It
was observed that the sensitivity of fungal isolates was
increased with increase of the studied metal ions
concentrations and the mixture of these ions decrease this
sensitivity comparable with the same concentration in
single state. The studied fungal isolates could be classified
according to their sensitivity in the following order A. nig.
P. chr. > A. tam. > R. sto. Also, the studied metal ions
could be classified according to their toxicity in the
following order Cd2+ > Cu2+ > Pb2+ > Cr6+ > mixture. The
tolerance towards mixture of metal ions with equal ratio at
different concentrations from 40 - 500 ppm showed that; A.
niger had the maximum tolerance toward metal ions
mixture till 400 ppm (100 ppm from each).
Also, the ability of most tolerated fungal species
(A. niger and P.chr.) to grow in liquid media was studied to

181
SUMMARY

determine the growth rate and best condition used for

fungal cultivation (static or shaking incubation). It was
found that growth rate of fungal isolates were affected with
metal ions concentration and the shaked incubation was the
best incubation for maximum growth rate. Training of A.
niger strain on the same metal ions before cultivation was
also affect the growth rate and the removal of this metal
ions than control strain. Results of training realized
Langmuir's and Freundlich's equation and this is an
indication that these biomasses were used as a good
biosorbent.
The biosorption capacity of the studied metal ions
by A. niger was investigated. The effect of environmental
conditions on the uptake of Cr6+ were studied to examine
the affinity and the capacity of A. niger strain and to
determine the optimum conditions for maximum uptake.
Experiments were done at different pH values showed that
the maximum uptake of the pH occurred at pH 5.0 0.2
and the pH of the solution was the most important factor
for the uptake of metal ions. Results showed that the
biosorption capacity differed from one metal ion to another.
The studied metal ions could be classified according to the
affinity of the A. niger toward the studied metal ions in the
following order Cr6+ > Pb2+ > Cu2+ > Cd2+. Results also

182
SUMMARY

indicated that temperature has a limited effect on the uptake

of Cr6+ ions at temperature range between 25C and 50C.
The rate of uptake increased with the increase in
temperature. Results also indicated that, the uptake of Cr6+
increased with the increase of stirring rates. But the rate of
250 rpm was chosen, since the high stirring rates caused
decay to the fungal cells on the long run. It was found that
with the increase of the biomass weights the uptake percent
from the solution increased but the density and efficiency
of the uptake decreased. It was also found that the best
biomass weights in the solution were 0.1g. Results also
showed that the uptake amount and density increased with
the increase of ion concentration in the solution (until the
fungal biomass reach saturation state) while the uptake
percent from the solution decreased. The capacity for metal
ion depend on; 1) Chemistry of the metal ion which include
(chelate formation, hydrolysis state and co-precipitation),
2) metal ion toxicity.
The effect of physical and chemical pretreatments of
A. niger biomass on biosorption capacity of Cr6+ ions was
studied. The chemical treatments included washing with
sodium hydroxide, glutraldehyde and CaCl2. The physical
treatments included boiling and autoclaving. Results
showed that the maximum uptake had the following order

183
SUMMARY

0.1N NaOH > Autoclaved > 0.1% CaCl2 > 0.1%

glutraldehyde > Control biomass > boiling.
The treatment of tannery wastewater using 0.1N
NaOH treated FC was investigated. Also, the effect of
immobilization on the treatment process was investigated.
Results show that the treatment by Ca-alginate-biomass had
the maximum removal for all toxic constituents then by Ca-
alginate then by alkali-treated FC. Results indicated that
immobilization of treated biomass with alginate were
efficient in removal by polymerization of alginate by Ca2+
ions than Al3+ and Fe3+ ions, respectively. Results showed
that the amount and capacity of uptake increased with the
increase of metal ion concentrations.
The reuse of control alginate beads and Ca alginate-
biomass beads was investigated for three cycles. Results
indicated that the uptake percent and efficiency decreased
at 2nd and 3rd cycles than the first cycle. This indicated the
possibility for reusing immobilizing systems to several
cycles to a certain levels.
SEM studies showed that there are semi-transparent
areas above the biomass either free or immobilized beads.
It shows also that control alginate bead were smooth and
clear, while the biomass immobilized beads showed
mycelia embedded in the beads. Both life and dead

184
SUMMARY

immobilized biomass beads showed unifier distributed

mycelial protrusion on the surface of the beads. EDAX
analysis showed that the immobilized biomass had higher
uptake for metal ions especially for chromium ions and for
chloride salts than control alginate and treated FC
respectively. EDAX analysis also showed that
immobilization of dead biomass had less uptake percent of
metal ions and for salts than that of alkali-immobilized
biomass. FT-IR studies were also done for A. niger and
NaOH treated A. niger It shows increase in the beaks by
treatment than control biomass. It was also done for Ca
alginate and Ca alginate-biomass. It showed also increase
in beaks and in free active sites. FT-IR was also done
before and after tannery WW treatment to show decrease of
the beak absorbance indicating the site of biosorption.
Finally, any biological treatment system can be modified
by insertion of treatment unit using immobilized system.
This unit was designated firstly being the sedimentation
tank for the first treatment step where settling of insoluble
salts and large molecules takes place. Then the effluents
were pumped to the alginate biomass immobilized system
tank with mixer to distribute the biomass beads with WW.
Then the effluent was then pumped to the rest of the
biological treatment or recycled to alginate biomass tank.

185
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