SIOKWU, STEPHEN CHUKWUDI
ISOLATION, CHARACTERIZATION AND BIOADSORPTION OF
HEAVY METALLIC ION TOLERANT FUNGI FROM UNIVERSITY OF
NIGERIA SEWAGE STABILIZATION POND
MICROBIOLOGY
BIOLOGICAL SCIENCES
MADUFOR, CYNTHIA
Digitally Signed by: Content manager’s Name
DN : CN = Webmaster’s name
University of Nigeria, Nsukka
C.O=
OU = Innovation Centre
i
ISOLATION, CHARACTERIZATION AND BIOADSORPTION OF
HEAVY METALLIC ION TOLERANT FUNGI FROM UNIVERSITY
OF NIGERIA SEWAGE STABILIZATION POND
BY
SIOKWU, STEPHEN CHUKWUDI
PG/M.Sc/08/49201
A DISSERTATION SUBMITTED IN PARTIAL FULFILMENT OF THE
REQUIREMENTS FOR THE AWARD OF MASTER OF SCIENCE
DEGREE IN MICROBIOLOGY
NOVEMBER, 2011
ii
CERTIFICATION
SIOKWU, STEPHEN CHUKWUDI, a postgraduate student of the Department of
Microbiology, with the registration number PG/M.Sc/08/49201, has satisfactorily completed
the research work for the Award of the Master of Science Degree in Microbiology. The work
embodied in this dissertation is original and has not been submitted in part or full for any
other diploma or degree of this or any other institution.
---------------------------------------
------------------------------------
DR. C. U. ANYANWU
PROF. (MRS.) I. M. EZEONU
Supervisor,
Head,
Department of Microbiology
Department of Microbiology
University of Nigeria, Nsukka.
University of Nigeria, Nsukka.
iii
DEDICATION
This work is dedicated to Almighty God for his guidance and protection throughout my
studies, my late father, Mr. Eugene Siokwu and my mother, Mrs. Tina Siokwu.
iv
ACKNOWLEDGEMENT
I express my heartfelt gratitude to my supervisor, Dr. C. U. Anyanwu for his invaluable
assistance and guidance throughout the course of my research work. I shall remain ever
grateful to him for his care, concern and sincere interest in my welfare. I must mention that
without his timely help in writing and correction, this thesis could not have been submitted in
time.
I am greatly indebted to the Head of Department, Prof. (Mrs.) I. M. Ezeonu and all my
lecturers as well as the administrative and laboratory staff of the Department of
Microbiology, University of Nigeria, Nsukka. I acknowledge the assistance of Miss Ifeoma
Nwajiaku, a friend whose advice and assistance has made this work a success.
I am also thankful to my colleagues for their encouragement and supports in making this
work a success.
Finally, my profound gratitude goes to Mr. Austin Mowah and every member of my family;
Blessing, Ann, Elizabeth, Rita, Benedicta and Benedict for their prayers, moral and financial
assistance. May the good Lord reward them abundantly.
v
TABLE OF CONTENTS
Title page
i
Certification
ii
Dedication
iii
Acknowledgment
iv
Table of contents
v
List of tables
ix
List of figures
x
Abstract
xi
CHAPTER ONE: INTRODUCTION AND LITERATURE REVIEW
1
1.1 Introduction
1
1.1.1 Statement of problem
6
1.1.2 Research objective
7
1.2 Literature review
7
1.2.1 Heavy metals
7
1.2.2 Heavy metal pollution
10
1.2.3 Methods of remediating metal polluted soil
11
1.2.3.1 Physiochemical methods of remediating metal polluted soil
11
1.2.3.2 Biological approaches of remediating metal polluted soils
13
CHAPTER TWO: MATERIALS AND METHODS
24
2.1 Sample collection
24
2.2 Preparation of samples
24
2.3 Isolation of microorganisms
24
2.4 Tolerance to heavy metals
25
vi
2.4.1 Preparation of stock solution
25
2.4.2 Tolerance study
25
2.5 Selection of working isolates
26
2.6 Characterization and identification of the working isolates
26
2.7 Preparation of biomass
26
2.7.1 Live biomass
26
2.7.2 Dead biomass
27
2.8 Preparation of standard curves
27
2.9 Biosorption studies
27
2.9.1 Biosorption study for copper
27
2.9.2 Biosorption study for zinc
28
2.9.3 Biosorption study for manganese
29
2.9.4 Effects of environmental factors on biosorption
30
2.9.4.1 Effect of pH
30
2.9.4.2 Effect of biomass concentration
30
2.9.4.3 Effect of metal ion concentration
31
2.10 Data evaluation
31
2.11 Data analysis
31
CHAPTER THREE: RESULTS
32
3.1 Isolation of microorganisms
32
3.2 Tolerance to heavy metals
32
3.2.1 Tolerance of fungi for copper
32
3.2.2 Tolerance of fungi for zinc
34
3.2.3 Tolerance of fungi for manganese
37
3.3 Selection of working isolate for biosorption
39
vii
3.4 Biosorption studies
43
3.4.1 Biosorption study using live biomass
43
3.4.1.1 Biosorption study for zinc
43
3.4.1.1.1 Effect of metal ion concentration
43
3.4.1.1.2 Effect of biomass
43
3.4.1.1.3 Effect of pH
47
3.4.1.2 Biosorption study for copper
47
3.4.1.2.1 Effect of metal ion concentration
47
3.4.1.2.2 Effect of biomass
47
3.4.1.2.3 Effect of pH
48
3.4.1.3 Biosorption study for manganese
48
3.4.1.3.1 Effect of metal ion concentration
48
3.4.1.3.2 Effect of biomass
52
3.4.1.3.3 Effect of pH
52
3.4.2 Biosorption study using dead biomass
56
3.4.2.1 Biosorption study for zinc
56
3.4.2.1.1 Effect of metal concentration
56
3.4.2.1.2 Effect of biomass
56
3.4.2.1.3 Effect of pH
56
3.4.2.2 Biosorption study for copper
60
3.4.2.2.1 Effect of metal ion concentration
60
3.4.2.2.2 Effect of biomass
60
3.4.2.2.3 Effect of pH
64
3.4.2.3 Biosorption study for manganese
64
3.4.2.3.1 Effect of metal concentration
64
viii
3.4.2.3.2 Effect of biomass
64
3.4.2.3.3 Effect of pH
65
3.4.3 Comparison between live and dead biomass in biosorption
69
CHAPTER FOUR: DISCUSSION
70
CONCLUSION
76
REFERENCES
77
APPENDIX
93
ix
LIST OF TABLES
Tables
Pages
1. Identification and morphological characteristics of the isolates
33
2. The mean effect of concentration on biomass
40
3. The mean effect of metal type on biomass
41
4. The mean effect of type of organism on biomass
42
x
LIST OF FIGURES
Figures
Pages
1. Tolerance of the isolates for CuSO4
35
2. Tolerance of the isolates for ZnSO4
36
3. Tolerance of the isolates for MnSO4
38
4. Biosorption of Zn using live biomass (40 mg) for 30 mins
44
5. Biosorption of Zn using live biomass (60 mg) for 30 mins
45
6. Biosorption of Zn using live biomass (80 mg) for 30 mins
46
7. Biosorption of Cu using live biomass (40 mg) for 30 mins
49
8. Biosorption of Cu using live biomass (60 mg) for 30 mins
50
9. Biosorption of Cu using live biomass (80 mg) for 30 mins
51
10. Biosorption of Mn using live biomass (40 mg) for 30 mins
53
11. Biosorption of Mn using live biomass (60 mg) for 30 mins
54
12. Biosorption of Mn using live biomass (80 mg) for 30 mins
55
13. Biosorption of Zn using dead biomass (40 mg) for 30 mins
57
14. Biosorption of Zn using dead biomass (60 mg) for 30 mins
58
15. Biosorption of Zn using dead biomass (80 mg) for 30 mins
59
16. Biosorption of Cu using dead biomass (40 mg) for 30 mins
61
17. Biosorption of Cu using dead biomass (60 mg) for 30 mins
62
18. Biosorption of Cu using dead biomass (80 mg) for 30 mins
63
19. Biosorption of Mn using dead biomass (40 mg) for 30 mins
66
20. Biosorption of Mn using dead biomass (60 mg) for 30 mins
67
21. Biosorption of Mn using dead biomass (80 mg) for 30 mins
68
xi
ABSTRACT
Conventional methods for removing heavy metals from aqueous solutions have been
uneconomical and are weak processes. An alternative technique is the use of microorganisms
as bioremediating agents. Some fungal species isolated from sewage effluent at the
University of Nigeria, Nsukka sewage treatment plant were studied for their tolerance to
some heavy metals namely, Copper (Cu), Zinc (Zn), and Manganese (Mn). The isolates were
exposed to various concentrations (100, 200, 500, 1000 and 1500 µg/ml) of the different
metals. The degree of tolerance was measured by the biomass yield in each metal
concentration and compared with control containing no heavy metals. Biosorption
characteristics of both live and dead biomass were examined for the metals as a function of
solution pH (2, 4, 6, 7 and 9), contact time of 30 mins, metal ion concentration (10, 20, 50
and 100 µg/ml) and biomass dosage (40, 60 and 80 mg/100 ml). Several species of fungi
which included Aspergillus niger, Aspergillus fumigatus, Penicillum notatum, and
Cladosporium species were isolated, characterized and identified. Results of the tolerance
study, showed decrease in biomass as metal concentration increases from 100 to 1500 µg/ml
for A. niger and Clado. sp while for A. fumigatus increase in biomass was observed as the
metal concentration (Cu) increased from 100 to 200 µg/ml and then decreased with further
increase in metal concentration from 500 to 1500 µg/ml. Also, P. notatum showed increase in
biomass as the metal concentration (Zn) increased from 100 to 1000 µg/ml with a decrease in
biomass as the concentration was further increased to 1500 µg/ml. In relation to the different
metals used, ZnSO4 was most tolerated by the isolates at 100 µg/ml. A. niger was the most
tolerant among all the tested isolates, hence its choice for the biosorption studies. Metal ion
concentration, biomass dosage and solution pH affected biosorption significantly. The
maximum metal biosorption was found to be at 100 µg/ml with a solution pH of 6 and
exposure time of 30 mins using 40 mg of biomass. There was no significant (p < 0.05) effect
xii
of cell viability on biosorption of the metal ions. The ability of A. niger biomass for
biosorption of the metal ions in aqueous solution runs in the order Zn2+> Cu2+> Mn2+ for both
live and dead biomass. This study indicates that the fungus A. niger is a potential candidate
for further investigation in bioremediation.
xiii
CHAPTER ONE
INTRODUCTION/ LITERATURE REVIEW
1.1 Introduction
Rapid industrial development has led to an increased discharge of industrial
effluents, which may contain heavy metals in concentrations well beyond the permissible
limits, into the environment (Ahuja et al., 2001). The pollutants of serious concern
include lead, chromium, mercury, uranium, selenium, zinc, arsenic, manganese,
cadmium, gold, silver, copper, nickel etc, due to the pollutants carcinogenic and
mutagenic nature (Ahalya et al., 2005). These toxic materials may be derived from
mining operations, refining ores, sludge disposal, fly ash from incinerators, the
processing of radioactive materials, metal plating, or the manufacture of electrical
equipment, paints, alloys, batteries, textile dyeing, leather tanning, pesticides or
preservatives (Ahalya et al., 2005).
Modern industry is, to a large degree, responsible for contamination of the environment.
Lakes, rivers and oceans are being overwhelmed with many toxic contaminants. Among
toxic substances reaching hazardous levels are heavy metals (Vieira and Volesky, 2000).
Heavy metals are the group of contaminants of concern, which comes under the inorganic
division. Some strong toxic metal ions such as Hg are very toxic even in lower
concentration of 0.001 - 0.1 mg/ L. The presence of metal ions in final industrial effluents
is extremely undesirable, as they are toxic to both lower and higher organisms. Under
certain environmental conditions, metals may accumulate to toxic levels and cause
ecological damage (Jefferies and Firestone, 1984). Of the important metals, mercury,
lead, cadmium, arsenic and chromium (VI) are regarded as toxic; whereas, others, such as
1
copper, nickel, cobalt, manganese and zinc are not as toxic, but their extensive usage and
increasing levels in the environment are of serious concerns (Brown and Absanullah,
1971; Moore, 1990; Volesky, 1990b).
Conventional methods for removing dissolved heavy metals from aqueous
solution have been studied in detail, such as chemical precipitation and sludge separation,
chemical oxidation or reduction, ion exchange, electrochemical treatment, membrane
technologies, reverse osmosis, filtration, adsorption on activated carbon and evaporative
recovery (Lopez and Vazquez, 2003). However, these methods have problems associated
with its application and are not economically feasible (Volesky, 2001; Sharma, 2003;
Okoronkwo et al., 2007).
Therefore, the search for efficient, eco-friendly and cost effective remedies for
wastewater treatment has been initiated. It was only in the 1990s that a new scientific
area developed that could help to recover heavy metals and it was bioremediation. The
early reports described how abundant biological materials could be used to remove, at
very low cost, even small amounts of toxic heavy metals from industrial effluents. The
principal advantages of biological technologies for the removal of pollutants are that they
can be carried out in situ at the contaminated site, usually environmentally benign (no
secondary pollution) and they are cost effective. Of the different biological methods,
bioaccumulation and biosorption have been demonstrated to possess good potential to
replace conventional methods for the removal of metals (Volesky and Holan, 1995;
Malik, 2004).
Biological process like biosorption has acquired due attention owning to a number
of advantages which has engaged scientists the world over to identify the potent biomass
2
type (Khan et al., 2009; Al-Masri et al., 2010; Xiao et al., 2010). Environmentally
ubiquitous fungi are structurally unique organisms that contribute to significant removal
of metal ions from wastewater than other microbes. This is because of their great
tolerance towards heavy metals and other adverse conditions such as low pH, and their
intracellular metal uptake capacity (Gadd, 1987). Fungal biomass, both living and dead,
has been used as suitable biosorbent for metal uptake (Sharma et al., 2002; El-Sayed et
al., 2004). There are two modes of metal ion uptake, the first mode is independent of cell
metabolic activity, and is referred to as biosorption or passive uptake. It involves surface
binding of metal ions to cell wall. The functional groups involved in the binding of heavy
metals to microbial cells are phosphates, carboxyl, and hydroxyl groups (Akthar et al.,
1995; Karna et al., 1999). This mode is common to both living and dead cells. The
second mode of metal uptake into cell across the cell membrane is dependent on the cell
metabolism, and is referred to as intracellular uptake, active uptake or bioaccumulation.
This mode occurs in living cells only (Garnham et al., 1992).
Copper (Cu) is a ubiquitous metal present in the environment and is the most
common contaminant of industrial effluents such as those produced by mining and metal
processing (Anand et al., 2006) or those used in vineyards, ranging from the application
of fertilizers, dumping of agricultural and municipal wastes. Cu is also an element
essential for all living organisms as 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). Experimental studies in humans suggest that ingestion
of drinking water with > 3 mg Cu/l will produce gastrointestinal symptoms including
nausea, vomiting and diarrhea (Pizzaro et al., 1999).
3
Zinc (Zn) is also an environmentally ubiquitous heavy metal. It is an essential
trace element that is needed by normal metabolism of living organisms. However,
anthropogenic inputs could cause elevated Zn concentrations in the environment due to
man-induced activities (Yap et al., 2005). High exposure to Zn in humans can cause
nephritis, anuria and extensive lesion in kidney. It is dissolved in aquatic ecosystems and
transported by water and taken up by aquatic organisms or can be stored and transported
in sediments. (Yap et al., 2005).
Manganese (Mn) is a transitional metal and can exist in 11 oxidation states, from 3 to +7. The most common valence in biological systems is +2 and +4 (which is present
as MnO2). Cycling between Mn2+ and Mn3+ may be potentially deleterious to biological
systems because it can involve the generation of radicals (ATSDR, 1997). Mn is an
essential element and is a co-factor for a number of enzymatic reactions, particularly
those involved in phosphorylation, cholesterol, and fatty acid synthesis, Mn is present in
all living organisms (Keen and Zidenberg- Cherr, 1996). The industrial use of the Mn has
expanded in recent years as a ferroalloy in the iron industry and as a component of alloys
used in welding and also in the application of Mn pesticides (Apostoli et al., 2000). Mn
derived from these human sources can contaminate surface water, ground water and
sewage water (Lenntech, 2009).
Effects of toxic metals on fungal growth have shown intra and interspecific
variability and dependence on metal species and speciation (Gadd 1993; Plaza et al.
1998). Copper was reported to be toxic and cadmium very toxic to cultures of 15
decomposer basidiomycetes (Hoiland 1995). However, a similar toxicity of both metals
was shown on a solid medium with cadmium and copper reducing radial growth of most
4
strains of aquatic hyphomycetes by 50 % at concentrations between 150 and 400 mM
(Miersch et al. 1997). For Trichoderma virens and Clonostachys rosea colonizing
spatially discrete toxic metal containing domains, colonization distance, hyphal extension
rates and the efficacy of carbon substrate utilization decreased considerably with
increasing concentrations of copper and cadmium (Fomina et al. 2003).
A decrease in metal toxicity is correlated with an increase in available carbon
source (Ramsay et al. 1999; Fomina et al. 2003). For Stereum hirsutum and Trametes
versicolor, cadmium and mercury toxicity was lower in rich, complex media (Baldrian
and Gabriel 1997), although metal binding to medium constituents would contribute in
this case (Gadd and Griffiths 1978). This was also reported for T. virens grown with
copper, cadmium and zinc where radial extension rate was commensurate with the
availability of carbon, revealing a decrease in metal toxicity with increasing levels of
glucose (Ramsay et al. 1999). The tolerance of decomposer basidiomycetes to cadmium
was higher in fungi from rich, basophilous soils than from poor, acidic soils, whereas
resistance to aluminium was highest in fungi from poor, acidic soils (Hoiland 1995). A
decrease in growth rate in the presence of toxic metals is sometimes accompanied by an
increase in the lag phase (or growth delay) (Gadd and Griffiths 1980; Baldrian and
Gabriel 2002; Baldrian 2003). A considerable increase in the lag period was observed for
T. virens and C. rosea grown with copper and cadmium (Fomina et al. 2003).
Toxic metal treatment was reported to reduce the sporulating ability of
Aspergillus niger (Magyarosy et al. 2002; Liao et al. 2003). Spore germination was found
to be more sensitive to Ni2+, Co2+, Fe2+, Mn2+ and Mg2+ than mycelia growth (Amir and
Pineau 1998). However, a proportion of the spores of metal-sensitive strains of
5
Curvularia sp. and Fusarium sp. were able to germinate and grow moderately well in the
presence of relatively high metal concentrations (Amir and Pineau 1998). Toxic metals
can be potent inhibitors of enzymatic reactions. Cadmium, copper, lead, manganese,
nickel and cobalt decreased cellulase and amylase production by several fungi, with
reduced enzyme activity correlating with increasing metal concentration (Falih 1998a,
1998b).
The purpose of this study was to evaluate various fungi isolated from the sewage
effluent at the University of Nigeria, Nsukka sewage treatment plant as potential biomass
for the removal of heavy metal ions from aqueous solution.
1.1.1 STATEMENT OF PROBLEM
Our environment has been facing a lot of water pollution problems which arises from
lack of treatment of industrial wastewater which may contain metals before discharging
into water bodies. There exist some difficulties in the recovery of metal at low
concentration and from wastewater, which is partly as a result of high cost of
conventional methods for removal of heavy metals from wastewater hence the need for
biosorption.
1.1.2 OBJECTIVES OF THE STUDY
The objectives of the study are outlined below:

Isolation and characterization of fungi from environmental sources.

To determine the tolerance of the isolates for some heavy metals.

To determine the biosorption ability of heavy metals by the fungal isolates.
6

To determine the effects of some environmental factors on the biosorption ability
of the isolates.
1.2 LITERATURE REVIEW
1.2.1 Heavy metals
A heavy metal is a member of an ill-defined subset of elements that exhibit
metallic properties, which would mainly include the transition metals, some metalloids,
lanthanides and actinides. Many different definition have been proposed, some based on
density, atomic number or atomic weight, and some on chemical properties or toxicity
(John, 2002.). There is an alternative term “toxic metal”; for which no consensus of exact
definition exist either. (Wikipedia Heavy metal, 2011).
Heavy metals occur naturally in the ecosystem with large variations in
concentrations. In modern times, anthropogenic sources of heavy metals, i.e. pollution,
have been introduced to the ecosystem. Waste-derived fuels are especially prone to
contain heavy metals so they should be a central concern in a consideration of their use.
Living organisms require trace amounts of some heavy metals, including cobalt,
copper, iron, manganese, molybdenum, vanadium, strontium, and zinc, but excessive
levels can be detrimental to the organism. These metals, or some form of them, are
commonly found naturally in foodstuffs, in fruits and vegetables, and in commercially
available multivitamin products (Schrauzer, 1984). Other heavy metals such as mercury,
plutonium, and lead are toxic metals that no known vital or beneficial effect, or
organisms and their accumulations overtime in the bodies of animals can cause serious
illness. Certain metals that are normally toxic are for certain organisms or under certain
7
conditions, beneficial. Examples include vanadium, tungsten and even cadmium (Lane
and Morel, 2000).
Heavy metals to a small extent can enter our bodies via food, drinking water and
air. As trace elements, some heavy metals (e.g. copper, selenium, zinc) are essential to
maintain the metabolism of the human body. Iron, for example, prevents anaemia, and
zinc is a cofactor in over 100 enzyme reactions. High levels of zinc can result in a
deficiency of copper, another metal required by the body. Trace metals therefore, are
metals in extremely small quantities, almost at the molecular level, that reside in or are
present in animal and plant cells and tissue. They are a necessary part of good nutrition,
although they can be toxic if ingested at excess quantities (Underwood, 1977).
Heavy (Toxic) metals sometimes imitate the action of an essential element in the
body, interfering with the metabolic process to cause illness. Many metals, particularly
heavy metals are essential; have a low toxicity, and bismuth (the heaviest non-radioactive
element) is non-toxic. Most often the definition includes at least cadmium, lead, mercury
and the radioactive metals. Metalloids (arsenic, polonium) may be included in the
definition. Radioactive metals have both radiation toxicity and chemical toxicity. Metals
in an oxidation state abnormal to the body may also become toxic: chromium III is an
essential trace element, but chromium VI is carcinogenic. The toxicity of any metal
depends on its ligands. Toxicity is a function of solubility; insoluble compounds as well
as the metallic forms often exhibit negligible toxicity. In some cases, organometallic
forms, such as dimethyl mercury and tetraethyl lead, can be extremely toxic. In other
cases, organometallic derivatives are less toxic such as cobaltocenium cation (Wikipedia
Toxic metal, 2011).
8
Decontamination for toxic metals is different from organic toxins; because toxic
metals are elements, they cannot be destroyed. Toxic metal may be made insoluble or
collected, possibly by the aid of chelating agents. Toxic metals can bioaccumulate in the
body and in the food chain. The exceptions are barium and aluminum. Therefore, a
common characteristic of toxic metals is the chronic nature of their toxicity (Wikipedia
Toxic metal, 2011).
Trace metals or micronutrients include iron, magnesium, zinc, copper, chromium,
nickel, cobalt, vanadium, molybdenum, and selenium. Trace metals are depleted through
the expenditure of energy by a living organism. They are replenished in animals by eating
plants, and replenished in plants through the uptake of nutrients from the soil in which the
plant grows. Human vitamin pills and plant fertilizers both contain trace metals as
additional sources for trace metals (Schrauzer, 1984).
1.2.2 HEAVY METAL POLLUTION
Heavy metals can enter a water supply by industrial and consumer waste, or even
from acidic rain breaking down soils and releasing heavy metals into streams, lakes,
rivers, and ground water
Motivations for controlling heavy metal concentration in gas streams are diverse.
Some of them are dangerous to health or to the environment (Hg, Cd, As, Pb, Cr), some
may cause corrosion (e.g Zn, Pb), some are harmful in other ways (e.g. Arsenic may
pollute catalysts) (Michael, 2010).
Heavy metals are found naturally in the soil mostly in its complexed or bound
form such as in ZnSO4, ZnCl and zinc oxides. They enter the environment by human
activities such as mining, purification of zinc, lead and cadmium, steel production, coal
9
burning, burning of waste, discharges from industrial effluents, excessive use of
fertilizers, pesticide application and use of raw sewage waste for farming (Lone et al.,
2008; Okoronkwo et al., 2005; Jing et al., 2007).
One of the biggest problems associated with the persistence of heavy metals is the
potential for bioaccumulation and biomagnifications causing heavier exposure for some
organism than is present in the environment alone. Coastal fish (such as the smooth
toadfish) and seabirds (such as the Atlantic puffin) are often monitored for the presence
of such contaminants (Wikipedia Heavy metal, 2011).
In view of the inadvertent toxicity of heavy metals to man and plants, efforts are
being made to remediate already polluted environment and to check against further
pollution of the environment by indiscriminate disposal of wastes, use of sewage sludge
in farming.
Lone et al. (2008) classified the different approaches used to reclaim already
metal polluted soils into physiochemical and biological approaches.
1.2.3 METHODS OF REMEDIATING METAL POLLUTED SOIL
There are different methods of remediating soil polluted by heavy metals and they
include;
1.2.3.1 PHYSIOCHEMICAL METHODS OF REMEDIATING METAL
POLLUTED SOIL.
The physiochemical approaches involved in soil remediation includes:
1. Excavation method
This involves the excavation and reburial of polluted soils in special landfills
(Conder et al., 2001; Jing et al., 2007; Lombi et al.,2001; Neilson et al., 2003; Bennett et
10
al., 2003). This even has the commonest means of reclaiming contaminated soil (Lombi
et al., 2001) and does not actually remediate the soil (Neilson et al., 2003).
2. Capping of the polluted soil
This involves top soiling of the polluted soils with uncontaminated soils from
offsite to a depth that would minimize uptake of metals by vegetation (Okoronkwo et al.,
2005). Still, this does not give a permanent solution to the problem since the metal can
still be leached into the underground water (Neilson et al., 2003).
3. Fixation and inactivation (stabilization) of the polluting heavy metals.
This involves the conversion of the polluting heavy metals to form that which are
less mobile and available for plants and microflora. Usually, the essence of stabilization
is to reduce the amount of phytoavailable metal and thus reduce toxicities to plants,
animals and soil microorganisms. Some commonly used chemical immobilization agent
includes zeolite, gravel, sludge, beringite, alkaline materials, organic material (sewage
sludge and compost), phosphate and lime stabilized municipal biosolids (Conder et al.,
2001). Even with these chemical immobilization agents, the polluting substances are still
present in the soil and could become available overtime as agents that can enhance their
phytoavailability could be introduced into the soil (Lone et al., 2008; Conder et al.,
2001).
4. Soil washing
This technique involves the use of acid (HCl and HNO3), chelators (EDTA,
nitriloacetic acid, DTPA etc) and other anionic surfactant (biosurfactant) (Neilson et al.,
2003) to solubilize the polluting metals. It takes the form of in situ treatments which
involves soil flushing with pumps (Neilson et al., 2003) or ex situ treatment which
11
involves washing an excavated portion of the contaminated site with these agents
followed by the return of clear soil residue to the site (Lone et al., 2008). This method is
generally expensive with lots of side effects such as pollution of groundwater (Lone et
al., 2008).
Other physiochemical methods include: thermal treatment, precipitation or
flocculation followed by sedimentation, ion exchanges, reverse osmosis and
microfiltration (Lone et al., 2008). These physicochemical approaches are not suitable for
their high cost, low efficiency, destruction of soil structure and fertility (Lone et al.,
2008; Jing et al., 2007).
1.2.3.2 BIOLOGICAL APPROACHES OF REMEDIATING METAL POLLUTED
SOILS.
1. Phytoremediation
Use of special type of plants to decontaminate soil or water by inactivating metals
in the rhzosphere or translocating them in their aerial parts, this approach is called
phytoremediation. This technique involves the use of green plants to decontaminate soils,
water and air. Its application spans through both the remediation of both organic and
inorganic pollutants (Lone et al., 2008). The phytoremediation of heavy metal
contaminated site essentially aims to extract or inactivate metals in the soil (Lombi et al.,
2001; Bennett et al., 2003). There are different categories of phytoremediation, these
include: phytoextraction, phytofiltration, phytostabilization and phytovolatisation (Lone
et al., 2008).
12
In recent times, efforts are being made to increase the efficiency of
decontaminating polluted soils with plants, such strategies as suggested by Bennette et
al., (2003) include
 Indentification of novel plants capable of hyper accumulating heavy metals
through screening studies.
 Optimization of agronomic practices for enhanced biomass production and metal
uptake.
 Breeding of selected plant species for the desired property through classical
breeding or genetic engineering.
The first is continuous or natural phytoextraction. This involves the use of natural
hyper accumulating capacity to remediate the soil. More than 400 plants species are
known to hyper accumulate heavy metals of more than half are nickel hyperaccumulator
(Lone et al., 2008). The setbacks in using this method includes the production of low
biomass by these plant species, the long time required to clean up a polluted site and the
reduced bioavailability of metals in polluted site (Lombi et al., 2001)
The second approach as suggested by Lombi and his colleagues is the chemically
enhanced phytoextraction. This involves the use of high biomass crops that are induced to
take up large amount of metal when their mobility in soil is enhanced by chemical
treatment. The chemicals employed are mostly chelating agent such as EDTA, NTA,
citric acid (Lombi et al., 2001).
Even though lots of successes have been recorded with this latter method, there is
concern over enhanced mobility of metals in the soil after chelates application and also
13
the potential risk of leaching of these metals into ground water (Lombi et al., 2001).
Researches are ongoing to discover new chelating agents that would not cause the
contamination of ground water.
2. Bioremediation
Bioremediation is an option that offers the possibility to destroy or render
harmless various contaminants using natural biological activity. As such, it uses
relatively low-cost, low-technology techniques, which generally have a high public
acceptance and can often be carried out on site (Vidali, 2001). Compared to other
methods, bioremediation is a more promising and less expensive way for cleaning up
contaminated soil and water (Kamaludeen, 2003). Bioremediation uses biological agents,
mainly microorganisms, e.g. yeasts, fungi or bacteria to clean up contaminated soil and
water (Strong and Burgess, 2008). Bioremediation, i.e. the use of living organisms to
control or remediate polluted soils and water, is an emerging technology. It is defined as
the elimination, attenuation or transformation of polluting or contaminating substances by
the use of biological processes. Some tests make an exhaustive examination of the
literature of bioremediation of organic and inorganic pollutants (King et al., 1997), and
another test takes a look at pertinent field application case histories (Flathman et al.,
1993).
Most bioremediation systems are run under aerobic conditions, but running a
system under anaerobic conditions may permit microbial organisms to degrade otherwise
recalcitrant molecules. Most important parameters for bioremediation are i) the nature of
pollutants, ii) the soil structure, pH, moisture contents and hydrogeology, iii) the
nutritional state, microbial diversity of the site and iv)Temperature and oxidation-
14
reduction (Redox-Potential) (Dua et al., 2002). In bioremediation processes,
microorganisms use the contaminants as nutrient or energy sources (Tang et al., 2007).
Bioremediation activity through microbe is stimulated by supplementing nutrients
(nitrogen and phosphorus), electron acceptors (oxygen), and substrates (methane, phenol,
and toluene), or by introducing microorganisms with desired catalytic capabilities (Ma et
al., 2007; Baldwin et al., 2008). Plant and soil microbes develop a rhizospheric zone
(highly complex symbiotic and synergistic relationships) which is also used as a tool for
accelerating the rate of degradation or to remove contaminants.
The search for new technologies involving the removal of toxic metals from
wastewater has directed attention to biosorption, based on metal binding capacities of
various biological materials. Biosorption can be defined as the ability of biological
materials to accumulate heavy metals from wastewater through metabolically mediated or
physico-chemical pathways of uptake (Fourest and Roux, 1992). Algae, bacteria, fungi
and yeasts have proved to be potential metal biosorbents. The major advantages of
biosorption over conventional treatment methods include; low cost, high efficiency,
minimization of chemical and biological sludge, no additional nutrient requirement,
regeneration of biosorbent and possibility of metal recovery (Kratochvil and Volesky,
1998).
Some types of biosorbents would be broad range, binding and collecting the
majority of heavy metals with no specific activity, while others are specific for certain
metals. Some laboratories have used easily available biomass whereas others have
isolated specific strains of microorganisms and some have also processed the existing raw
biomass to a certain degree to improve their biosorption properties. Microbial biomass
15
was used as an adsorbing agent for the removal and recovery of uranium present in
industrial effluents and mine wastewater (Nakajima and Sukaguchi, 1986). Strong
biosorbent behavior of certain microorganisms towards metallic ions is a function of the
chemical make up of the microbial cells. This type of biosorbent consists of dead and
metabolically inactive cells.
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 the 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 and liquid phases.
(Kratochvil and Volesky, 1998)
Biosorption by fungi, as an alternative treatment option for wastewater containing
heavy metal, has been reviewed by Kapoor and Viraghavan (1995) and Modak et al.,
(1996). Any fungus can tolerate high concentration of potentially toxic metals; this may
be correlated with decreased intracellular uptake or impermeability of the cell membrane.
A close relation between toxicity and intracellular uptake has been shown for Cu2+, Cd2+,
Co2+ and Zn2+ in yeast Saccharomyces cerevisiae (Gadd, 1986; White and Gadd; 1986).
Waste mycelia from industrial fermentation plants (Aspergillus niger, Penicillium
chrysogenum and Claviceps paspali) were used as a biosorbent for removal of Zn ions
from aqueous environments, both batch-wise as well as in column mode. Under
optimized conditions A. niger and C. paspali were found superior to P. chrysogenum
(Luef et al., 1991). Removal of lead ions from aqueous solution by non-living biomass of
P. chrysogenum was studied and observed that Pb2+ was strongly affected by the pH in
16
the range of 4 - 5. Uptake of Pb2+ was 116 mg/g dry biomass, which was higher than that
of activated carbon and some other microorganisms (Niu et al., 1993).
Chromium biosorption by non-living biomass of Chlorella vulgaris, Clodophora
crispate, Zoogloea ramigera, Rhizopus arrhizus and Saccharomyces cerevisiae was
studied and observed that optimum initial pH (1.0 - 2.0) of the metal ion solution affected
the metal uptake capacity of the biomass for the entire microorganism. Maximum
adsorption rates of metal ions to microbial biomass were obtained at temperature in the
range of 25-350C. The adsorption rates increased with increasing the metal concentration
of C. vulgaris, C. crispate, Z. ramigera, R. arrhizus and S. cerevisiae up to 200, 200, 75,
125 and 100 mg/l respectively (Nourbakhsh et al., 1994). Dead cells of Saccharomyces
cerevisiae removed 40% more uranium or zinc than the corresponding live cultures.
Biosorption of uranium by Saccharomyces cerevisiae was a rapid process
reaching 60% of the final uptake value within the 15 min contact. The deposition
differing from that of other heavy metals more associated with the cell wall, uranium was
deposited as fine needle-like crystal both on inside and outside the S. cerevisiae cells
(Volesky and Holan, 1995). The yeast biomass Saccharomyces cerevisiae which is a byproduct from brewery industry was used for purifications of water polluted by uranium
ions, which had an efficiency to absorb up to 2.4 mMol μg/dry biomass (Omar et al.,
1996).
For the removal Hg and Cd several brown seaweeds were tested for their ability to
remove metal ions from aqueous solution by biosorption and 90 - 95% removal level was
found from industrial wastewater (Wilson and Edyvean, 1995). Lemna minor, duckweed
has been studied to remove the soluble lead from water. Result showed that viable
17
biomass removed 85 - 90% of lead while non-viable removed the 60 - 75% of lead
(Rahmani and Sternberg, 1999).
Biosorptive capacity of different biosorbent including dried mycelium of some
species of fungi, baggase, rice rusk and fermented baggase by selected fungal species or
natural microflora was examined to remove cyanide from industrial effluent. The biomass
of Rhizopus sexualis and the fermented baggase by Rhizopus sexualis or Aspergillus
terreus showed higher sorption capacity than activated charcoal. The biomass of
Rhizopus sexualis and Mortierella ramanniana exhibited higher cyanide sorptive
capacity than ascomycetes e.g. Aspergillus terreus and Penicillium capsulatum (Azab et
al., 1995).
Maximal removal of Ni from electroplating industries occurred by 2.5 gm of
biomass of Saccharomyces cerevisae within 5 hr. Ni uptake capacity from aqueous
solution was also studied in filamentous fungi such as Rhizopus sp., Penicillium sp. and
Aspergillus sp. The metal uptake was highest by Rhizopus sp. (Gill et al., 1996).
Dead biomass of actinomycetes, which is the waste product from industrial
fermentation, was mixed with wastewater as a free bacterial suspension and biosorption
occurred. Cadimum cations bound to negative charged sites on bacterial cell wall could
also be recovered from the cell wall (Butter, et al., 1995). Cd (II) biosorption to nonliving biomass of Rhizopus arrhizus and Schizomeris leiblenii was studied in batch
reactor. Maximum adsorption rates of Cd (II) ions to microbial biomass were at 30 OC and
at the optimum pH 5.0 for both microorganisms. The adsorption rates increased with
increasing Cd (II) concentration for Rhizopus arrhizus and Schizomeris leiblenii up to
100 - 150 mg/l respectively. The adsorption for Rhizopus arrhizus was higher than that of
18
Schizomeris leiblenii (Ozer et al., 1997a). Dry cells of Rhizopus arrhizus has been used
for the removal of Iron (II), Pb (II) and Cd (II) ions from the industrial wastewater.
Higher adsorption rates and adsorption capacities were obtained at initial metal
concentration up to 100 mg/l in batch reactor. High concentration of heavy metal ions
may be purified by using multistage batch reactor in series (Ozer et al., 1997b).
Biosorption studies were also carried out with the white-rot fungi Polyporous
versicolor and Phanaerochaete chrysoporium for Cu (II), Cr (III), Cd (II), Ni (II) and
Pb(II) under same operating conditions. Result showed that both were effective in
removing Pb (II) from aqueous solutions with maximum biosorption capacity of 57.5 and
110 mg Pb (II)/g dry biomass (Yeti et. al., 1998).
Mucor meihi was found to be an effective biosorbent for the removal of
hexavalent chromium from industrial tanning effluents. Sorption levels of 1.15 and 0.7
mmol/g were observed at pH 4 and 2, respectively. In comparative studies with the ion–
exchange resins, Mucor biomass demonstrated chromium biosorption levels that
correspond closely to those of commercial strongly acidic exchange resins, while the pH
behaviour mirrored that of the weakly acidic resins in solutions. However, the chromium
elution characteristic from the Mucor biomass was similar to those of both the weakly
and strongly acid resins (Tobin and Roux, 1998).
Waste biomass from the pharmaceutical fermentation industry, i.e. non living
Rhizopus nigricans has been used for adsorption of lead over a range of metal ions
concentration, adsorption time, pH and co-ions. The process of uptake obeys the
Langmuir and Freundlich isotherms. Comparison of uptake between NaOH-treated and
19
untreated biomass shows that the adsorption takes place in the chitin structure of the cell
wall (Zhang et al., 1998).
The biosorption of Cu (II), Ni (II) and Cr (VI) from aqueous solution on dried
algae (Chlorella vulgaris, Scenedesmus obliquus and synechocystis sp.) were tested under
laboratory conditions as a function of pH, initial metal ion and biomass concentration.
Results from the experiment showed the influence of the alga concentration on the metal
uptake for all the species. Both Freundlich and Langmuir adsorption models were found
to be suitable for describing the short term biosorption of Cu (II), Ni (II) and Cr (VI) by
all algal species (Donmez et al., 1999).
Non-living waste biomass from Aspergillus niger along with wheat bran was used
as a biosorbent for the removal of Zn and Cu from aqueous solution. The binding
capacity of the biomass for Cu was observed to be higher than that of Zn. The metal
uptake was found to be a function of the initial metal concentration, the biomass dosage
and pH. The metal uptake of Cu by the biomass decreased in the presence of Cobalt ion.
The uptake of Cu by the biomass decreased in the presence of Zn and vice versa. The
decrease in the metal uptake was dependent on the concentration of metal ions in two
compounds in aqueous solution (Modak et al., 1996).
Dried, nonliving, granulated biomass of Streptoverticillium cinnamoneum was
used for the recovery of Pb and Zn from the solution. The optimum pH of Zn and Pb was
3.5 - 4.5 and 5.0 - 6.0, respectively. The maximum loading capacity of S. cinnamoneum
biomass was 57.7 mg/g for Pb and 21.3 mg/g for the Zn with boiling water pretreatment.
The loaded metals could be desorbed effectively with dilute HCl, nitric acid and 0.1 M
EDTA. Treatment with 0.1 M Na carbonate permitted reuse of desorbed biomass
20
although the loading capacity in subsequent cycles decreased by 14 - 37% (Puranik and
Paknikar, 1997).
Non-living free and immobilized biomass of Rhizopus arrhizus was used to study
the biosorption of Cr (VI). Chromium removal rate was slightly more in free biomass
conditions over immobilized state. Stirred tank reactor studies indicated maximum
chromium biosorption at 100 rpm and at 1:10 biomass –liquid ratio. Fluidised bed reactor
is more efficient in chromium removal over stirred tank reactor. Immobilization of
biomaterial has little effect on the chromium biosorption by Rhizopus arrhizus
(Prakasham et al., 1999).
Living or dead fungal biomass and fungal metabolites have been used to remove
metal
or
metalloid
species,
compounds
and
particulates,
radionuclides
and
organometalloid compounds, from solution by biosorption (Gadd and White, 1989, 1990,
1992, 1993; Gadd, 1990; Kadoshnikov et al. 1995; Wang and Chen, 2006). These
processes are best suited for use in bioreactors (Gadd, 2000). Biosorption occurs via a
variety of mechanisms of a chemical and physical nature, including ion exchange,
complexation, hydrogen bonding, hydrophobic and van der Waals forces, and entrapment
in fibrillar capillaries and spaces of the mycelial network (Gadd, 1990, 1999). Certain
pretreatments and immobilization of fungal biosorbents may make metal sorption more
efficient. For example, growth of melanin-producing fungi in a medium containing
bentonite significantly enhanced the copper sorption ability of the resultant fungal
biomineral sorbent (Fomina and Gadd, 2002). Potential binding sites on fungal biomass
can include acetoamino groups from chitin, amino groups from proteins, sulphydryl
groups from proteins and peptides, hydroxyl groups, phosphate groups, and carboxyl
21
groups from organic acids, polysaccharides, (poly)phenols/quinones, and melanin (Sarret
et al. 1998; Gadd, 1999; Tobin, 2001; Fomina and Gadd, 2002). Phosphate and carboxyl
groups are of principal importance in metal biosorption to fungal hyphal walls. Phosphate
groups were responsible for 95% of the lead bound to Penicillium chrysogenum, carboxyl
groups for 55 % of the zinc sorbed to P. chrysogenum and 70 % of the zinc sorbed to
Trichoderma reesei (Fourest et al. 1996; Sarret et al. 1999).
All these mechanisms are highly dependent on the metabolic and nutritional status
of the organism, as this will affect expression of energy-dependent resistance
mechanisms, as well as synthesis of wall structural components, pigments and
metabolites, which gratuitously affect metal availability and organism response (Gadd
1992, 1993; Ramsay et al. 1999).
Keeping all these points in view, our study is directly related to check for efficient
fungi biosorbents (live and dead biomass) isolated from sewage effluent to be used in the
bioremediation of heavy metals from aqueous environment.
22
CHAPTER TWO
MATERIALS AND METHODS
2.1 Sample Collection
The sewage water samples were collected from the oxidation pond of the sewage
treatment plant at the University of Nigeria Nsukka using sterile bottles. The site is
located by latitude 06o 52’ N and longitude 07o 24’ E within the derived savanna zone of
eastern Nigeria. The minimum and maximum temperatures are about 22 oC and 30oC,
respectively, while the average relative humidity is rarely below 60% (Asadu et al.,
2002). The effluent of the sewage plant is from the hostels, staff quarters, laboratories,
offices and other parts of the University.
2.2 Preparation of samples
Sterile distilled water was used for serial dilution of the sewage water. Serial
dilution was prepared by adding 1.0 ml of sewage sample to 9.0 ml distilled water in a
test tube which was thoroughly mixed and 1.0 ml was then transferred to the next test
tube containing 9.0 ml of distilled water in other to reduce the microbial load. This was
done for up to 5 dilutions.
2.3 Isolation of microorganisms
From the serial dilution, 0.1 ml of the appropriate dilution was inoculated into
potato dextrose agar (PDA) in duplicate using the spread plate method. The sample was
evenly distributed on the PDA using a sterile glass rod in other to obtain distinct colonies.
The inoculated plates were incubated at room temperature (30 ± 2oC) for 7 days. The
medium used for the isolation and all glass wares were properly sterilized at 121 oC for 15
mins. After 7 days of incubation, standard techniques were used for isolation of the
23
different morphological colonies to obtain pure cultures. The pure isolates obtained were
further subcultured on PDA amended with 50 µg/ml of the metals (CuSO 4, ZnSO4 and
MnSO4) considered to obtain isolates tolerant to the metals and was then stored on PDA
slants at 4oC.
2.4 Tolerance to heavy metals
2.4.1 Preparation of stock solution
Stock solutions of CuSO4, ZnSO4 and MnSO4 were prepared by dissolving 1.0 g
of each salt in 1000 ml of deionized water.
2.4.2 Tolerance study
The fungal isolates obtained were tested for their ability to tolerate different
concentrations of the metals using potato dextrose broth. From the slant, the organisms
were picked with a sterile wire loop and inoculated into freshly prepared PDA, incubated
at room temperature for 7 days. Each of the isolates was inoculated using a sterile cork
borer (6 mm in diameter) into 250 ml Erlenmeyer flasks containing 100 ml of potato
dextrose broth amended with different concentrations (100, 200, 500, 1000, and 1500
µg/ml) of CuSO4, ZnSO4 and MnSO4. The flasks were incubated at room temperature (30
± 2oC) for 7 days and observed for growth. The experiment was set up in duplicates. The
growth of the isolates was determined by weighing the fungi biomass harvested from
broth by filtration using Whatman filter paper No. 1 and dried in a hot air oven at 60oC
until a constant dry weight was obtained.
2.5 Selection of working isolates
The isolates that were able to grow at different concentrations of the metals
studied were selected for characterization and identification. The fungus that was most
24
tolerant to the different concentrations of the metals was selected for the biosorption
studies.
2.6 Characterization and identification of the working isolates
The selected fungal isolates were identified and characterized by microscopy and
cultural morphology using lactophenol cotton blue staining technique and viewed under
x40 magnification objective lens. The isolates were identified using the definitions of
Smith (1971) and Beneke and Rogers (1971).
2.7 Preparation of biomass
2.7.1 Live biomass
From the stock culture (slants) a sterile wire loop was used to inoculate the
organism onto PDA and incubated at room temperature for 7 days. Potato dextrose broth
(100 ml) was prepared in a 250 ml conical flask and autoclaved at 121oC for 15 mins and
allowed to cool. A sterile cork borer (6 mm) was used to inoculate the organism grown
on PDA into the broth and incubated at room temperature for 7 days. Thereafter, the
fungal biomass was harvested by filtration using Whatman filter paper No.1. The biomass
was then transferred into a 500 ml beaker, oven dried at 60oC for about 2 hours and
powdered in a mortar. The powdered biomass was used as the biosorbent for biosorption
studies.
2.7.2 Dead biomass
From the stock culture a sterile wire loop was used to inoculate the organism onto
PDA and incubated at room temperature for 7 days. Potato dextrose broth (100 ml) was
prepared in a 250 ml conical flask and the media autoclaved at 121 oC for 15 mins and
allowed to cool. A sterile cork borer (6 mm) was used to inoculate the organism grown
25
on PDA into the broth and incubated at room temperature for 7 days. Thereafter, the
fungi biomass was harvested by filtration using Whatman filter paper No.1. The biomass
was then transferred into a 500 ml beaker and killed by autoclaving at 121oC for 20 mins.
The biomass was oven dried at 60oC for about 2 hours and powdered in a mortar. The
powdered biomass was used as the biosorbent for biosorption studies.
2.8. Preparation of standard curves
Metal concentrations of 10, 20, 40, 50, 60, 80 and 100 µg/ml of CuSO4, ZnSO4
and MnSO4 were prepared in 250 ml conical flasks. The concentration of CuSO4, ZnSO4
and MnSO4 was analyzed using spectrophotometric method of Shishehbore et al (2005),
dithizone method (APHA, 1995) and AOAC method (AOAC, 1995), respectively. The
calibration curve was then constructed by plotting the absorbance against the
concentrations of the metals.
2.9 Biosorption studies
2.9.1 Biosorption study for CuSO4
For CuSO4, the amount biosorbed by the fungal isolates was determined by
incorporating the dried biosorbent (40, 60 and 80 mg/100 ml) for each of both live and
dead biomass into the metal solutions in a 250 ml conical flask. The concentrations of the
metal solutions prepared for the biosorption studies were 10, 20, 50 and 100 µg/ml. After
the incorporation of the dried biosorbent into the different concentrations of metal
solutions, the reaction mixture was shaken on an orbital shaker at 120 rpm for 30 mins at
room temperature. At the end of the contact time, the reaction mixture was filtered using
Whatman filter paper No.1 and the residual metal determined spectrophotometrically by
the method of Shishehbore et al (2005).
26
In the method, 1.0 ml of the filtrate was placed in a test tube and adjusted to pH
7.5 with 2.0 ml buffer solution prepared by mixing appropriate ratios (1 : 4) of 0.5 M
sodium acetate solution and 10 M sodium dihydrogen phosphate solution, respectively.
The mixture was diluted with 10 ml of deionized water and 0.5 ml of 0.01% 1, 5diphenylcarbazone (DPC) (0.01 g dissolved in 100 ml of 95% ethanol) was added. The
solution was mixed very well and allowed to stand for one minute; 0.4 ml of 20%
solution of naphthalene in acetone (20 g of naphthalene dissolved in 100 ml acetone) was
added with continuous shaking. The solid mass formed consisting of naphthalene and the
metal complex was separated by filtration using Whatman filter paper No.1. The amount
of residual metal was ascertained by reading its absorbance using a spectrophotometer
(Spectrum Lab 23A, USA) at 542 nm against the reagent blank and extrapolating from
the standard curve. The experiment was done in duplicate.
2.9.2 Biosorption study for ZnSO4
For ZnSO4, the experimental set up was as described for CuSO4. At the end of the
contact time, the reaction mixture was filtered using Whatman filter paper No.1 and the
residual metal determined spectrophotometrically by the dithizone method (APHA,
1995).
In the dithizone method, 2.0 ml of the filtrate was added in a 250 ml conical flask
and 5.0 ml of acetate buffer (pH 5) and 5.0 ml of 10% (w/v) sodium thiosulphate solution
were added. Acetate buffer (pH 5) was prepared by dissolving in water 50 g of anhydrous
sodium acetate and 30 g of acetic acid and then diluted with water to 250 ml. Thereafter,
the solution containing the filtrate, acetate buffer and sodium thiosulphate was shaken
with portions of dithizone solution in carbon tetrachloride (0.05% of dithizone in CCl4)
27
until the green layer no longer changes colour. Each shaking was not less than 2 mins.
The solution with the dithizone in CCl4 was further shaken with two 5.0 ml portions of
wash solution (10.0 ml of the acetate buffer (pH 5) and 10.0 ml of the thiosulphate
solution diluted with deionized water to 100 ml; this solution was prepared just before
use) in other to separate the dithizone from the CCl4. The free dithizone from the CCl4
layer (green layer) was washed out using 5.0 ml dilute ammonia (one drop of
concentrated NH3 solution in 25 ml of water). Thereafter, the pink solution of Zn(HDz)2
with CCl4 formed was diluted in a 25 ml standard flask and mixed well. The solution was
measured at 538 nm against the reagent blank using the same spectrophotometer as stated
above. The amount of residual metal was extrapolated from the standard curve.
2.9.3 Biosorption study for MnSO4
For MnSO4, experimental set up was as described for CuSO4. At the end of the
contact time, the reaction mixture was filtered using Whatman filter paper No.1 and the
residual metal remaining determined spectrophotometrically by the AOAC method
(AOAC, 1995).
In the method, 2.0 ml of the filtrate was added into a test tube and 3.0 ml of
deionized water was also added. Thereafter, 0.5 ml of concentrated sulphuric acid
(H2SO4) was added and boiled for 1 hour. To the boiled solution, a spatular tip of
potassium periodate was added and heated for 10 mins. It was allowed to cool and made
up to 10.0 ml with deionized water. The reaction mixture was read at 520 nm against
reagent blank using the same spectrophotometer as stated earlier. The amount of residual
metal was extrapolated from the standard curve.
28
2.9.4 Effects of environmental factors on biosorption
2.9.4.1 Effect of pH
The various metal ion concentrations of 10, 20, 50, and 100 µg/ml for CuSO4,
ZnSO4 and MnSO4 were adjusted with 1M HCl or 1M NaOH to five different pH levels
(2, 4, 6, 7 and 9). After incorporation of the biomass (40, 60 and 80 mg/100 ml), the
flasks were placed on an orbital shaker (120 rpm) for 30 mins. The residual metal was
measured using the different spectrophotometric methods for each metal as described
earlier.
2.9.4.2 Effect of biomass concentration
The different weights of the biomass (40, 60 and 80 mg/100 ml) were dispersed in
solutions containing 10, 20, 50 and 100 µg/ml of each metal concentration. The solutions
were adjusted to different pH levels of 2, 4, 6, 7 and 9, in which maximum biosorption of
the metal ion occurred. The residual metal after biosorption by the biomass was
determined using the different spectrophotometric methods for each metal described
earlier.
2.9.4.3 Effect of metal ion concentration
To study the effect of each metal ion concentration on biosorption, metal
solutions of 10, 20, 50 and 100 µg/ml at different solution pH of 2, 4, 6, 7 and 9 were
prepared in 250 ml flasks. To each flask, 40, 60 and 80 mg dried biomass was added and
kept on an orbital shaker (120 rpm) at room temperature. Samples were drawn at 30 mins
of contact time. The residual metal was determined spectrophotometrically for each metal
as described earlier.
29
2.10 Data Evaluation
The amount of adsorbed metal ion (Cu, Zn and Mn) per gram of the biomass was
obtained by using the general equation:
q = [(Ci - Cf) . V] / M
where q is the amount of metal ion biosorbed by the biomass (mg/g); Ci is the initial
metal ion concentration in solution (mg/l); C f is the final metal ion concentration in
solution (mg/l); V is the volume of the medium (l); M is the amount of the biomass used
in the reaction mixture (g).
2.11 Data Analysis.
Factorial in complete randomized design was used for the ANOVA test of the
generated data from the tolerance and biosorption studies using GENSTAT discovery
edition 3 (2010). Separation of means for statistical significance was done using F-LSD
at 5% probability level according to Obi (2002).
30
CHAPTER THREE
RESULTS
3.1 Isolation of microorganisms
A total of eight fungi were isolated from the sewage water samples following
plating on PDA and subculturing until pure cultures were obtained. These isolates were
coded as OP2, OP3, XQ, XP, GC2, GC3, YQ and YP. Further subculturing into PDA
amended with 50 µg/ml of each of the metal salts, reduced the number of isolates
selected to four (OP2, OP3, XQ and XP), which were able to grow at 50 µg/ml
concentration of each metal salt. The other four (GC2, GC3, YP and YQ) showed no
visible growth after 7 days of incubation at room temperature. The four selected isolates
were characterized and identified and shown in Table 1.
3.2 Tolerance to heavy metals
3.2.1 Tolerance of fungi for CuSO4
In this study, the highest biomass was observed at 0 µg/ml (no metal
concentration or the control) for Penicillium notatum, Cladosporium species, Aspergillus
niger and Aspergillus fumigatus with 510 mg, 385 mg, 575 mg and 540 mg dry weight,
respectively. For Aspergillus niger, the growth (biomass) decreased as the concentration
of CuSO4 increased from 100 µg/ml to 1500 µg/ml with 405 mg being the highest at 100
µg/ml and 300 mg the lowest at 1500 µg/ml (Fig.1). Biomass increased for Aspergillus
fumigatus as the concentration of metal increased from 100 µg/ml (385mg) to 200 µg/ml
(490mg). With further increase in concentration from 500 µg/ml to 1500 µg/ml, a
31
Table 1. Identification and morphological characteristics of the isolates
Isolates
Morphological
characteristics on
the media (PDA)
OP2
Fast growing
colonies with
powdery white and
green surface colour.
OP3
Morphological
characteristics
under the
microscope
Possesses septate
hyphae with
unbranched
conidiosphoes. The
conidia are round,
smooth and in
tangled chains
The colonies are slow The hyphae are dark
growers with flat and green, septate and the
velvety green colour. spores forms a dense
tree-like heads.
XQ
Colonies spreads
rapidly with woolly
appearance,
originally displaying
a white colour that
turns black at
maturity.
XP
Fast growing
colonies with dark
smoky green and
velvety in nature.
Organisms
Penicillium notatum
Cladosporium
species
(Hormodendrom sp)
Septate hyphae with
Aspergillus niger
globular vesicle
which is black and
possesses phialides
that cover the entire
surface of the vesicle
forming a radiate
head.
The conidia appears
Aspergillus fumigatus
green in colour and
are globose which are
in chains from the tip
of the phialides.
32
decrease in biomass was observed from 490 mg to 230 mg dry weight (Fig.1). The
biomass of Penicillium notatum was 390 mg at 100 µg/ml which decreased as the
concentration increased from 100 µg/ml to 1500 µg/ml (fig.1). The highest biomass of
380 mg was produced by Cladosporium species at 100 µg/ml which decreased as the
concentration increased from 100 µg/ml to 1500 µg/ml with 55 mg being the lowest at
1500 µg/ml (fig.1).
3.2.2 Tolerance of fungi for ZnSO4
In the study of fungi tolerance for ZnSO4, the production of biomass for
Cladosporium species, Aspergillus niger and Aspergillus fumigatus, followed the same
pattern as in CuSO4 with the exception of Penicillium notatum which increased in growth
as the concentration increased from 100 µg/ml to 1000 µg/ml with 1000 µg/ml the
highest at 490 mg and a decline in the growth at1500 µg/ml (485 mg) (Fig. 2). At 100
µg/ml concentration of ZnSO4, Aspergillus niger had its highest biomass of 455 mg
which decreased as the concentration increased from 100 µg/ml to 1500 µg/ml, with the
lowest being 195 mg at 1500 µg/ml (Fig. 2). The biomass of Aspergillus fumigatus was
340 mg at 100 µg/ml which decreased as metal concentration increased from 100 µg/ml
to 1500 µg/ml with 205 mg as the lowest biomass at 1500 µg/ml. In Penicillium notatum,
the biomass increased with increase in concentration from 100 µg/ml to 1000 µg/ml and
decreased at 1500 µg/ml with 490 mg the highest biomass at 1000 µg/ml and 360 mg the
lowest at 100 µg/ml. Cladosporium species also showed the same trend as Aspergillus
niger and Aspergillus fumigatus, with 335 mg the highest biomass at 100 µg/ml and 145
mg the lowest at 1500 µg/ml (Fig. 2).
33
600
Biomass (mg)
500
400
0 µg/ml
100 µg/ml
300
200 µg/ml
500 µg/ml
200
1000 µg/ml
1500 µg/ml
100
0
P. notatum
Clado. species
A. niger
A. fumigatus
Organism
Fig 1. Tolerance of the isolates for CuS04
34
600
Biomass (mg)
500
400
0 µg/ml
100 µg/ml
300
200 µg/ml
500 µg/ml
200
1000 µg/ml
1500 µg/ml
100
0
P. notatum
Clado. species
A. niger
A. fumigatus
Organism
Fig 2. Tolerance of the isolates for ZnS04
35
3.2.3 Tolerance of fungi for MnSO4
For the fungi tolerance of MnSO4, the biomass of Aspergillus niger, at 100 µg/ml
was 570 mg which decreased with increase in concentration from 100 µg/ml to 1500
µg/ml being 345 mg at 1500 µg/ml. The biomass of Aspergillus fumigatus was 435 mg at
100 µg/ml concentration which decreased with increase in concentration from 100 µg/ml
to 1500 µg/ml with 255 mg as the lowest biomass at 1500 µg/ml. Penicillium notatum
and Cladosporium species also followed the same trend as Aspergillus niger and
Aspergillus fumigatus having 305 mg and 310 mg dry weight at 100 µg/ml with 85 mg
and 130 mg dry weight at 1500 µg/ml, respectively (Fig. 3).
At the end of the tolerance study, analysis showing the mean effects of the factors,
namely, metal concentration, type of metal and type of organism, was carried out. The
results are shown in Tables 2, 3 and 4.
Table 2 shows the concentration effect of the metals on the growth of the
organisms. The results show that at 100 µg/ml, the organisms produced the highest mean
biomass of 389.20 mg which is significantly (P≤ 0.05) different from those at 200, 500,
1000, and 1500 µg/ml with mean biomass of 372.10 mg, 319.20 mg, 251.20 mg and
206.70 mg, respectively. The effect of the different metal types on the growth of the
organisms, shows that mean biomass produced by the organisms in ZnSO4 (336.80mg)
was higher than that produced by the same organisms in MnSO 4 and CuSO4 with
biomass of 298.30 mg and 288.00 mg, respectively (Table 3).
Among the four organisms used for the tolerance study, Aspergillus niger
significantly (P≤ 0.05) showed the best tolerance than the rest organisms with a mean
36
600
Biomass (mg)
500
400
0 µg/ml
100 µg/ml
300
200 µg/ml
500 µg/ml
200
1000 µg/ml
1500 µg/ml
100
0
P. notatum
Clado. species
A. niger
A. fumigatus
Organism
Fig 3. Tolerance of the isolates for MnS04
37
biomass of 385.30 mg. Aspergillus fumigatus, Penicillium notatum and Cladosporium
species had mean biomass of 337.30 mg, 298.70 mg and 209.30 mg, respectively (Table
4).
3.3 Selection of working isolate for biosorption
Four fungal isolates that were tolerant to the heavy metals (Cu, Zn and Mn) were
identified as Penicillium notatum, Cladosporium species (Hormodendrum species),
Aspergillus niger and Aspergillus fumigatus using standard mycological techniques
(Table 1). Aspergillus niger showed the greatest ability to tolerate the heavy metals
considered having the highest mean biomass of 385.30 mg. Hence, its choice for
biosorption studies.
38
Table 2. The mean effect of concentration on biomass.
Concentration (µg/ml)
Biomass (mg)
100
389.20
200
372.10
500
319.20
1000
251.20
1500
206.70
F-LSD (0.05)
9.02
39
Table 3. The mean effect of metal type on biomass
Metal
Biomass (mg)
CuSO4
288.00
ZnSO4
336.80
MnSO4
298.30
F-LSD (0.05)
6.98
40
Table 4. The mean effect of type of organism on biomass
Organism
Biomass (mg)
Penicillium notatum
298.70
Cladosporium species
209.30
Aspergillus niger
385.30
Aspergillus fumigates
337.30
F-LSD (0.05)
8.07
41
3.4 Biosorption Studies
The biosorption of metals (Zn, Cu and Mn) using different weights of Aspergillus
niger (biomass) was tested at different metal concentrations and pH values for 30 mins of
contact time.
3.4.1 Biosorption study using live biomass
3.4.1.1 Biosorption study for Zn
3.4.1.1.1 Effect of metal ion concentration
The metal ion concentrations in the range of 10 to 100 µg/ml showed significant
(P≤ 0.05) effect on the rate of biosorption. Figures 4 to 6 showed the biosorption of Zn
using live biomass for 30 mins of contact time, which increases as the metal ion
concentration increases. For instance, from the results of analysis of variance at 30 mins
contact time, the least rate of biosorption of 5.71 mg/g was observed at 10 µg/ml of metal
ion concentration while the highest rate of biosorption of 74.73 mg/g was at 100 µg/ml,
irrespective of the solution pH (appendix 1).
3.4.1.1.2 Effect of biomass
The effect of adsorbent dose was studied by varying the amount of adsorbents
(40, 60 and 80 mg/100 ml). The results showed significant (P≤ 0.05) difference with
maximum biosorption of Zn obtained at minimum dose of 40 mg which shows that with
an increase in dose there was decrease in the rate of biosorption. At 40 mg, the maximum
rate of biosorption was 95.18 mg/g whereas 60 mg and 80 mg biomass had their
maximum rate of biosorption at 93.55 mg/g and 91.34 mg/g, respectively (Figs. 4 to 6).
42
Quantity adsorbed (mg/g)
100
90
80
70
60
pH 2
50
pH 4
40
pH 6
30
pH 7
20
pH 9
10
0
10
20
50
100
Metal ion concentration (µg/ml)
Fig. 4 Biosorption of Zn using live biomass (40 mg) for 30 mins.
43
Quantity adsorbed (mg/g)
100
90
80
70
60
pH 2
50
pH 4
40
pH 6
30
pH 7
20
pH 9
10
0
10
20
50
100
Metal ion concentration ( µg/ml)
Fig. 5 Biosorption of Zn using live biomass (60 mg) for 30 mins.
44
Quantity adsorbed (mg/g)
100
90
80
70
60
pH 2
50
pH 4
40
pH 6
30
pH 7
20
pH 9
10
0
10
20
50
100
Metal ion concentration ( µg/ml)
Fig. 6 Biosorption of Zn using live biomass (80 mg) for 30 mins.
45
3.4.1.1.3 Effect of pH
The effect of pH (2, 4, 6, 7 and 9) on the biosorption of Zn was investigated at 10,
20, 50 and 100 µg/ml which showed significant (P≤ 0.05) effect. In Figs. 4 to 6, the rate
of biosorption of Zn as a function of solution pH is shown. Biosorption of Zn ions
increases as the solution pH increased from 2 to 6 with pH 6 having the maximum rate of
biosorption while biosorption of the Zn ions decreased as the solution pH was further
increased from 6 to 9. For instance, in Fig. 4, the maximum rate of biosorption of Zn ions
is at pH 6 for all concentrations with 10 µg/ml (7.18 mg/g), 20 µg/ml (15.00 mg/g), 50
µg/ml (37.83 mg/g) and 100 µg/ml (94.50 mg/g).
3.4.1.2 Biosorption study for Cu
3.4.1.2.1 Effect of metal ion concentration
The biosorption capacity at different concentrations of Cu ions was determined
which showed significant (P≤ 0.05) effect. The rate of biosorption of Cu ions increases as
the concentration increases from 10 to 100 µg/ml (Figs. 7 to 9). For instance, from the
results of analysis of variance at 30 mins contact time, 10 µg/ml showed the least rate of
biosorption of 4.24 mg/g while 100 µg/ml showed the highest rate of biosorption of 69.47
mg/g, irrespective of the solution pH (appendix 2).
3.4.1.2.2 Effect of biomass
Cu biosorption on biomass were studied at various biosorbent concentrations (40,
60 and 80 mg/100 ml). The results showed significant (P≤ 0.05) difference with
maximum biosorption of Cu obtained at minimum dose of 40 mg which shows that with
an increase in dose there was decrease in the rate of biosorption. At 40 mg, the maximum
46
rate of biosorption was 93.09 mg/g whereas 60 mg and 80 mg biomass had their
maximum rate of biosorption at 91.23 mg/g and 88.89 mg/g, respectively (Figs. 7 to 9).
3.4.1.2.3 Effect of pH
The effect of pH (2, 4, 6, 7 and 9) on the biosorption of Cu ions on A. niger
biomass was investigated at 10, 20, 50 and 100 µg/ml which was significant (P≤ 0.05). In
Figs. 7 to 9, the rate of biosorption of Cu as a function of solution pH is shown.
Biosorption of Cu ions increased with an increase in solution pH from 2 to 6 with pH 6
having the maximum rate of biosorption while biosorption of the Cu ions decreased as
the solution pH was further increased from 6 to 9. For instance, in Fig. 7, the maximum
rate of biosorption of Cu ions is at pH 6 for all concentrations with 10 µg/ml (5.87 mg/g),
20 µg/ml (12.23 mg/g), 50 µg/ml (31.33 mg/g) and 100 µg/ml (92.57 mg/g).
3.4.1.3 Biosorption study for Mn
3.4.1.3.1 Effect of metal ion concentration
The biosorption capacity at different concentrations of Mn ions was determined
which showed significant (P≤ 0.05) effect. The rate of biosorption of Mn ions increases
as the concentration increases from 10 to 100 µg/ml (Figs. 10 to 12). For instance, from
the results of analysis of variance at 30 mins contact time, 10 µg/ml showed the least rate
of biosorption of 3.32 mg/g while 100 µg/ml showed the highest rate of biosorption of
63.98 mg/g, irrespective of the solution pH (appendix 3).
47
Quantity biosorbed (mg/g)
100
90
80
70
60
pH 2
50
pH 4
40
pH 6
30
pH 7
20
pH 9
10
0
10
20
50
100
Metal ion concentration (µg/ml)
Fig. 7 Biosorption of Cu using live biomass (40 mg) for 30 mins.
48
Quantity adsorbed (mg/g)
100
90
80
70
60
pH 2
50
pH 4
40
pH 6
30
pH 7
20
pH 9
10
0
10
20
50
100
Metal ion concentration (µg/ml)
Fig. 8 Biosorption of Cu using live biomass (60 mg) for 30 mins.
49
Quantity adsorbed (mg/g)
100
90
80
70
60
pH 2
50
pH 4
40
pH 6
30
pH 7
20
pH 9
10
0
10
20
50
100
Metal ion concentration (µg/ml)
Fig.9 Biosorption of Cu using live biomass (80 mg) for 30 mins.
50
3.4.1.3.2 Effect of biomass
The effect of adsorbent dose was studied by varying the amount of adsorbents
(40, 60 and 80 mg/100 ml). The results showed significant (P≤ 0.05) difference with
maximum biosorption of Mn obtained at minimum dose of 40 mg which shows that with
an increase in dose there was decrease in the rate of biosorption. At 40 mg, the maximum
rate of biosorption was 88.90 mg/g whereas 60 mg and 80 mg biomass had their
maximum rate of biosorption at 84.90 mg/g and 82.84 mg/g, respectively (Figs. 10 to 12).
3.4.1.3.3 Effect of pH
The effect of pH (2, 4, 6, 7 and 9) on the biosorption of Mn was investigated at
10, 20, 50 and 100 µg/ml which showed significant (P≤ 0.05) effect. In Figs. 18 to 23, the
rate of biosorption of Mn as a function of solution pH is shown. Biosorption of Mn ions
increases as the solution pH increased from 2 to 6 with pH 6 having the maximum rate of
biosorption while biosorption of the Mn ions decreased as the solution pH was further
increased from 6 to 9. For instance, in Fig. 10, the maximum rate of biosorption of Mn
ions is at pH 6 for all concentrations with 10 µg/ml (4.90 mg/g), 20 µg/ml (11.46 mg/g),
50 µg/ml (29.79 mg/g) and 100 µg/ml (87.97 mg/g).
51
Quantity biosorbed (mg/g)
100
90
80
70
60
pH 2
50
pH 4
40
pH 6
30
pH 7
20
pH 9
10
0
10
20
50
100
Metal ion concentration (µg/ml)
Fig. 10 Biosorption of Mn using live biomass (40 mg) for 30 mins.
52
Quantity biosorbed (mg/g)
100
90
80
70
60
pH 2
50
pH 4
40
pH 6
30
pH 7
20
pH 9
10
0
10
20
50
100
Metal ion concentration (µg/ml)
Fig. 11 Biosorption of Mn using live biomass (60 mg) for 30 mins.
53
Quantity biosorbed (mg/g)
100
90
80
70
60
pH 2
50
pH 4
40
pH 6
30
pH 7
20
pH 9
10
0
10
20
50
100
Metal ion concentration (µg/ml)
Fig. 12 Biosorption of Mn using live biomass (80 mg) for 30 mins.
54
3.4.2 Biosorption study using dead biomass
3.4.2.1 Biosorption study for Zn
3.4.2.1.1 Effect of metal ion concentration
The biosorption capacity at different concentrations of Zn ions was determined
which showed significant (P≤ 0.05) effect. The rate of biosorption of Zn ions increases as
the concentration increases from 10 to 100 µg/ml (Figs. 13 to 15). For instance, from the
results of analysis of variance at 30 mins contact time, 10 µg/ml showed the least rate of
biosorption of 5.96 mg/g while 100 µg/ml showed the highest rate of biosorption of 74.96
mg/g, irrespective of the solution pH (appendix 4).
3.4.2.1.2 Effect of biomass
The effect of adsorbent dose was studied by varying the amount of adsorbents
(40, 60 and 80 mg/100 ml). The results showed significant (P≤ 0.05) difference with
maximum biosorption of Zn obtained at minimum dose of 40 mg which shows that with
an increase in dose there was decrease in the rate of biosorption. At 40 mg, the maximum
rate of biosorption was 95.50 mg/g whereas 60 mg and 80 mg biomass had their
maximum rate of biosorption at 93.78 mg/g and 91.50 mg/g, respectively (Figs. 13 to 15).
3.4.2.1.3 Effect of pH
The effect of pH (2, 4, 6, 7 and 9) on the biosorption of Zn was investigated at 10,
20, 50 and 100 µg/ml which showed significant (P≤ 0.05) effect. In Figs. 13 to 15, the
rate of biosorption of Zn as a function of solution pH is shown. Biosorption of Zn ions
increases as the solution pH increased from 2 to 6 with pH 6 having the maximum rate of
55
Quantity biosorbed (mg/g)
100
90
80
70
60
pH 2
50
pH 4
40
pH 6
30
pH 7
20
pH 9
10
0
10
20
50
100
Metal ion concentration (µg/ml)
Fig. 13 Biosorption of Zn using dead biomass (40 mg) for 30 mins.
56
Quantity biosorbed (mg/g)
100
90
80
70
60
pH 2
50
pH 4
40
pH 6
30
pH 7
20
pH 9
10
0
10
20
50
100
Metal ion concentration (µg/ml)
Fig. 14 Biosorption of Zn using dead biomass (60 mg) for 30 mins.
57
Quantity biosorbed (mg/g)
100
90
80
70
60
pH 2
50
pH 4
40
pH 6
30
pH 7
20
pH 9
10
0
10
20
50
100
Metal ion concentration (µg/ml)
Fig. 15 Biosorption of Zn using dead biomass (80 mg) for 30 mins.
58
biosorption while biosorption of the Zn ions decreased as the solution pH was further
increased from 6 to 9. For instance, in Fig. 13, the maximum rate of biosorption of Zn
ions is at pH 6 for all concentrations with 10 µg/ml (7.50 mg/g), 20 µg/ml (15.33 mg/g),
50 µg/ml (38.00 mg/g) and 100 µg/ml (94.83 mg/g).
3.4.2.2 Biosorption study for Cu
3.4.2.2.1 Effect of metal ion concentration
The biosorption capacity at different concentrations of Cu ions was determined
which showed significant (P≤ 0.05) effect. The rate of biosorption of Cu ions increases as
the concentration increases from 10 to 100 µg/ml (Figs. 16 to 18). For instance, from the
results of analysis of variance at 30 mins contact time, 10 µg/ml showed the least rate of
biosorption of 4.46 mg/g while 100 µg/ml showed the highest rate of biosorption of 69.98
mg/g, irrespective of the solution pH (appendix 5).
3.4.2.2.2 Effect of biomass
The effect of adsorbent dose was studied by varying the amount of adsorbents
(40, 60 and 80 mg/100 ml). The results showed significant (P≤ 0.05) difference with
maximum biosorption of Cu obtained at minimum dose of 40 mg which shows that with
an increase in dose there was decrease in the rate of biosorption. At 40 mg, the maximum
rate of biosorption was 93.30 mg/g whereas 60 mg and 80 mg biomass had their
maximum rate of biosorption at 91.36 mg/g and 89.05 mg/g, respectively (Figs. 16 to 18).
59
Quantity biosorbed (mg/g)
100
90
80
70
60
pH 2
50
pH 4
40
pH 6
30
pH 7
20
pH 9
10
0
10
20
50
100
Metal ion concentration (µg/ml)
Fig. 16 Biosorption of Cu using dead biomass (40 mg) for 30 mins.
60
Quantity biosorbed (mg/g)
100
90
80
70
60
pH 2
50
pH 4
40
pH 6
30
pH 7
20
pH 9
10
0
10
20
50
100
Metal ion concentration (µg/ml)
Fig. 17 Biosorption of Cu using dead biomass (60 mg) for 30 mins.
61
Quantity biosorbed (mg/g)
100
90
80
70
60
pH 2
50
pH 4
40
pH 6
30
pH 7
20
pH 9
10
0
10
20
50
100
Metal ion concentration (µg/ml)
Fig 18 Biosorption of Cu using dead biomass (80 mg) for 30 mins.
62
3.4.2.2.3 Effect of pH
The effect of pH (2, 4, 6, 7 and 9) on the biosorption of Cu was investigated at 10,
20, 50 and 100 µg/ml which showed significant (P≤ 0.05) effect. In Figs. 16 to 18, the
rate of biosorption of Cu as a function of solution pH is shown. Biosorption of Cu ions
increases as the solution pH increased from 2 to 6 with pH 6 having the maximum rate of
biosorption while biosorption of the Cu ions decreased as the solution pH was further
increased from 6 to 9. For instance, in Fig. 16, the maximum rate of biosorption of Cu
ions is at pH 6 for all concentrations with 10 µg/ml (5.93 mg/g), 20 µg/ml (12.50 mg/g),
50 µg/ml (31.58 mg/g) and 100 µg/ml (92.78 mg/g).
3.4.2.3 Biosorption study for Mn
3.4.2.3.1 Effect of metal concentration
The biosorption capacity at different concentrations of Mn ions was determined
which showed significant (P≤ 0.05) effect. The rate of biosorption of Mn ions increases
as the concentration increases from 10 to 100 µg/ml (Figs. 19 to 21). For instance, from
the results of analysis of variance at 30 mins contact time, 10 µg/ml showed the least rate
of biosorption of 3.60 mg/g while 100 µg/ml showed the highest rate of biosorption of
70.09 mg/g, irrespective of the solution pH (appendix 6).
3.4.2.3.2 Effect of biomass
The effect of adsorbent dose was studied by varying the amount of adsorbents
(40, 60 and 80 mg/100 ml). The results showed significant (P≤ 0.05) difference with
maximum biosorption of Mn obtained at minimum dose of 40 mg which shows that with
an increase in dose there was decrease in the rate of biosorption. At 40 mg, the maximum
63
rate of biosorption was 89.05 mg/g whereas 60 mg and 80 mg biomass had their
maximum rate of biosorption at 85.87 mg/g and 83.83 mg/g, respectively (Figs. 19 to 21).
3.4.2.3.3 Effect of pH
The effect of pH (2, 4, 6, 7 and 9) on the biosorption of Mn was investigated at
10, 20, 50 and 100 µg/ml which showed significant (P≤ 0.05) effect. In Figs. 19 to 21, the
rate of biosorption of Mn as a function of solution pH is shown. Biosorption of Mn ions
increases as the solution pH increased from 2 to 6 with pH 6 having the maximum rate of
biosorption while biosorption of the Mn ions decreased as the solution pH was further
increased from 6 to 9. For instance, in Fig. 19, the maximum rate of biosorption of Mn
ions is at pH 6 for all concentrations with 10 µg/ml (5.78 mg/g), 20 µg/ml (12.75 mg/g),
50 µg/ml (30.53 mg/g) and 100 µg/ml (88.43 mg/g).
64
Quantity biosorbed (mg/g)
100
90
80
70
60
pH 2
50
pH 4
40
pH 6
30
pH 7
20
pH 9
10
0
10
20
50
100
Metal ion concentration (µg/ml)
Fig. 19 Biosorption of Mn using dead biomass (40 mg) for 30 mins.
65
Quantity biosorbed (mg/g)
100
90
80
70
60
pH 2
50
pH 4
40
pH 6
30
pH 7
20
pH 9
10
0
10
20
50
100
Metal ion concentration (µg/ml)
Fig. 20 Biosorption of Mn using dead biomass (60 mg) for 30 mins.
66
Quantity biosorbed (mg/g)
100
90
80
70
60
pH 2
50
pH 4
40
pH 6
30
pH 7
20
pH 9
10
0
10
20
50
100
Metal ion concentration (µg/ml)
Fig. 21 Biosorption of Mn using dead biomass (80 mg) for 30 mins.
67
3.4.3 Comparison between live and dead biomass in biosorption
The biosorption of Zn, Cu and Mn ions by A. niger biomass (live and dead), at
different metal ion concentrations, solution pH and 30 mins of contact time showed no
significant (P > 0.05) effect. (Appendix 7, 8 and 9).
68
CHAPTER FOUR
DISCUSSION
From the three metals considered (CuSO4, ZnSO4 and MnSO4), a gradual
decrease was observed in the growth (biomass) of fungi (Aspergillus niger,
Cladosporium sp) from 100 µg/ml to 1500 µg/ml with the exception of Aspergillus
fumigatus in CuSO4 and Penicillium notatum in ZnSO4. In this study, the tolerance of the
fungi for different metal ions was probably affected not only by the surface properties of
the organisms involved but also by various other physico-chemical parameters of the
metals.
The increase in growth of A. fumigatus as the concentration of CuSO4 increased
from 100 µg/ml to 200 µg/ml might be that the metal stimulates the growth of the
organism at those concentrations. This is in line with the report of Englander and Corden
(1971), where they observed that CuSO4 can stimulate the mycelial growth of Endothia
parasitica.
As the concentration of ZnSO4 increased from 100 µg/ml to 1000 µg/ml, the
weight of the biomass of Penicillium notatum increased. This could be because zinc is
associated with enzymes particularly metalloenzymes which are essential for fungal
growth (El-Sharouny et al., 1988; Ross 1994). Work done by Al-Obaid and Hashem
(1997) showed that Penicillium citrinum was resistant to zinc at 500 µg/ml. Colpaert and
Van Assche, (1992) and Sintuprapa et al. (2000) showed a maximum Zn tolerance at
1000 µg/ml for ectomycorrhizal fungi and Penicillium species. However, other fungi are
less tolerant, for example Aspergillus flavipes tolerated 200 mg/l of Zn (Lopez and
Vazquez, 2003) and arbuscular ectomycorrhizal mycellum (Glomus species) tolerated
69
100 mg/l of Zn (Joner et al., 2000). At higher concentrations of zinc, it could become
toxic to the organism (Aronson, 1982), which could be the reason for the reduction of the
weight of biomass at 1500 µg/ml.
The decline in the weight of the biomass of the fungi as the concentrations
increased from 100 µg/ml to 1500 µg/ml, may be as a result that at higher concentrations,
the metal may become toxic to the organisms (Gadd, 1993) which inhibits their growth.
Although fungi might require some metals at low concentrations as essential
micronutrients which could act as cofactor for metalloproteins and certain enzymes
(Gadd, 1993; Nies, 1999). However, at higher concentrations it has also been reported
that metals interact with nucleic acids and enzyme’s active site which can lead to rapid
decline in membrane integrity and cell death (Cervantes and Gutierrez-Corana, 1994;
Hazel and Williams, 1990; Ohsumi et al., 1988; Stohs and Bagchi, 1995). The different
reported ranges of heavy metal tolerance may be because of the diverse ability of the
different fungi (Lopez and Vazquez, 2003).
For the uptake capacity of A. niger (both live and dead biomass) for Zn, Cu and
Mn, the increase in the concentration of these metals from 10 µg/ml to 100 µg/ml saw an
increase in the rate of biosorption. This might be due to the fact that at high metal ion
concentration more binding sites are free for interaction resulting in high metal uptake at
higher concentration than at low metal ion concentration (Mausumi et al., 2007). Another
explanation could be that a higher initial concentration provides an important driving
force to overcome all mass transfer resistances between the metal solution and fungal cell
wall, thus the biosorption capacity increased. In addition, the number of collisions
between metal ions and biosorbent increased with increasing initial metal concentration,
70
so the biosorption process was enhanced (Aksu and Tezer, 2005). Similarly, Wang and
Chen (2006) found that the uptake rate of the metal (Cu) had increased along with
increasing the initial concentration (0 to 100 µg/ml) if the amount of biomass was kept
unchanged. Park et al. (2005) used dead fungal biomass for the detoxification of
hexavalent chromium and observed an increase in the removal rate of Cr(VI) with
increasing initial Cr(VI) concentration. Also Yazdani et al. (2010) observed that from
100 to 300 mg/l Trichoderma atroviride showed a sudden increase in the uptake of Zn
ions with its maximum uptake at 300 mg/l. Colpaert and Van Assche (1992) reported
different biosorption of Zn for Suillus bovines isolated from unpolluted and Zn-polluted
sites in Belgium. The strain isolated from the Zn-polluted area showed 2.8, 7.9, 10.8 and
17.4 mg/g of Zn uptake in media containing Zn concentrations of 10, 100, 500 and 1000
mg/l, respectively while the strain isolated from an unpolluted area showed 0.654, 3.6
and 9.4 mg/g of Zn uptake in media with 10, 100 and 500 mg/l of Zn, respectively. In
both cases they observed increase in the rate of adsorption with an increase in
concentration of Zn. Chaudhary (2011) used millet husk as biosorbent for the removal of
chromium and manganese ions from aqueous solution, where he observed an increase in
the rate of adsorption of Mn ions as the concentration was increased from an initial
concentration of 20 mg/l to 80 mg/l and then after no further increase in the rate of
adsorption was noticed (at 100 mg/l).
The study of the effect of dose of adsorbent is necessary and very useful to find
out the optimum amount of A. niger biomass required for the removal of Zn, Cu and Mn
ions. The effect of adsorbent dose studies was observed between 40 mg, 60 mg and 80
mg for each of the metals using both live and dead biomass. The equilibrium value of the
71
quantity of metal adsorbed decreases with increasing concentrations of the biomass. This
might be due to the interference between binding sites at higher concentrations or
insufficiency of metal ions in solution with respect to available binding sites (de Rome
and Gadd, 1987). Similar observations were made in studies on chromium (VI)
biosorption using husk of Bengal gram by Ahalya et al. (2005), where they observed a
progressive decrease in the percentage biosorption of chromium from 99.65% to 75% as
the biomass concentration was increased from 1 to 40 g/l. Other studies which also
followed similar trend includes, metal sorption by immobilized Phormidium laminosum
(Blanco et al., 1999), biosorption characteristics of Aspergillus fumigatus in removal of
cadmium from aqueous solution (Al-Garni et al., 2009), biosorption of copper from
aqueous solution using algal biomass (Narsi et al., 2004) and also copper and chromium
uptake by Aspergillus carbonarius (Al-Asheh and Duvnjak, 1995). Higher uptake at
lower biomass concentrations could also be due to an increased metal to biosorbent ratio,
which decreases upon an increase in biomass concentration (Mausumi et al., 2007)
In the study of the effect contact time for the efficient removal of the metal ions, it
was observed that equilibrium for the biosorption of metals were reached at 30 mins of
contact time using both live and dead biomass. The rate of Zn, Cu and Mn ions binding
with A. niger biomass increases and remains almost constant at 30 mins. The rate of
removal of metal became almost insignificant above 30 mins, this might be due a quick
exhaustion of the adsorption site (Singanan, 2011) and also that adsorption and
desorption balance each other (Narsi et al., 2004). Similar results have been reported for
removal of Mn ion from aqueous solution using Bombax malabaricum fruit shell
substrate (Emmanuel et al., 2007), removal of lead (II) and cadmium (II) from waste
72
water using activated biocarbon (Singanan, 2011) and biosorption uptake of copper by
Pseudomonas aeruginosa (Chang et al., 1997)
The results of the influence of pH on the biosorption of Cu, Zn and Mn ions using
both live and dead biomass showed that there was a gradual increase in the rate of
adsorption as the solution pH increases from 2 to 6 with the maximum rate of adsorption
at pH 6. This might be attributed to the competition of hydrogen ion with metal ion on the
sorption site. Thus at lower pH (ie below pH 5), the rate of adsorption was low due to the
protonation of the binding site resulting to the high concentration of protons which
reduces the negative charge intensity on the site and resulting in the reduction or
inhibition for the binding of metal ion. Most of the microbial surfaces are negatively
charged due to the ionization of the functional groups, thereby contributing to the metal
binding. Fungal surfaces have a negative charge at pH range 2 to 6. At low pH, some of
the functional groups will be positively charged and may not interact with metal ions
(Yan and Viraraghavan, 2003). Similar result was reported (Omar, 2008) for the
biosorption of copper, nickel and manganese using non living biomass of marine alga,
Ulva lactuca, where maximum biosorption capacity was observed at pH 5. Also Narsi
and Garima (2005) observed that Rhizopus arrhizus exhibited higher Zn uptake at a
solution pH 5.8. Maximum rate of biosorption between pH range of 5 to 6 for copper and
5 to 5.5 for manganese has been reported (Vegliό et al., 1997).
As the pH increased above the maximum adsorption rate of pH 6 (pH 6 to 9),
there was a gradual decrease in the rate of adsorption by both live and dead biomass. This
might be due to a reduction in the electrostatic attraction between the metal and the
sorbent surface. At alkaline pH values (8 and above), a reduction in the solubility of
73
metals may contribute to lower uptake rates (Congeevaram et al., 2007). Similar results
were reported by Nasseri et al. (2002), who showed the removal of chromium using
Aspergillus oryzae, where maximum removal was observed at pH 5 and with further
increase in pH beyond 5, the chromium removal rate decreased.
After analyzing the two nature of biomass (live and dead), the efficiency of metal
sorption by dead cells was reported to be statistically equivalent to that of the live cells.
Although, in some cases the opposite result had been reported (Werner and Morschel,
1978; Al-Garni et al., 2009). Paris and Lewis (1976), Lederman and Rhee, (1982) and
Mac Rae (1985) had reported that the accumulation of organic pollutants by dead
microbial biomass is greater than or equal to the accumulation by the same living
microorganisms. Zucconi et al. (2003) found that living cells of Paecilomyces lilacinus
showed higher capacity than dead cells. Mane et al. (2011) reported an increased
bioadsorption capacity of autoclaved biomass in comparison with live biomass which
they attributed to the exposure of the latent binding sites. Çabuk et al. (2005), has also
reported similar results.
This behavior might be due to the difference in the mechanism of biosorption in
both live and dead microbial biomass (Al-Garni et al., 2009) and also factors such as the
absence of metabolic protection against transport of pollutants into the cell, increased
permeability of the dead cell membrane (Davson and Danielli, 1952), and the change of
the surface adsorptive properties of the microbial cell following its death (Tsezos and
Bell, 1989). However, dead biomass has more advantages than live biomass (Volesky,
1990a; Gupta et al., 2000; Zhou and Kiff, 1991; Bayramoglu et al., 2003)
74
Moreover, some researchers have demonstrated that factors such as contact time,
biomass dosage and pH are known to influence the biosorption of metals (Zafar et al.,
2007). The variations in the metal tolerance for different or the same type of biosorbent
might be due to the presence of one or more types of tolerance strategies or resistant
mechanisms exhibited by different biosorbent (Zafar et al., 2007)
CONCLUSION
The present study showed that fungi isolated from sewage effluent from the
sewage treatment plant at the University of Nigeria have the ability to tolerate heavy
metals at high concentrations. From the experiment, Aspergillus niger showed better
ability to tolerate heavy metals than other fungi.
The results from the biosorption study indicate that dead A. niger biomass has
equivalent biosorption ability as the live biomass. In other words, both live and dead A.
niger biomass could be developed as an efficient metal removal biosorbent for Cu, Zn
and Mn ions from aqueous solutions. The biosorption process was influenced by
conditions such as pH, contact time, biomass dosage and metal ion concentration. The
results obtained from both tolerance and biosorption studies suggest that A. niger is a
potential organism for further studies in the bioremediation of heavy metals from aqueous
environment.
75
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APPENDIX 1
The mean effect of concentration, biomass and pH in the biosorption of Zn2+ using live
biomass at 30 mins contact time.
Concentration (µg/ml)
Quantity of metal biosorbed (mg/g)
10
5.71
20
12.62
50
34.88
100
74.73
F-LSD (0.05)
0.024
Biomass (mg)
Quantity of metal biosorbed (mg/g)
40
32.94
60
32.14
80
30.87
F-LSD (0.05)
0.021
pH
Quantity of metal biosorbed (mg/g)
2
28.33
4
29.75
6
37.61
7
32.85
9
31.39
F-LSD (0.05)
0.027
F-LSD (0.05)
Conc x Biomass (mg)
0.041
Conc x pH
0.053
Biomass (mg) x pH
0.046
Conc x Biomass (mg) x pH
0.092
Legend: Conc = Concentration (µg/ml)
91
APPENDIX 2
The mean effect of concentration, biomass and pH in the biosorption of Cu 2+ using live
biomass at 30 mins contact time.
Concentration (µg/ml)
Quantity of metal biosorbed (mg/g)
10
4.24
20
9.33
50
26.08
100
69.47
F-LSD (0.05)
0.026
Biomass (mg)
Quantity of metal biosorbed (mg/g)
40
28.64
60
27.20
80
25.99
F-LSD (0.05)
0.022
pH
Quantity of metal biosorbed (mg/g)
2
21.01
4
24.55
6
34.30
7
29.23
9
27.32
F-LSD (0.05)
0.029
F-LSD (0.05)
Conc x Biomass (mg)
Conc x Ph
Biomass (mg) x Ph
Conc x Biomass (mg) x Ph
Legend: Conc = Concentration (µg/ml)
0.045
0.058
0.050
0.100
92
APPENDIX 3
The mean effect of concentration, biomass and pH in the biosorption of Mn2+ using live
biomass at 30 mins contact time.
Concentration (µg/ml)
Quantity of metal biosorbed (mg/g)
10
3.32
20
8.11
50
24.09
100
63.98
F-LSD (0.05)
0.025
Biomass (mg)
Quantity of metal biosorbed (mg/g)
40
27.01
60
24.62
80
23.00
F-LSD (0.05)
0.022
pH
Quantity of metal biosorbed (mg/g)
2
4
18.20
21.41
6
31.41
7
27.92
9
25.44
F-LSD (0.05)
0.028
F-LSD (0.05)
Conc x Biomass (mg)
0.044
Conc x pH
0.057
Biomass (mg) x pH
0.049
Conc x Biomass (mg) x pH
0.098
Legend: Conc = Concentration (µg/ml)
93
APPENDIX 4
The mean effect of concentration, biomass and pH in the biosorption of Zn2+ using dead
biomass at 30 mins contact time.
Concentration (µg/ml)
Quantity of metal biosorbed (mg/g)
10
5.96
20
12.88
50
35.17
100
74.96
F-LSD (0.05)
0.024
Biomass (mg)
Quantity of metal biosorbed (mg/g)
40
33.21
60
80
32.38
31.13
F-LSD (0.05)
0.020
pH
Quantity of metal biosorbed (mg/g)
2
28.55
4
6
30.02
37.87
7
9
33.15
31.62
F-LSD (0.05)
0.026
F-LSD (0.05)
Conc x Biomass (mg)
0.041
Conc x pH
0.053
Biomass (mg) x pH
0.046
Conc x Biomass (mg) x pH
0.091
Legend: Conc =concentration (µg/ml)
94
APPENDIX 5
The mean effect of concentration, biomass and pH in the biosorption of Cu 2+ using dead
biomass at 30 mins contact time.
Concentration (µg/ml)
Quantity of metal biosorbed (mg/g)
10
4.46
20
9.60
50
26.63
100
69.98
F-LSD (0.05)
0.024
Biomass (mg)
Quantity of metal biosorbed (mg/g)
40
28.98
60
80
27.67
26.36
F-LSD (0.05)
0.020
pH
Quantity of metal biosorbed (mg/g)
2
21.30
4
6
7
24.90
34.65
29.70
9
27.79
F-LSD (0.05)
0.026
F-LSD (0.05)
Conc x Biomass (mg)
0.041
Conc x pH
0.053
Biomass (mg) x pH
0.046
Conc x Biomass (mg) x pH
0.091
Legend: Conc =concentration (µg/ml)
95
APPENDIX 6
The mean effect of concentration, biomass and pH in the biosorption of Mn2+ using dead
biomass at 30 mins contact time.
Concentration (µg/ml)
Quantity of metal biosorbed (mg/g)
10
3.60
20
9.04
50
24.95
100
70.09
F-LSD (0.05)
0.016
Biomass (mg)
Quantity of metal biosorbed (mg/g)
40
28.10
60
26.83
80
25.83
F-LSD (0.05)
0.014
pH
Quantity of metal biosorbed (mg/g)
2
19.59
4
23.63
6
33.43
7
29.85
9
28.07
F-LSD (0.05)
0.018
F-LSD (0.05)
Conc x Biomass (mg)
0.028
Conc x pH
0.036
Biomass (mg) x pH
0.031
Conc x Biomass (mg) x pH
0.062
Legend: Conc =concentration (µg/ml)
96
APPENDIX 7
Comparison between live and dead biomass in the biosorption of Zn2+
Nature of Biomass
Quantity of metal biosorbed (mg/g)
Dead
33.21
Live
32.94
t (0.05)
Ns
97
APPENDIX 8
Comparison between live and dead biomass in the biosorption of Cu2+
Nature of Biomass
Quantity of metal biosorbed (mg/g)
Dead
28.98
Live
28.64
t (0.05)
Ns
98
APPENDIX 9
Comparison between live and dead biomass in the biosorption of Mn2+
Nature of Biomass
Quantity of metal biosorbed (mg/g)
Dead
28.10
Live
27.01
t (0.05)
Ns
99
APPENDIX 10
Zn (II) Standard Curve
1.6
1.4
y = 0.0141x
R2 = 0.9859
Absorbance
1.2
1
0.8
0.6
0.4
0.2
0
0
20
40
60
80
100
120
Concentration (µg/ml)
100
APPENDIX 11
Cu (II) Standard Curve
2
1.8
y = 0.0176x
R2 = 0.9781
1.6
Absorbance
1.4
1.2
1
0.8
0.6
0.4
0.2
0
0
20
40
60
80
100
120
Concentration (µg/ml)
101
APPENDIX 12
Mn (II) Standard Curve
2.5
y = 0.0216x
Absorbance
2
R2 = 0.9459
1.5
1
0.5
0
0
20
40
60
80
100
120
Concentration (µg/ml)
102