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Chapter 1
INTRODUCTION
Rapid unabated increase in human population coupled with rapid
industrialization has resulted in multidimensional adverse impact on the
biosphere.
Pollution represents
one
such
complex
dimension.
The
management of pollution is one of the prime concerns of human society
today.
Considering
the
economic
constraints
of
existing
pollution
management strategies, affordable and effective methods for the purpose
are required without much delay.
Untreated or partially treated wastewaters and industrial effluents
discharged into the environment pose aq serious threat to the ecosystem
including the life forms (Bhole et al., 2004). The industrial wastes are one of
the greatest causes of water pollution. The industrial wastes may have
pollutants of almost all kinds ranging from simple nutrients and organic
matter to complex toxic substances. The industrial wastes affect the ground
water quality also, and render it unfit for drinking purpose. Ground water
pollution, in turn, causes irreparable damage to soil, plants and animals
including human beings. Polluted ground water is a major cause for the
spread of epidemics and chronic diseases in human beings (Srinivas et al.,
2002). Heavy metals constitute one significant group of pollutants.
The pollution by heavy metals has come to be recognized as one of the major
environmental problems (Dushenkov et al., 1995; Crusberg et al., 2004;
Rani et al., 2007; Das et al., 2008; Piccardo et al., 2009; Nawachukwu et al.,
2010). As a result of their mobility in natural water ecosystems, heavy
metals easily contaminate the aquifers so much so that these are emerging
as major inorganic contaminants of water (Atkinson et al., 1998). Not only
this, these metals get accumulated in soils (Hiroki, 1992) and thereby
further aggravate the problems.
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The term ‘heavy metals’, in strict sense, refers to the metallic elements
which have an atomic density higher than 6gm/cm3 (Akpore et al., 2010)
and which are able to forms sulphide (Adrino, 1986). The metals with
atomic number 23 onwards (except R, Sr, Y, Cs, Ba, and Fr) are generally
referred to as heavy metals. About forty elements fall into this category.
Though a number of heavy metals are essential micronutrients for both
plants and animals (Eichenberger, 1986; Wintz et al., 2002), these are
potentially toxic at elevated concentrations (Gadd and White, 1989). Al, Cr,
Mn, Fe, Co, Ni, Cu, Zn, Cd, Hg and Pb are believed to be the metals of
immediate concern to the mankind specially with respect to pollution
caused by them more specially due to their discharge into the aquatic
environment (Son et al., 2004). Heavy metals, once released into the soil
matrix, also find their way into the food web through ground water aquifers
(Walton, 1995; Athar and Ahmad, 2002).
In a natural undisturbed ecosystem, the primary source of most heavy
metals is the underlying bedrock (Peterson, 1978; Adrino, 1986) or surface
material transported via atmosphere from another location (Davidson et al.,
1985). During weathering of bedrock, heavy metals from the parent
material are incorporated into nearly forming soil. Emissions of heavy
metal as particulates and gases from volcanoes, forest fires and dust have
always been a natural input to soils and ecosystems (Salmons and Forstner,
1984). However, during the last 5000 years, human-related emissions of
heavy metals have become increasingly important (Settle Patterson, 1980).
Modern agricultural ecosystems have been dependent upon a substantial
input of a variety of agrochemicals many of which contain heavy metals.
Fertilizers applied over last century have added appreciable quantities of
Cd, Cr, Ni, and Zn to the soilo (Jones et al., 1988). Mining, smelting,
printing, battery-manufacturing, electroplating, tanning etc. are some of
the
other
examples
of
anthropogenic
activities
leading
to
high
concentrations of these metals. In fact, any industrial activity involving
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metals might lead to deposition of metals in the environment which, in the
long run, cause serious threat to the environment (Britto and Geetha, 1997;
Lata et al., 2005). These generate aqueous effluents containing metal
pollutants the concentration of which need to be reduced to the levels so
that these do not cause much harm to the environment (Joseph, 1995;
Lovely and Covas, 1997). Many agricultural and industrial activities have
resulted in the contamination of large areas across the world (Smith et al.,
1996; Shallari et al., 1998; Herawati et al., 2000). Even if these metals are
present
in
minute
undetectable
quantities,
these
ultimately
get
biomagnified to toxic concentration through the food chain (Smith et al.,
1996; Herawati et al., 2000). Once these reach the soil matrix, these also
affect soil microbial populations, which are important for biogeochemical
cycle and plant growth. Almost every aspect of microbial metabolism and
activity including primary productivity, methanogenesis, N 2-fixation,
respiration, motility, mineralization, decomposition and enzyme synthesis
are affected (Gadd, 1992; Rani et al., 2008).
The harmful effects of metal ions can be placed in five categories depending
upon the molecular mechanisms of their toxicity (Ochiai, 1987). These
include: (a) displacement of essential metal ions from biomolecules and
other biologically functionally units; (b) blockage of essential functional
groups of biomolecules, including enzymes and polynucleotides; (c)
modification of active conformation of biomolecules, especially enzymes of
polynucleotides; (d) disruption of the integrity of biomolecules; (e)
modification of some other biologically active agents.
A number of factories possess their own wastewater treatment plants but
quite often the effluents are discharged into the water bodies without any
treatment. Tightening government legislations forcing industries to treat
waste effluents before releasing these into the environment. Several
strategies are currently available to remove metals from the industrial
effluents. The environmental techniques for the removal of metals from
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industrial effluents include precipitation, ion-exchange and electrolytic
techniques (Blanco et al., 1999; Hai et al., 2007). Small and medium scale
industries in India usually do not keep sufficient budget of these and are,
therefore, the major sources of pollution because of two reasons: (a) use of
obsolete technologies in manufacturing processes; (b) lack of financial
ability to invest on conventional waste water treatment systems which are
quite expensive (Horsefall et al., 2003; Vieira, 2000; Volesky, 2006; Crini et
al., 2007).
Chemical precipitation of metals is achieved by the addition of coagulants
such as alum, lime, iron salts and other organic polymers. However, a large
amount of sludge containing toxic compounds are produced during the
process. In ion-exchange process, metal ions from dilute solutions are
exchanged with ions held by electrostatic forces on the exchange resin. This
process has two major disadvantages: (i) high cost, and (ii) partial removal
of certain ions (Vijayraghvan et al., 2008). In electrolysis, with the help of
semi-permeable ion-selective membranes, the heavy metals are separated
out. Application of electrical potential between the two electrodes causes a
migration of cations and anions towards respective electrodes; but results in
the formation of metal hydroxides which clog the membrane.
In 1990s, a new scientific area of biosorption has come into limelight with
the objective of recovering heavy metals from metal-containing effluents. It
is based on the metal binding capacities of various biological materials.
Biosorption techniques hold great promise for effective use in the effluent
treatment processes mainly for the heavy metals (Carpentor and Price,
1976; Borstad et al., 1992). Several studies have revealed that non-living
and living biomass can be used effectively for removing metals from the
environment (Lujan et al., 1994; Sood et al., 1994; Mofa, 1995; GardeaTorresday et al., 1996; Ingole and Bhole, 2000; Santosh et al., 2002; Horsfall
et al., 2003; Mahvi et al., 2005). A number of workers I the past had
advocated the use of microorganisms for accumulating heavy metals and
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radionuclides from their external environment (Gadd and Griffiths, 1978;
Macaskie and Dean, 1989, 1990; Volesky, 1990; Birch and Bachofen, 1990;
Avery et al., 1991; Gadd, 1992; Hemapriya et al., 2010; Can and Jialong,
2010). It is possible to develop commercial products with microbes as a
major component for managing the heavy metals contamination. The
biological materials, mainly yeast and fungi, are major candidates for the
development of such devices which may be called biotraps (Crusberg, 2004).
The biotraps can be categorized into three types on the basis of functions(A) Biosorption or the process by which individual cell or cell component bind or take
up metals- (a) adsorption of ions onto a surface including ion-exchange and
complexing with ligands; (b) the uptake into the cell and sequestration by
cytoplasmic components;
(B) transformation by oxidation or reduction into a less toxic or volatile form;
(C) precipitation by forming insoluble salts.
The major advantages of biosorption over conventional treatment methods include:
(i) low cost, (ii) high efficiency, (iii) minimization of chemical or biological sludge,
(iv) no additional nutrient requirement, (v) regeneration of biosorbent, and (vi)
possibility of metal recovery (Kratochvil and Volesky, 1998).
The application of fungal biomass as adsorbent or ion-exchanger for the removal of
heavy metals from polluted environment has opened a novel area of biotechnology
(Siegel et al., 1990). This potential of fungi had been recognized much earlier
(Shumate et al., 1978), as is already implicit in the literature concerning the
biogeochemistry of heavy metals (Purner and Siegel, 1978).
The ability to accumulate micronutrients in high parts per million (ppm) ranges in
common fungal attribute (Standburg et al., 1981; Gadd, 1986). By virtue of their
aggressive growth, greater biomass production and extensive hyphal reach in the
environment, and high surface : cell ratio of the filaments are likely to perform
better than bacteria.
The various aspects of the toxicity of the heavy metals as also trapping and
biosorption of heavy metals by the fungi have been reviewed by a number of
workers including Tien and Kirk (1983), Bumpus et al. (1988), Aust,
(1990),
Akamatsu et al. (1990), Volesky and Holan (1995), Blackwell et al. (2002), Eisler
(2003), Lacina et al. (2003), Gaur and Adholeya (2004), Ramteke (2003), Sharma
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(2005), Bellion et al. (2006), Fomina et al. (2005, 2006), Swami and Buddhi (2006),
Chander et al. (2007), Duribe et al. (2007), Zapotozny et al. (2007), Das et al.
(2008), Wang and Chen (2009) and Lesmano et al. (2009), Sutherland and
Venkobachar (2010), Javaid et al. (2010), Pan et al. (2010), Ramasamy et al. (2011),
Achal et al. (2011), Pocsi (2011), Marandi (2011), Hemambika et al. (2011),
Dhankhar et al. (2011), Ahmad et al. (2011), Naja and Volesky (2011), Javaid et al.
(2011) and Liu et al. (2011).
The ability of the microbes to grow and survive under high metal concentrations is
well documented (Sen, 2008; Tiwari, 2010). It is attributed to stress-induced
selection of these microbes through molecular mechanisms. Metal microbe
interactions leading to the removal of metals from solutions is based on
mechanisms ranging from purely physiochemical interactions such as adsorption
to cell wall or other components (Bhagat and Srivastava, 1993; Taniguchi et al.,
2000); or to the mechanisms that rely upon cellular metabolism (such as
intracellular compartmentation and extracellular precipitation). Some of these
mechanisms confer upon the microbes the capacity to remove, immobilize or
detoxify metals and radionuclides (Gadd, 1990; Ji and Silver, 1995; Srivastava,
2000). While a lot of emphasis has been laid down in the recent years on
metabolism-independent biosorption using non-living biomass, a number of
workers believe that the novel capabilities of microbes and their products in the
removal of hazardous metals from industrial effluents and waste water might also
be immensely useful in mitigating metal pollution etc. It is hoped that some of the
physiological processes and genetic adaptations which protect organisms against
toxic metals and other inorganic contaminants can be identified, and it might be
possible to exploit these for the removal and recovery of these metals from aqueous
water streams (Ow, 1997; Nies, 1999; Crusberg, 1996, 2004). Thus, attempts at
isolation of metal-resistant microbes (including fungal strains) from natural
resources could yield some strains more efficient in metal removal as compared to
those available at present. The selection of metal-resistant microbes, therefore, is
useful especially due to the following reasons: (i) a resistant strain would thrive
and grow under metal stress conditions; (ii) the performance of a microbe is always
dependent upon its genetic make up; and (iii) any other types of biosorptive
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pathway in addition to adsorption may also operate in a resistant strain
(Srivastava et al., 2002).
A number of metal-resistant fungal strains have been isolated from soils
contaminated by mine draining (Tatsuyama et al., 1975). Various studies have
revealed that fungi are more resistant than bacteria to long-term heavy metal
contamination
(Baath,
1991;
Fliesbach
et
al.,
1994;
Frostegard
et
al.,
1996).Concentrations of heavy metals which could cause severe toxicity in higher
plants commonly occur in fungi without causing ill-effect. This may indicate a
greater toxic threshold in the fungi. The above discussed properties of fungi have
led to the innovation of a number of biotechnological applications. These include
the use of microbes for remediation of toxic metals and synthesis of metal
nanoparticles. A comparative assessment of living and non-living biomass with
respect to their capacities to remove metals from aquatic solutions would be
helpful in framing strategies in this direction.
Another interesting aspect of the biosorption process is that some serious
treatment regimen may drastically increase the potential of a given biomass to
remove
metals.
Physical
pretreatment
methods
include
heat
treatment,
autoclaving, freeze-drying and boiling. Chemical pretreatment methods such as
exposing fungal cells to acids, bases and organic chemicals have led to
enhancement of metal biosorption by fungal biomass (Huang et al., 1988; Kapoor
and Viraraghavan, 1997, 1998; Yan and Viraraghavan, 2001; Bai and Abraham,
2002; Loukidou et al., 2003; Ilhan et al., 2004; Wilke et al., 2006; Vijayaraghvan;
Yeoung-Sang, 2007; Alluri et al., 2007; Kumar, 2008; Javaid et al., 2010). An
increase in biosorption of metal ions as a result of pretreatment could be due to an
exposure of active metal binding sites embedded in the cell wall, or due to chemical
modifications of cell wall components or due to the removal of surface impurities
(Huang and Huang, 1996). However, the response to pretreatment varies with the
types of fungal biomass and metal (Brady et al., 1994; Zhao and Duncan, 1998).
Therefore, the effect of various physical and chemical pretreatments on the metal
biosorption capacity of fungal biomass also needs to be investigated extensively.
Beveridge and coworkers (1980, 1996, 2000) demonstrated that gold particles of
nano-scale dimensions are precipitated when bacterial cells are incubated with
Au+3 ions. Pseudomonas steiltizri, isolated from silver mine, when placed in
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concentrated aqueous solution of AgNO3, cause the reduction of Ag ions leading to
the formation of Ag nano-particles. Nair and Pradeep (2002) demonstrated that for
large-scale production of nanoparticles of a given metal it is not necessary to use
bacteria normally exposed to large concentration of that particular metal ions. A
number of workers have demonstrated that fungi are also extremely good
candidates for synthesis of metals and metal sulphide nano-particles (Sastry et al.,
2003). Verticillum spp. and Fusarium oxysporum exposed to aqueous Ag and Au
ions reduced the metal ion rapidly leading to the formation of the Au (Mukherjee et
al., 2001, 2002) and Ag (Mukherjee et al., 2002; Ahmed et al., 2003) nano-particles.
Fusarium oxysporum, when challenged with CdSO4
solution,
caused enzymatic
reduction of sulphate to sulphide ions leading to the formatin of CdS ions (Ahmad
et al., 2002). Thermonospora sp. (an actinomycete) is shown to be capable of
forming Au nano-particles extracellularly (Ahmed et al., 2003). Fusarium
oxysporum, when challenged to aqueous solutions of metal salts or a mixture of
metaln salts, resulted in extracellular formation of nanoparticles of dimensions 550 nm and alloy nanoparticles of dimensions 8-14 nm (Mukherjee et al., 2001,
2002; Ahmad et al., 2003; Sastry et al., 2003). Some workers demonstrated
extracellular production of gold, silver and bimetallic Au-Ag alloy nanoparticles by
Fusarium oxysporum (Mukherjee et al., 2002; Ahmad et al., 2003; Souza et al.,
2004; Senapati et al., 2005; Duran et al., 2005). Bhainsa and D’souza (2006)
reported fastest reduction of silver nitrate by Aspergillus fumigatus and produced
monodispersed silver nanoparticles of size 5−25 nm within 10 minutes. Vahabi et
al. (2011) demonstrated that Trichoderma reesei reduced toxic Ag+ ions to nontoxic metallic silver nanoparticles by catalytic effect on silver nitrate. The
production of nanoparticles by the fungi have been reviewed by a number of
workers including Medentsev and Alimenko (1998), Oksanen et al. (2000), Chen et
al. (2003), Souza et al. (2004), Duran et al. (2005), Senapati et al. (2005), Bhainsa
and D’Souza (2006), Vigneshwaran et al. (2007), Basavaraja et al. (2008), Balaji et
al. (2009), Verma et al. (2010), Naveen et al. (2010), Mansoori (2010), CastroLongoria et al. (2011) and Vahabi et al. (2011).
Many new techniques based upon vibrational spectroscopy are fast emerging as
promising approaches for obtaining a clearer picture of chemical functional groups
in cells. FT-IR spectrum, based-upon vibrations leading to change in the dipole
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moment, represents a fingerprint which is characteristic of a given chemical
substance (Guizer and Hisse, 1996). Since a given biological material is composed
of a specific set of chemical substance, it must have a characteristic fingerprint.
Absorption of light by cellular compounds results in a finger-like spectrum which
provides information regarding the molecular composition and structure as well as
molecular interaction in cells and tissues (Jackson and Mantsch, 1990; Wolthuis et
al., 1999). It would be interesting to know (a) the type of changes occurring in the
nature of fungal biomass as a result of treatment with different chemicals like
formaldehyde and gluteraldehyde, in term of changes in FT-IR spectra and; (b) to
work-out the relationship, if any, between the changes in the biomass composition
with the changes in the capacity of the biomass to adsorb metals.
In view of the above considerations, it was decided to work out the following
aspects of metal-fungal relationships:
(a) isolation and identification of fungal strains from soil samples− control as well
as those treated with different concentrations of salts of chromium and lead;
(b) assessment of the potential of selected metal−tolerant fungal species to adsorb
metals from aqueous solutions;
(c) effect of metal concentrations on the biosorption efficiency;
(d) effect
of
different
chemical
pretreatments
of
fungal
biomass
(with
gluteraldehyde and formaldehyde) on their efficiency to adsorb metals;
(e) changes in the FT-IR spectra of biomass of selected fungi as a result of
pretreatment with gluteraldehyde and formaldehyde;
(f) possible training of microbes for better biosorption of metals;
(g) an attempt was also made to synthesize silver nanoparticles using Apergillus
fumigatus and Aspergillus niger biomass.