INTRODUCTION

Environmental pollution has become a key focus of concern for all the nations worldwide, as not only the developing countries but developed nations as well are affected by and suffer from it. Pollution has many forms, the air we breathe, the water we drink, the ground where we cultivate our food crops and even the increasing noise we hear everyday-all contribute to health problems and lower quality of life. Among all the environmental pollutions, pollution of water resources is a matter of great concern. Poor and developing countries are at high risk due to lack of waste water treatment technologies. Increasing contamination of aquatic sources with large number of pollutants is not only endangering the aquatic biota but creating a worldwide shortage of recreational waters (Rai et al., 1998). The water of aquatic systems gets polluted by domestic activities, mining activities, municipal wastes, modern agricultural practices, marine dumping, radioactive wastes, oil spillage, underground storage leakages and industries. But the major culprits causing the pollution of water resources are different industrial units. Indiscriminate discharge of toxic chemicals through effluents from a wide range of industries (i.e. textile, steel, oil, tanneries, canneries, refineries, mines, fertilizers production units, detergent production units, electroplating units and sugar mills) into water bodies pollutes these resources and causes hazardous effects on flora and fauna (Singh and Singh, 2000; Gavrilescu, 2004; Iqbal and Edyvean, 2004; Akar and Tunali, 2005).

I.

HEAVY METAL POLLUTION Among all the pollutants, heavy metals are most dangerous one as these are non

biodegradable and persist in environment. These enter into the water resources through both natural and anthropogenic sources. More attention is being given to the potential health hazards posed by heavy metals. The term heavy metal refers to any metallic chemical element that has a relatively high density. The density of heavy metals is usually more than 5.0 g/cm3. Examples of heavy metals include mercury (Hg), cadmium (Cd), arsenic (As), chromium (Cr), thallium (Tl), lead (Pb), Copper (Cu), Zinc (Zn), Cobalt (Co), Nickel (Ni), and Iron (Fe). These metals are classified in to three categories: toxic metals (such as Hg, Cr, Pb, Zn, Cu, Ni, Cd, As, Co, Sn, etc), precious metals (such as Pd, Pt, Ag, Au, Ru etc.) and radionuclides (such as U, Th, Ra, Am, etc.) (Volesky, 1990; Bishop, 2002). Toxic metals cause toxicity to organisms even at ppm level of concentration.

Heavy metals are natural components of the earth's crust. To a small extent they enter our bodies via food, drinking water and air. As trace elements, some of these heavy metals (e.g. copper, selenium, zinc) are essential to maintain the metabolism of the human body. However, at higher concentrations they can lead to poisoning. Heavy metal poisoning could result from drinking-water contamination, high ambient air concentrations near emission sources, or intake via the food chain.

Heavy

metals

are

dangerous

because

these

tend

to

bioaccumulate.

Bioaccumulation means an increase in the concentration of a chemical in an organism over time, compared to its concentration in the environment. Compounds accumulate in

living systems when these are taken up and are stored faster than these are broken down (metabolized) or excreted.

Heavy metals may enter a water supply through industrial or consumer wastes releasing heavy metals into streams, lakes, rivers, and groundwater. Unlike organic pollutants, heavy metals, being non-biodegradable pose a different kind of challenge for remediation. A well known environmental disaster associated with heavy metals is the Minamata disease caused by Mercury pollution in Japan.

a) Sources of Heavy Metal Pollution

Heavy metal pollution mainly arises from the effluents of industrial units. Some of the common industrial units releasing toxic heavy metals into environment are listed in Table 1. Irrigation by effluents released from paper mills and fertilizer factories are adding various alkalies, ammonia, cyanides and heavy metals into the water resources (Singh, 1994; Fazeli et al., 1998). The waste water from the dyes and pigment industries, film and photography, galvanometry, metal cleaning, electroplating, leather and mining industries contains considerable amounts of heavy metal ions.

In contrast to herbicides, pesticides and other potential toxicants which undergo break down, albiet extremely slowly, heavy metals can not be eliminated from a water body and thus persist in sediments where these are slowly released in to the water.

Table 1: Common industrial units releasing heavy metals into water bodies.

Metal Chromium

Common sources Chrome plating, petroleum refining, electroplating industry, leather, tanning, textile manufacturing and pulp processing units. It exists in both hexavalent and trivalent forms.

Nickel Lead Copper Zinc

Galvanization, paint and powder, batteries processing units, metal refining and super phosphate fertilizers. Petrol based materials, pesticides, leaded gasoline, and mobile batteries. Electroplating industry, plastic industry, metal refining and industrial emissions. Rubber industries, paints, dyes, wood preservatives and ointments. Batteries, electroplating industries, phosphate fertilizers, detergents,

Cadmium

refined petroleum products, paint pigments, pesticides, galvanized pipes, plastics, polyvinyl and copper refineries.

Iron Aluminium Arsenic Mercury

From metal refining, engine parts. Industries preparing insulated wiring, ceramics, automotive parts, aluminum phosphate and pesticides. Automobile exhaust/industrial dust, wood preservatives and dyes. Electric/light bulb, wood preservatives, leather tanning, ointments, thermometers, adhesives and paints.

b)

Health Risks of Heavy Metals

At low concentrations some of the heavy metals stimulate some biological processes, but at threshold concentration these become toxic. Being nonbiodegradable, these metals accumulate at various trophic levels through food chain and can cause human health problems (He et al., 1998). In humans these metals accumulate in living tissues and thus multiply the danger. Some common harmful effects and health risks of some heavy metals to human beings are given in Table 2. The health risks of heavy metals ingestion thus are of wide range. Some metals cause physical discomfort while others may cause life-threatening illness, damage to vital body system, or cause other damages. Thus, it is very necessary to control emission of heavy metals into the environment.

II.

CONVENTIONAL REMOVAL

TECHNIQUES

USED

FOR

HEAVY

METAL

Several physico-chemical methods like chemical precipitation, electrodialysis, ion-exchange, ultra-filtration, reverse osmosis etc. are commonly employed for stripping toxic heavy metals from waste waters (Sandau et al., 1996b; Eccles, 1999; Mehta and Gaur, 2001a; Ahalya et al., 2003). Brief description of each method is presented below:

a) Reverse Osmosis

It is a process in which heavy metals are separated through a semi-permeable membrane at a pressure greater than osmotic pressure caused by the dissolved solids in wastewater. The disadvantage of this method is that it is expensive (Ozaki et al., 2002).

Table 2: Common health problems caused to humans by heavy metals.

Metal
Chromium

Health Risks
Irritant, nausea and vomiting, carcinogen, and cause ulceration. long-term exposure can cause kidney and liver damage, and damage to circulatory and nervous tissue. Dermatitis, chronic rhinitis, hypersensitivity reactions in the immune system

Nickel

which in turn cause hyper allergenic reactions to various substances, Long-term toxicity may lead to liver necrosis and carcinoma, myocardial infarction, respiratory illnesses such as asthma, heart and liver damage, skin irritation. Effects on the kidneys, gastrointestinal tract, joints and reproductive system, and acute or chronic damage to the nervous system, anemia, headache, fatigue,

Lead

weight loss, cognitive dysfunction and decreased coordination, memory loss, nerve conductions. The central nervous system is most sensitive to the effects of lead. Biliary obstruction (inability to excrete excess copper), liver disease, renal dysfunction, fibromyalgia symptoms, muscle and joint pains, depression,

Copper

chronic fatigue symptoms, irritability, tumor, anemia, learning disabilities and behavioral disorders, stuttering, insomnia, niacin deficiency, leukemia, high blood pressure.

Zinc

Nausea and vomiting. Hypertension or high blood pressure, dulled sense of smell, anemia, joint

Cadmium

soreness, hair loss, dry scaly skin, loss of appetite, decreased production of Tcells and, therefore, a weakened immune system, kidney diseases and liver damage, emphysema, cancer and shortened lifespan Gastrointestinal disturbance, fatigue, headache, poor calcium metabolism,

Aluminium

decreased liver and kidney function, forgetfulness, speech disturbances and memory loss, weak and aching muscles, seizures, vertigo and loss of balance Headache, confusion, drowsiness, convulsions, changes in fingernail

Arsenic

pigmentation, vomiting, diarrhea, bloody urine, muscle cramps, convulsions, gastrointestinal upsets and coma.

Mercury

Anxiety, depression, confusion, irritability, insecurity and fatigue.

b) Electrodialysis

In this process, the ionic components (heavy metals) are separated through the use of semi-permeable ion-selective membranes. Application of an electric potential between the two electrodes causes the migration of cations and anions towards respective electrodes. Because of the alternate spacing of cation and anion permeable membranes, cells of concentrated and dilute salts are formed. The disadvantage is the formation of metal hydroxides, which clog the membrane and thus cost involved is high (Mohammadi et al., 2005).

c) Ultrafiltration

This is a pressure driven membrane operation that uses porous membranes for the removal of heavy metals. The main disadvantage of this process is the generation of sludge.

d) Ion-exchange

In this process, metal ions from dilute solutions are exchanged with ions held by electrostatic forces on the exchange resin. The disadvantages include high cost and partial removal of certain ions.

e) Chemical Precipitation

Precipitation of metals is achieved by the addition of coagulants such as alum, lime, iron salts and other organic polymers. The large amount of sludge (containing toxic compounds) produced during the process is the main disadvantage.

f)

Phytoremediation

Phytoremediation is the use of certain plants to clean up soil, sediment, and water contaminated with metals. The disadvantages include that it takes a long time for removal of metals and the regeneration of the plant for further biosorption is difficult.

The above stated methods are effective for removal of metals from water with high concentration of metals while for low concentrations (ppm level) of metals these techniques are not very fruitful. These methods also have other several disadvantages, such as incomplete metal removal, limited tolerance to pH change, expensive equipment and monitoring system requirements, high reagent or energy requirements and generation of toxic sludge or other waste products that require disposal (Yan and Viraraghavan, 2001; Aksu et al., 2002; Gavrilescu, 2004; Alimohamadi et al., 2005; Wang and Chen, 2006). Further, these techniques may be ineffective or extremely expensive when metal concentration in waste water is in the range 1-100 ppm (Mehta and Gaur, 2005).

The Need for Novel Technology The increasing concern about the contamination of water bodies by heavy metals has stimulated a large number of researches to find possible ways to remove these toxic substances from the environment. To overcome some of the limitations of physicochemical treatments, there is a need for inexpensive and efficient technology for the treatment of metal containing wastes so that metal concentration can be reduced to environmentally acceptable levels (Wilde and Benemann, 1993; Sandau et al., 1996a; Aksu, 1998; Gavrilescu, 2004). Use of biomass for metal removal/recovery is considered to be a viable alternative to conventional methods.

III.

REMOVAL OF HEAVY METALS THROUGH BIOSORPTION

Biosorption of heavy metals is defined as the ability/use of the biological materials to remove heavy metals from wastewater through metabolically mediated or physico-chemical uptake of metal (Fourest and Roux, 1992). The most prominent features of biosorption are low cost and highly efficient materials to adsorb heavy metals even when present at very low concentrations (Yu et al., 2001).

a) Removal by Low Cost Sorbents and Industrial Wastes

The use of low cost sorbents has been investigated as a replacement for current costly methods of removing heavy metals from solutions. The application of anaerobic granular biomass (Hawari and Mulligan, 2006), peat biomass (Ma and Tobin, 2003), waste brewery biomass (Marques et al., 2000), husk of Bengal gram (Ahalya et al., 2005), rice husk and maize cobs (Abdel-Ghani et al., 2007), paper mill sludge (Battaglia et al., 2003), sewage sludge (Zhai et al., 2004), agro based waste materials (Qaiser et al., 2007), chitosan (Babel and Kurniawan, 2003), agarose (Pandey et al., 2007), crab shell and chitin (Barriada et al., 2007), rose waste biomass (Iftikhar et al.,2009), waste sludge (Slevaraj et al., 2003; Li et al., 2004), tea factory waste biomass (Malkoc et al., 2006) for heavy metal removal has been explored. But these materials are not easily available at all the places and all the time.

b)

Removal by Plant Biomass

Plant biomass has also been used by various workers for metal removal purposes (Cimina et al., 2000; Elifantz and Tel-Or, 2002; Li et al., 2004; Shekhar et al., 2004;

Sarin and Pant, 2006; Ahluwalia and Goyal, 2007; Bhatti et al., 2007; Zubair et al., 2008, Anand Kumar and Mandal, 2009; Romero-Gonzalez et al., 2009).

c)

Removal by Microorganisms

The ability of microbial biomass to remove heavy metal ions from polluted aquatic systems has been reported and has also attracted much interest in recent years. Microorganisms which have been tested for biosorption of heavy metals include bacteria (Scott and Palmer, 1990; Chang et al., 1997; Nurba et al., 2002; Srinath and Verma, 2002; Ilhan et al., 2004; Selatnia et al., 2004; Iyer et al., 2005; Shaker and Hussein, 2005; Elangovan et al., 2006; Srivastava and Thakur, 2007; Rehman et al., 2008; Rajkumar et al., 2009), Yeast (Huang et al., 1990; Volesky et al., 1993; Goksungur et al., 2005; Seki et al., 2005; Wang and Chen, 2006; Chergui et al., 2007; Ashwini et al., 2009), fungi (Lewis and Kriff, 1988; Fourest et al., 1994; Sudha and Abraham, 2001; Dursun et al., 2003; Ahmet et al., 2005; Park et al., 2005; Pal et al., 2006; Tunali et al., 2006; Liu et al., 2007; Coreno-Alonso et al., 2009; Khambhaty et al., 2009) and algae (Gupta et al., 2001; Donmez and Aksu, 2002; Terry and Stone, 2002; Pagnanelli et. al., 2003; Bishnoi and Garima, 2004; Yun, 2004; Loderio et al., 2005; Tuzun et al., 2005; Vijayaraghavan et al., 2005, 2006; Vilar et al., 2005; Bishnoi et al., 2007; Chojnacka, 2007; Khattar et al., 2007; Doshi et al., 2008; Deng et al., 2009; Gupta and Rastogi, 2009; Gupta et al., 2010; Lodi et al., 2010) etc. These biosorbents possess metal sequestering properties and decrease the concentration of heavy metal ions in solution from ppm to ppb level. They can effectively sequester dissolved metal ions out of dilute complex solutions quickly and with high efficiency. Therefore biosorption involving microorganisms is an ideal technique for the treatment of high volume and low metal concentration complex waste waters.

d)

Removal by Algae Of all the microbes, algae are able to take up, accumulate and concentrate heavy

metals in significant amounts from the aqueous solution. Algae have also been considered to be potential biosorbents because of their easy handling, cheap availability, relatively high surface area and high binding affinity. Microalgae are often preferred for bioremediation process owing to their high photosynthetic efficiency coupled with simple nutritional requirements and these can be easily cultured and grown rapidly in both industrial and laboratory circumstances (Radway et al., 2001; Tien et al., 2005; Khattar et al., 2007). Many studies have demonstrated metal biosorption by seaweeds such as Sargassum sp. (Esteves et al., 2000; Cruz et al., 2004; de Franca et al., 2006; Vijayaraghavan et al., 2006; Sheng et al., 2007; Pahlavanzadeh et al., 2010), Laminaria sp. (Yin et al., 2001; Hashim and Chu, 2004; Lodeiro et al., 2005; Luo et al., 2006) and Fucus sp. (Herrero et al., 2006), Ulva (Sheng et al., 2004 a, b; Suzuki et al., 2005; Vijayaraghavan et al., 2005; Kumar et al., 2006), cyanobacteria such as Synechococcus sp. (Gardea-Torresdey et al., 1998; Satoh et al., 2005), Spirulina sp. (Chojnacka et al., 2004; Rangsayatorn et al., 2004; Gong et al., 2005; Doshi et al., 2007), Nostoc sp. (Prashad and Pandey, 2000; El-Sheekh et al., 2005), Lyngbya sp. (Klimmek et al., 2001; Lee et al., 2004; Kiran and Kaushik, 2008), Oscillatoria sp. (Mohapatra and Gupta 2005), Phormidium sp. (Sadettin and Donmez, 2007), Anacystis sp. (Khattar et al., 2002, 2007). Biosorption by green algae such as Chlorella sp. (Aksu and Acikel, 2000; Mehta et al., 2002; Fraile et al., 2005; Doshi et al., 2008), Cladophora sp. (Ozer et al., 1994,

2004; Deng et al., 2006), Eklonia sp. (Park et al., 2004), Chaetophora sp. (Andrade et al., 2005), Sphaeroplea (Rao et al., 2005) has also been reported.

The major advantages of biosorption over conventional treatment methods include (Kratochvil and Volesky, 1998):

Low operating cost High efficiency Minimization of chemical and biological sludge No additional nutrient requirement Regeneration of biosorbent Possibility of valuable metal recovery Successful operation over a wide range of pH and temperature Resins are hard ligands and are less effective in adsorbing minute quantities of metals when compared to soft ligands of biological origin

Microbial biomass required for biosorption may be available as a fermentation waste product or specifically grown one, using cheap substrates

Biosorption processes may serve as polishing system to existing processes

All these studies have demonstrated that algae have enormous capacity to bind/accumulate heavy metals. Thus, algae can hopefully be exploited for removal of heavy metal ions from polluted waste waters and industrial effluents to get fruitful results. Biosorption capacities of some algae are summarized in Table 3.

Table 3: Reports on heavy metal removal potential of some algae. Sorption (mg g1) 75.28 0.08 15.32 0.01 45.97 12.48 146.12 125.89 41.59 123.64 84.30 37.09 11.24 112.40 120.27 85.42 7.19 9.62 5.41 54.60 31.72 89.02 190.62 76.76 420.63 94.0 249.71

Metal Al

Algae Laminaria japonica Spirulina sp.

Reference Lee et al. (2004) Chojnacka et al. (2004) Mohapatra and Gupta (2005) Chojnacka et al. (2004) Vijayaraghavan et al. (2005) Sandau et al. (1996) Yin et al. (2001) Lee et al. (2004) Klimmek et al. (2001) Ofer et al. (2003) Sheng et al. (2004b) Rangsayatorn et al. (2004) Chojnacka et al. (2004) Ofer et al. (2003) Cruz et al. (2004) Sheng et al. (2004b) Gardea-Torresdey et al. (1998) Chojnacka et al. (2005) Gardea-Torresdey et al. (1998) Sheng et al. (2004b) Sheng et al. (2004b) Mehta and Gaur (2001a) Mehta and Gaur (2001b) Mehta and Gaur (2001c) Mehta et al. (2002) Feng and Aldrich (2004) Pradhan et al. (1998)

Co

Oscillatoria angustissima Spirulina sp. Ulva reticulata

Cd

Chlorella vulgaris L. japonica L. japonica Lyngbya taylorii Padina pavonia Padina sp. Spirulina platensis Spirulina sp. S. vulgaris Sargassum sp. Sargassum sp. Synechococcus sp. PCC 7942

Cr (III) Spirulina sp. Synechococcus sp. PCC 7942 Cr (VI Padina sp. Sargassum sp. Cu C. vulgaris C. vulgaris C. vulgaris C. vulgaris ( acid pretreated E. maxima Microcystis aeruginosa

Oscillatoria angustissima Sargassum sp. Spirulina sp. Synechococcus sp. PCC 7942 Ni Chlorella miniata C. sorokiniana C. vulgaris C. vulgaris C. vulgaris (Acid pretreated) Lyngbya taylorii Microcystis aeruginosa Scenedesmus obliquus Spirulina sp. Synechococcus sp. PCC 7942 Ulva reticulata Laminaria japonica Lyngbya taylorii Spirulina platensis Spirulina sp. Synechococcus sp. PCC 7942 Zn C. vulgaris Laminaria japonica Lyngbya taylorii Microcystis sp. Oscillatoria anguistissima O. anguistissima Spirulina sp.

7.63 45.75 12.45 11.31 0.04 0.12 23.48 205.48 437.98 38.16 249.98 30.18 0.18 3.17 46.51 349.09 304.56 16.98 0.01 30.45 0.26 56.87 32.03 632.12 21.57 641.28 0.20

Mohapatra and Gupta (2005) Chaisuksant (2003) Chojnacka et al. (2005) Gardea-Torresdey et al. (1998) Chong et al. (2000) Chong et al. (2000) Mehta and Gaur (2001a) Mehta and Gaur (2001b) Mehta et al. (2002) Klimmek et al. (2001) Pradhan et al. (1998) Donmenz et al. (1999) Chojnacka et al. (2004) Gardea-Torresdey et al. (1998) Vijayaraghavan et al. (2005) Lee et al. (2004) Klimmek et al. (2001) Sandau et al. (1996) Chojnacka et al. (2004) Gardea-Torresdey et al. (1998) Chong et al. (2000) Lee et al. (2004) Klimmek et al. (2001) Singh et al. (1998) Mohapatra and Gupta (2005) Ahuja et al. (1999) Chojnacka et al. (2004)

IV.

METAL TOLERANCE

Presence of some organisms in metal contaminated waters leads to a conclusion that somehow, these organisms are able to resist against metal toxicity. Several microorganisms are able to grow in presence of toxic heavy metals by keeping intracellular concentrations of toxic forms of metals well below the lethal threshold levels. Baker (1981) suggested two basic strategies of tolerance to metals a) b) Metal exclusion, whereby metal uptake and transport is restricted and Metal accumulation, when there is no such restriction then metals are accumulated in the cells in a detoxified form. Several possible mechanisms have been suggested for metal tolerance. These are: i) Adsorption or metal binding to the surface of cell wall (Lee et al., 2000; Mehta and Gaur, 2005). ii) Intracellular compartmentalization of metals in vacuole, cell walls, polyphosphate bodies (Sufia et al., 1999). iii) iv) v) Efflux of heavy metals (Rouch et al., 1989; Nies, 1999, 2003). Reduced transport (Baker, 1981). Chelation i.e. synthesis of metallothioneins and phytochelatins (Gekeler et al., 1988; Verma and Singh, 1991; Singh et al., 1992; Gardea Torresdey et al., 1998; Marijana and Raspor, 1998). vi) Secretion of extracellular exudates e.g. polysaccharides and siderophores of algae also have good metal binding capacity (Plude et al., 1991; Decho and Herndl, 1995; De Philippis et al., 2001, 2003, 2007). Siderophores are iron chelating compounds released by the microorganisms. In cyanobacteria,

siderophores are produced as defensive mechanism against heavy metal pollution. Metal uptake event includes i) A passive (rapid uptake) mechanism, where cell surface of a microorganism functions as an ion exchange site for metal cations. ii) Active intracellular transport mechanism (slower uptake).

Sandau et al. (1996a) have suggested following types of extracellular sorptive mechanisms: a) Electrostatic interactions of positively charged heavy metal ions with negatively charged groups/ligands of the cell walls (adsorption, ion exchange) b) c) d) e) Microprecipitation Surface complexing Covalent bonds between heavy metals cations and proteins Binding to other polymers

Various intracellular accumulative mechanisms have also been suggested by Sandau et al. (1996a). These include: i. Complexation of the accumulated heavy metal ions and reduction in toxicity of metal concentrations by several species-specific processes (e.g. phytochelatin reactions, hypertrophying and multiplication of the polyphosphate bodies) ii. Heavy metal incorporation into vacuoles (demobilization and concentration)

iii. Binding to proteins, lipids, DNA etc. iv. Efflux of heavy metal complexed substances

v.

Membrane reinforcement of cell walls, mitochondria, chloroplasts and nuclei as heavy metal barriers in adapted cells

These mechanisms, thus provide tolerance to microbes, including algae and cyanobacteria, towards toxic heavy metal ions. Due to this resistance/tolerance towards heavy metals, these organisms can be used as indicators and scavengers of heavy metal pollution (Bilgrammi et al., 1996). Thus, due to significant characteristics of microorganisms making them tolerant to metal toxicity, various microorganisms can be exploited to solve the world wide problem of heavy metal pollution.

V.

CLASSIFICATION OF METAL BINDING MECHANISMS

Metal biosorption mechanisms vary as there are many ways for the metals to be taken up by the microbial cells. These mechanisms may be classified following various criteria. Depending upon whether cellular metabolism is involved or not, biosorption mechanism can be divided in to

i) ii)

Non- metabolism dependent and Metabolism dependent

During the passive uptake, metal ions are adsorbed on to the cell surface within a relatively short span of time (few seconds or minutes) and the process is metabolism independent. In this type of biosorption, metal uptake is through physico-chemical interaction between the metal ions and the functional groups present on the microbial cell surface. This is based on physical adsorption, ion exchange and chemical sorption, which is not dependent on the cellular metabolism.

An initial faster (passive) uptake is followed by a much slower (active) uptake (Bates et al., 1982; Mehta and Gaur, 2005). Active uptake is metabolism dependent, causing the transport of metal ions across the cell membrane into the cytoplasm for intracellular accumulation. This means that this kind of biosorption is possible only with viable cells. It is often associated with an active defense system of microorganisms, which is stimulated in the presence of toxic metals. According to the location where the metal ions are located after their removal from solution, biosorption can be classified as i) ii) Extracellular accumulation/ Cell surface sorption Intracellular accumulation Volesky (1990) suggested following metal binding mechanisms which are involved in biosorption process:i) ii) iii) Chemisorption by ion exchange, complexation, coordination and chelation Physical adsorption Micro- precipitation These mechanisms may be acting simultaneously to varying degrees depending on the biosorbent and metal solution in the environment. A systematic presentation of the relationships between different mechanisms is compiled in Fig. 1. The classification of bond type (Myers, 1991) was used.

Ion Exchange

Adsorption

Micro precipitation

Sorbate/Sorbent

Metal/legand

Solute/Solvent

Complexation

Chemical forces

Physical forces

Covalent Electrostatic ionion ion -dipole London-vander waals dipoledipole-induced dipole London dispersion

Figure 1: Metal Biosorption mechanisms: bold lines, mechanism probably important in biosorption; dashed lines, biosorption binding relations of secondary importance.

Binding Sites Biosorption of metals has been attributed to presence of different types of groups on the cell. The surface of algal cell wall is composed of macromolecules having an abundance of charged functional groups, such as hydroxyl, phosphoryl, amino, carboxyl, sulphydryl, amine, imidazole, sulphate, phosphate, carbohydrate etc. Whether any given group is important for biosorption of a particular metal ion by a certain biomass depends on factors such as: Quantity of sites in the biosorbent material Accessibility of the sites Chemical state of the site, i.e. availability Affinity between site and metal, i.e. binding strength

Usually, the net charge on cell surface is negative because of the abundance of carboxylate and phosphate residues (Hamdy, 2000; Andrade et al., 2005; Gong et al., 2005; Mehta and Gaur, 2005; Apiratikul and Pavasant, 2006; Singh et al., 2007). Since metal ions in water are generally in cationic form, thus, these are passively adsorbed onto the cell surface. However, because of the presence of amine and imidazole groups, which are positively charged when protonated, cell surface may also bind negatively charged metal complexes. Some binding sites are also present inside the algal cells. Here intracellular accumulation takes place by binding with cytoplasmic ligands, phytochelatins, metallothioneins and other intracellular molecules. Thus, algal cells contain many polyfunctional metal-binding sites for both cationic and anionic metal complexes. Figure 2 shows the probable sites in an algal cell for the binding of metal ions.

Each functional group has specific pKa (dissociation constant) (Niu and Volesky, 2000; Volesky, 2007) and it dissociates into corresponding anions and protons at a specific pH. Structural formulae and pKa values of the binding groups are summarized in Table 4.

VI.

ADSORPTION ISOTHERMS

Biosorption has been studied as a simplified sorption system, usually containing one heavy metal. This is an appropriate simplification for effective experimentation. In order to evaluate feasibility and effectiveness of biosorption in waste water treatment, it is essential to make predictions of the sorption performance (e.g., for facilitating process design). Extensive studies have also been carried out on biosorption and its dependence on solution chemistry, ionic competition by other metals, influence of pH and ionic concentration (Bai and Abraham, 2002). Therefore, it is necessary to develop appropriate mathematical models of biosorption. Modeling the biosorption-binding equilibrium is a pre-requisite for understanding and evaluating the feasibility of the biosorption process. Different adsorption isotherms have been used to quantify and contrast between the performances of different biosorbents (Davis et al., 2003). Adsorption isotherms are the way of presentations of amount of solute adsorbed per unit of adsorbent.

The amount of metal M (sorbate) bound per mass of sorbent is called the uptake (qe). The binding is not only dependent on the sorbent material but also on the equilibrium concentration (Ce) of the sorbate in the solution and on other parameters such as pH and equilibrium concentration of other ions in the solution. The relationship between equilibrium binding and the concentration of ions (at constant temperature) is

Table 4: Major functional groups present on the surface of algal cell wall and involved in metal binding process.

Binding group Hydroxyl Carbonyl (ketone) Carboxyl

Structural formula -OH >C=O -C=O OH -SH O -S=O O >S -NH2 >NH - C=O NH2 =NH -C-N-H <CH H-C-N OH -P=O OH > P=O OH -OH

pKa 9.5-13 1.7 - 4.7

Ligand atom O O O

Sulfydryl (thiol) Sulfonate

8.3-10.8 1.3

S O

Thioether Amine Secondary amine Amide

8-11 13 -

S N N N

Imine Imidazole

11.6-12.6 6.0

N N

Phosphonate

0.9-2.1 6.1-6.8

Phosphodiester

1.5

Phenolic

10

depicted in an isotherm plot of qe versus Ce. With increasing metal concentration in solution its binding increases from zero to the maximum. It is desirable that the sorbent should possess a high sorption capacity and high affinity for the sorbate species, which is reflected in a steep slope of the isotherm curve at low equilibrium concentrations. Table 5 summarizes some of the simple sorption isotherm models that are most frequently applied. A particular model may not apply to a particular situation, and in some cases more than one model may explain the biosorption mechanism. There is no critical reason to use a more complex model if a two-parameter model (such as the Langmuir and Freundlich isotherm models) can fit the data reasonably well.

The field of biosorption is challenging one. The main objectives are the elucidation of binding mechanisms, the relative affinity of heavy metals for the biomass and how both are affected by varying environmental conditions. Ultimately, the goal is the successful implementation of a remediation programme. The model used to describe the results should be capable of predicting heavy metal binding at both low and high concentrations, ideally a model should not only be predictive but should rest on our understanding of the mechanism of biosorption (Davis et al., 2003). Among all isotherms, the Langmuir and Freundlich models are most frequently used to describe metal biosorption (Ledin, 2000).

a) Langmuir Isotherm (Langmuir, 1918)

Langmuir adsorption isotherm has traditionally been used to quantify and compare the performance of different biosorbents. However, in order to evaluate the appropriateness of this model, we must look at its underlying assumptions. The Langmuir

Table 5: Adsorption models used frequently for metal removal processes. Model Langmuir Equation Advantages Interpretable parameters Disadvantages Not Structured: monolayer sorption

Freundlich Simple expression Not Structured

Combination of Langmuir and Freundlich Radke and Prausnitz

Combination of above two

Unnecessarily complicated

Simple expression

Empirical; Requires three parameters.

Radlich Peterson

Approaches at higher concentration

No significant advantages

Where: qe = Amount of metal adsorbed (experimental value) Ce = residual concentration of metal qmax =Maximum amount of metal ions that can be adsorbed (theoretical value) b = Biosorption affinity

Kf = Freundlich isotherm constant related to adsorption capacity of biomass 1/n = Freundlich isotherm constant related to adsorption intensity Cf = Final concentration of metal = Metal sorption ability of biomass a = Model constant

model assumes: (i) a monolayer adsorption of metals on binding sites; (ii) all adsorption species (metal ions) interact only with a site and not with each other; and (iii) adsorption energy of all the sites is identical and independent of the presence of adsorbed species on neighbouring sites. The Langmuir model shows a good fit where passive sorption of metals on biosorbents prevails (Aksu, 1998). An important point to be noted here is that most of the above assumptions are not fulfilled in biosorption. Opposite to Langmuir assumptions, biological surfaces have more than one type of binding sites contributing to biosorption process, each of which may have different affinity for sorbing heavy metal ions. This isotherm represents one of the first theoretical treatments of nonlinear sorption and suggests that uptake occurs on a homogeneous surface by monolayer sorption with interaction between adsorption molecules.

b) Freundlich Isotherm (Freundlich, 1907)

The Freundlich isotherm is empirical and describes multi-layered adsorption of metal ions on sorbent surface. In metal biosorption studies, the Freundlich model describes metal sorption as a function of metal concentration in solution at equilibrium, without reference to pH or other ions in the same aqueous system. The Freundlich model provides a more realistic description of metal adsorption by organic matter because it accounts for sorption to heterogeneous surfaces or surfaces supporting sites of varied affinity. In contrast to Langmuir model, the Freundlich model does not assume saturation of metal sorption. The Freundlich model assumes that stronger binding sites on the biosorbent surface are occupied first and that the binding strength decreases with increasing degree of site occupation by metal ions. For fitting the model to experimental

data, the Freundlich model generally gives a better fit for higher equilibrium concentration of metal in solution.

c) Frequently Used Adsorption Models

Frequently used single-component adsorption models, their advantages and disadvantages (Kuyucak and Volesky, 1989) are given in Table 5. From the literature survey, it is clear that work has been done on different aspects of heavy metals in relation to algae. Metal tolerant algal species which adsorb, accumulate or chelate metal ions can be exploited to solve the problem of heavy metal pollution. Metal uptake studies have been conducted mainly employing laboratory grown algal species, using single metal ions. There are not much studies which employed algal species, naturally growing in polluted water to remove multimetal ions from solution. Since industrial effluents may contain more than one metal ion, and the algae growing in metal polluted water may have higher biosorption potential, the present study on Evaluation of Heavy Metal Bioremediation Potential of Algae Growing in Polluted Water was undertaken so that algal species are identified for their exploitation to remove multimetal ions from industrial effluents before their discharge into water bodies. The study was aimed to evaluate the potentialities of algae growing in polluted waters to scavenge heavy metal ions. Three metals, Cu2+, Cd2+ and Ni2+ were chosen as these metals are released in abundance in the effluents of dye, electroplating and metal refining industries.