bacterial biosorbents for detoxification of heavy metals...

International Journal of Advances in Science and Technology,

Vol. 2, No.6, 2011

Bacterial Biosorbents for Detoxification of Heavy

Metals from Aqueous Solution: A Review

Rajesh Dhankhar1 and Rachna Bhateria Guriyan2

Department of Environmental Sciences, M.D.University, Rohtak-124001, India.

Abstract

Discharge of industrial wastewater contaminated with heavy metals is a severe socio-environmental

problem. Since, it is impossible to degrade toxic metals by any means, only way left for their treatment is

their concentration and safe disposal in the environment. Literature shows vast array of biomaterials

such as bacteria, algae, fungi and yeasts which could be employed for heavy metal removal and recovery

due to their excellent performance, low cost and large available quantities. Biosorption is a typically

metal-biomass interaction process involving binding of sorbate to sorbent of biological origin. The

industrial effluents contaminated with heavy metals are the reservoir of unusual bacteria which are well

adapted to their environment and possess various mechanisms to resist metal toxicity. Bacteria are

capable of accumulating metal ions due to metal attracting composition on their cell wall or via metal-

dependent intracellular metal uptake mechanisms. This review mainly focus on bacteria’s cellular

structure, biosorption performance, their mechanisms towards metal tolerance, modeling of biosorption

and industrial applicability. Thus this article reviews the current status of bacteria as biosorbent and

hopes to enlighten the future research.

Keywords: Biosorption, Biosorbents, bacteria, microorganisms, heavy metals.

Contents 1. Introduction

1.1 Biosorption and biosorbents

1.1.1 Bacteria as biosorbent

1.1.2 Bacterial cell wall structure

2. Mechanism of metal resistance by bacteria

3. Preparation of bacterial biosorbents

3.1 physically/ chemically modified biosorbents

3.2 Genetically modified biosorbents

4. Biosorbent immobilization

5. Commercial biosorbents

6. Instrumentation for Biosorption Research

7. Biosorption experimental procedures

7.1 Factors influencing batch biosorption

7.2 Biosorption isotherms

7.3 Batch modeling

7.3.1 Emperical modeling

7.3.2 Mechanistic modeling

8. Desorption and Regeneration

9. Continuous Biosorption

10. Scope and Future directions

11. References

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1. Introduction

Due to excessive contamination of the environment by several inorganic and organic compounds such as

heavy metals, fuels and petroleum industry, the focus of research has been shifted towards better

decontamination methods. One such group of contaminants of concern, which comes under the inorganic

diversion are metals. The presence of metal ions in final industrial effluents is extremely undesirable, as

they are toxic to both lower and higher organisms [1]. Since it is impossible to degrade those pollutants, the

only way to remove them from the environment is to exclude metals from cycling through their

concentration, with possible recovery and reuse [2]. This would also reduce the consumption of non

renewable resources [3]. Thus, metal as a kind of resource is becoming shortage and also brings about

serious environmental pollution, threatening human health and ecosystem [4]. There are various industries

which discharge heavy metals into the environment via their effluents such as metallurgy, iron and steel,

electrolysis, textiles, tanneries, fertilizers and pesticide industry etc but among these, the following four

appear as the main priority targets, particularly in the industrial world [5,6].

1. acid mine drainage (AMD)—associated with mining operations;

2. electroplating industry waste solutions (growth industry);

3. coal-based power generation (throughput of enormous quantities of coal);

4. nuclear power generation (uranium mining/processing and special waste

generation).

In this endeavour, microbial biomass has emerged as a solution. Due to selective pressure from the

metal in the growth environment, microorganisms have evolved various mechanisms to resist the heavy

metal stress [7]. Microoorganisms react with metals and minerals in natural and synthetic environments,

altering their physical and chemical state, with metals and minerals also able to affect microbial growth,

activity and survival [6]. Microorganisms are a feasible solution because they can achieve different

transformation and immobilization processes [8].

1.1 Biosorption and biosorbents

Biosorption is defined as the process of concentration of sorbate and a prefix ‗bio‘ means that the sorbent

is of biological origin, a surface of biological matrix [3]. Biosorption is a property of both living and dead

organisms (and their components) and has been heralded as a promising biotechnology for pollutant

removal from solution, and/or pollutant recovery, for a number of years, because of its efficiency,

simplicity, analogous operation to conventional ion exchange technology, and availability of biomass [9].

In biosorption, metabolic processes in living organisms may affect physico-chemical biosorption

mechanisms, as well as pollutant bioavailability, chemical speciation and accumulation or transformation

by metabolism-dependent properties [9]. Biosorbents are cheaper, more effective alternative for the

removal of metallic elements, especially heavy metals from aqueous solution [4]. These biosorbents

possess metal-sequestering property and can be used to decrease the concentration of heavy metal ions in

solution from ppm to ppb level [4]. Biological processes can be carried out in situ at the contaminated site

and they are usually environmentally benign (no secondary pollution) [1]. The limitations include first of

all a shorter life time of biosorbents when compared with conventional sorbents [9]. A large array of

biomaterials has been investigated as biosorbents for the removal of metals or organics extensively [4]. The

biosorbents can be classified as bacteria, fungi, yeast, algae, industrial wastes and other polyssacharide

materials etc [1]. The factors which influence biosorption performance include the type of the biomass (and

resulting the composition of cell wall, pH, temperature, presence of other competing ions (both cations and

anions) [10]. Biosorption is possible with both living and non-living biomass [11]. The metal ion uptake by

living and dead cells consists of two modes [12]. The first uptake mode involves the surface binding of

metal ions to cell wall and extracellular material. The second mode of metal uptake into the cell across the

cell membrane is dependent on the cell metabolism and is referred to as intracellular uptake, active uptake

or bioaccumulation. The first mode is common to metal adsorption by both living and dead cells; the

second mode, which is metabolism dependent, occurs in living cells. For living cells metal uptake is also

facilitated by the production of metal binding proteins also called as metallothioneins (MTs) or low

molecular weight cysteine-rich proteins. Bacteria and higher organisms have developed resistance

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mechanisms to toxic metals to make them innocuous. They respond to heavy metal stress using different

defence mechanisms like exclusion, compartmentalization, making complexes and synthesis of binding

proteins such as metallothioneins (MTs) or phytochelatins (PCs). These systems can be employed for

remediation of polluted waters and soils. Biosorption is a typical, reversible physic-chemical process,

involving binding of sorbate to sorbent of biological origin [3] (Fig. 1) In biosorption, metallic ions remain

at the cellular surface by different mechanisms [1].

For industrial application of biosorption, it is necessary to propose a mathematical description.

Kinetics of biosorption is usually described with pseudo-first or pseudo-second order model and the

equilibrium more frequently with Langmuir than Freundlich eqution, because a clear plateau can be

distinguished on the sorption isotherm [3].

1.1.1 Bacteria as biosorbent: Bacteria make excellent biosorbents because of their high surface-to-

volume ratios and a high content of potentially active chemosorption sites such as on teichoic acid in their

cell walls [13]. Bacteria were used as biosorbents because of their small size, their ubiquity, their ability to

grow under controlled conditions, and their resilience to a wide range of environmental situations [14].

Bacterial species such as Bacillus, Pseudomonas, Streptomyces, Escherichia, Micrococcus, etc, have been

tested for uptake metals or organics. List of bacterial biosorbents known for metal removal is shown in

Table 1. The most relevant work on true bacterial biosorption has been done by the Brierleys [50-52] who

took the metal biosorption concept all the way to the commercial stage [41]. Various advantages of using

dead cells can be summarized as:

● absence of toxicity limitations

● absence of requirements for growth media and nutrients in the feed solution

● easy absorbance and recovery of biosorbed metals

● easy regeneration and reuse of biomass

● possibility of easy immobilization of dead cells

● avoidance of sudden death of the biomass population

● easy mathematical modelling of metal uptake reactors.

1.1.2 Bacterial Cell wall structure: Bacteria are microscopic organisms whose single cells have neither a

membrane-bounded nucleus nor other membrane-bounded organelles like mitochondria and chloroplasts.

Bacteria are the most abundant and versatile of microorganisms and constitute a significant fraction of the

entire living terrestrial biomass of ~1018 g [53]. Bacteria can be found in a variety of shapes, which includes

cocci (such as streptococcus), rods (such as bacillus), spiral (such as rhodospirillum) and filamentous (such

as sphaerotilus). Bacteria can be classified as Gram-positive and Gram-negative bacteria based on the

chemical compostion of their cell wall. The Gram positive cell envelope consists of two functional layers: a

cytoplasmic membrane surrounded by a thick cell wall of peptidoglycan. Negatively charged teichoic acids

(acidic polysaccharides) and teichuronic acids are attached to the cell wall. Teichoic acids give the Gram-

positive cell wall an overall negative charge, due to the presence of phosphodiester bonds between the

teichoic acid monomers. Peptidoglycan carboxyl groups are the main binding site for metal cations in

Gram-positive bacterial cell walls with phosphate groups contributing significantly in Gram-negative

species [54-56]. Other bacterial metal-binding components include proteinaceous S-layers, and sheaths

largely composed of polymeric materials including proteins and polysaccharides [9]. In many bacteria,

initial binding is followed by inorganic deposition of increased amounts of metal which leads to the

accumulation of greater than stoichiometric amounts of metals, even to levels approaching 50 % of the dry

weight. Contrarily the cell wall of Gram negative bacteria contains an additional outer membrane

composed of phospholipids and lipopolysaccharides which confers an overall negative charge on gram

negative cell wall.

2. Mechanism of metal resistance by bacteria

Microbes have various types of resistance mechanisms in response to heavy metals. Metal resistance in

particular is defined as the ability of an organism to survive metal toxicity by means of a mechanism

produced in direct response to the metal species concerned. A particular organism may directly and/or

indirectly rely on several survival strategies. Synthesis of metallothioneins or c-glutamyl peptides is a



Vol. 2, No.6, 2011

mechanism of Cu2+ resistance in Saccharomyces cerevisiae [57]. Bacterial communities in serpentine soil

were reported to tolerate spiking of metals, such as nickel and zinc, more than those of unpolluted soils

[58]. The Providencia sp. was isolated from the contaminated sites of chemical industries. The bacterial

isolate could grow and reduce chromate at a concentration ranging from 100–300 mg L-1 and at a

concentration of 400 mg L-1, pH 7 and temperature 37ºC. It also exhibited multiple heavy metal (Ni, Zn,

Hg, Pb, Co) tolerance [59].

In order to survive in the wild, bacteria need to develop different mechanisms to confer resistances

to these heavy metals. There is no general mechanism for resistance in bacteria for all heavy metal ions.

There are four possible known mechanisms of bacterial heavy metal resistances. These are:

The first mechanism is by keeping the toxic ion out of cell by altering a membrane transport system

involved in initial cellular accumulation.

The second mechanism is the intracellular or extracellular sequestration by specific mineral-ion

binding components (analogous to the metallothioneins of eukaryotes and the phytochelatins of plants,

but generally at the level of the cell wall in bacteria). Extracellular accumulation/ precipitation may be

facilitated by using viable microorganisms, cell surface sorption or complexation which can occur with

alive or dead microorganisms, while intracellular accumulation requires microbial activity [6].

The third method is the most commonly found mechanism of plasmid-controlled bacterial metal ion

resistance, involving highly specific cation or anion efflux systems encoded by resistance genes

(analogous to multidrug resistance of animal tumor cells).

The fourth known mechanism involves detoxification of the toxic cation or anion by enzymatically

converting it from a more toxic to a less toxic form.

The last mechanism does indeed occur, as best known for detoxification of inorganic and organomercurials.

Though it is well known that both living and dead cells are capable of metal accumulation but there are

differences in the mechanism involved. Some major mechanisms of microbial metal transformations

between soluble and insoluble metal species include chemolithotrophic leaching, chemoorganotrophic

leaching, rock and mineral bioweathering and biodeterioration, biocorrosion, redox mobilization,

methylation, complexation (with microbial products such as extracellular polymers (EPS) and

metallothionein like proteins) in case of soluble metal species while for latter case we speak about

biosorption, accumulation, biomineral formation, redox immobilization, metal sorption to biogenic

minerals and formation of metalloid nanoparticles [60]. The key factors controlling and characterizing

these mechanisms are:

i. The type of biological ligands available for metal sequestration;

ii. The status of the biomass i.e. living or non living;

iii. The chemical, stereochemical and coordination characteristics of the targeted metals and metal species

[61];

iv. The characteristics of the metal solution such as pH, and presence of competing co-ions [62].

3. Preparation of Bacterial biosorbents

3.1 Physically/Chemically modified biosorbents

It is widely accepted that biosorption process involves mainly cell surface sequestration, the modification

of cell wall can greatly alter the binding of metal ions. Different methods can be employed for the

modification of cell wall in microbial cells. The pretreatment methods can be broadly categorized as

physical treatment and chemical treatment. The physical treatment includes heating/ boiling,

freezing/thawing, drying and lypholization and chemical treatment employs methods like washing the

biomass with detergents, cross-linking with organic solvents and alkali/acid treatment. The method of

pretreatment should be selected with utmost care and after careful screening. Citric acid to modify an

alkali-saponification biomass and they reported that biomass modified using 0.6 mol/L) citric acid at 800C

for 2 h exhibited cadmium uptake capacity twice than that of raw biomass [63]. Several chemical agents

(mineral acids, NaOH, Na2CO3, CaCl2 and NaCl) have been for the pretreatment of C. glutamicum in the

biosorption of Reactive black 5 [64]. Same modification can be introduced either during the growth of a

microorganism or in the pre-grown biomass because the condition in which microorganism grow effects its

cell components or surface phenol type, which in turn effects its biosorption potential [4, 65].



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3.2 Genetically modified biosorbents

Genetic engineering can offer a great advantage in biosorption process by improving or redesigning

microorganisms. Genetic modification is a potential solution to enhance the selectivity as well as the

accumulating properties of the cells [1]. Microorganisms have evolved several mechanisms that respond to

the toxic effects of heavy metals. One of the common mechanisms is the induction of metal binding

proteins following the uptake of metals into the cells. A well-studied class of metal binding proteins is

called metallothioneins (MTs) or low molecular weight cysteine-rich proteins. Similarly, glutathione (GSH)

and Phytochelatins(PCs) are also cystein rich peptides which are produced in response to the presence of

heavy metals. Metallothioneins (MTs) are ubiquitous class of proteins that have been isolated from a wide

range of animals and several microorganisms including mammals, yeast, algae, fungi, protozoa, neurospora

and cyanobacteria.. They are present in a vast range of taxonomic groups, ranging from prokaryotes (such

as Syneccococus spp.), protozoa (such as Tetrahymena genera), plants (such as Pisum sativum, Triticum

durum, Zea mays, Quercus suber), yeast (such as Saccharomyces cerevisiae, Candida albicans),

invertebrates (such as the nematode Caenorhabditis elegans, the insect Drosophila melanogaster, the

mollusc Mytilus edulis, or the echinoderm Strongylocentrotus purpuratus) and vertebrates (such as the

chicken, Gallus gallus, or the mammalian Homo sapiens or Mus musculus). The overexpression of MTs in

bacterial cells will result in an enhanced metal accumulation and thus offers a promising strategy for the

development of microbial-based biosorbents for the remediation of metal contamination [66]. In

mammalian cells MT genes are highly inducible by many heavy metals including Zn, Cd, Hg, and Cu.

Therefore, MT genes frequently used as a valuable marker of metal contamination [67]. A unique property

of this class of proteins is their inducibility in response to heavy metals [68]. They can accumulate one

particular metal to the nearly complete exclusion of all others and can be used for environmental

remediation [69].These proteins bind a variety of heavy metals, such as Cd2+ , Zn2+ , Cu2+, Co2+ , Pb2+ , Ni2+

and Fe2+ with high affinity through coordination bond to their cysteine residues [70, 71]. They are involved

in many diverse biological processes. e.g. MT transports metal ions in organism, or they bind and eliminate

toxic metal ions. Metal-binding proteins play important role in structural stability, signaling, regulation,

transport, immune response, metabolism control and metal homeostasis. MTs also display antioxidant

function, and are involved in Zn2+ homeostasis. MT gene transcription is induced by heavy metals through

metal response elements (MRE) that are present in multiple copies in the proximal promoters of MT genes.

MREs were shown to mediate transcriptional response of MT to Zn, Cd, oxidative stress, and to hypoxia

[72, 73]. These multifunction proteins constitute a superfamily. Their presence has been implicated in metal

homeostasis and detoxification. If this protein can be synthesized, it can be used for mining or processing

the metals. Bacteria have evolved a wide range of mechanisms for dealing with toxic levels of metals in

environment including methylation, precipitation, chelation and exclusion [74, 75]. Several attempts have

been made to create recombinant bacteria with improved metal binding capacity but so far success is

restricted mostly to Escherichia coli. The reason behind it is that E. coli greatly facilitates genetic

engineering experiments and is found to have more surface area per unit of cell mass, which potentially

should give higher rates of metal removal from solution [1].

4. Biomass immobilization

Microbial biosorbents are basically small particles with low density, poor mechanical strength and little

rigidity [1]. They are bestowed with various merits such as high biosorption capacity, rapid steady state

attainment, less process cost and good particle mass transfer. The immobilization of the biomass in solid

structures creates a material with the right size, mechanical strength and rigidity and porosity necessary for

metal accumulation [76]. The immobilized materials can be used in a manner similar to ion exchange resins

and activated carbons such as adsorption-desorption cycles (recovery of the adsorbed metal, reactivated and

re-use of the biomass) [4]. Although cell entrapment imparts mechanical strength and resistance to

chemical and microbial degradation upon the biomass, costs of immobilizing agents cannot be ignored.

Cost information is seldom reported and the expense of individual sorbent varies depending upon the

degree of processing required and local availability. Although immobilizing the biomass for sole purpose of

biosorption will enhance the process cost, it is often necessary for practical implementation of biosorption



Vol. 2, No.6, 2011

in real application. Free cells are not suitable for use in a column in that due to their low density and size

they tend to plug the bed resulting in large drops in pressure. Immobilization of microorganisms within a

polymeric matrix has exhibited greater potential, especially in packed or fluidized bed reactors, with

benefits including the control of particle size, regeneration and reuse of the biomass, easy separation of

biomass and effluent, high biomass and minimal clogging under continuous flow conditions [1, 77].

Support matrices suitable for biomass immobilization include sodium or calcium alginate, polysulfone,

polyacrylamide, polyurethane, silica, cellulose and Gluteraldehyde [78].

Biomass is usually mixed with immobilizing agents at densities of 4 to 6 % biomass to 1 % carrier (w/w)

[79] thereby reducing the amount of agent required. Proper care must be taken so that the above ratio

should not be exceeded which could result in poor quality beads with biomass ―leaching‖ into the

surrounding solution. Cross linkers also play vital role in the immobilization. The technique was used in

immobilization of algae [66]. The most common cross linkers are: Formaldehyde, glutaric dialdehyde,

divinylsulfone and formaldehyde- urea mixtures.

5. Commercial biosorbents

The process of biosorption has been recently commercialized and accepted by EPA [80]. Various

commercial microbial biosorbents available are ALGASORBTM, AMT-Bioclaim and Bio-fix. The

economics of these sorbents merit their commercialization, over chemical ion exchangers. ALGASORBTM

was produced by Bio-recovery Systems Inc. in Las Cruces, New Mexico. It was developed using a fresh

water alga Chlorella vulgaris to treat wastewater [81]. It can efficienty remove metallic ions from dilute

solutions and its performance was not affected by the presence of calcium and magnesium ions. The

biosorbent resembles an ion exchange resin and can undergo more than 100 biosorption-desorption cycles

[4, 82, 83]. ALGASORB® is powdered algal sorbent, 1-3 mm, which is sold for the price of 28 £. The

sorbent consists of the biofilm of immobilized in silica gel, consisting of filamentous multi cellular green

alga spirogyra [84, 3].

BIOCLAIM® (bacteria of the genus Bacillus treated with caustic soda (to enhance metal-binding),

washed, immobilized in extruded beads-polyethyleneimine (PEI) and glutaraldehyde) [3]. The metal

sorption agent AMT-BIOCLAIMTM (MRA) (Visa Tech Ltd) has employed Bacillus biomass to

manufacture granulated material for wastewater treatment and metal recovery [85]. This can accumulate

metal 2.90 mmol Pb g-1, 2.39 mmol Cu g-1, 2.09 mmol Zn g-1, 1.90 mmol Cd g-1 or 0.8 mmol Ag g-1 metal

cations with efficient removal of more than 99% from dilute solutions [4]. It is non-selective and metal(s)

can be stripped using H2SO4, NaOH or complexing agents and the granules can be regenerated for repeated

use [11]. Another biosorbent AMT-BIOCLAIMTM is able to accumulate gold, cadmium and zinc from

cyanide solutions, and is therefore suitable for metal-finishing operations [69].

The biosorbent BIO-FIX is made up of a variety of biomasses, including Sphagnum peat moss,

cyanobacteria (Spirulina), algae, yeast, bacteria, and plants (Lemna sp.). This biosorbent is selective for

toxic heavy metals over that of alkaline earth metals [1, 4]. U. S. Bureau of Mines (Golden, Colorado)

produced the granular Bio-fix, which has been tested extensively for the treatment of acid mine waste [72].

The results showed the Zn binding to the biosorbent BIO-FIX is about 4- fold higher than the ion exchange

resins. The metal affinity followed: Al3+ > Cd2+ > Zn2+ > Mn2+ and a much lower affinity for Mg2+ and Ca2+

.

There are two more commercialized biosorbents: ―MetaGeneR‖ and ―RAHCO Bio-beads‖. They are

effective to remove metal ions from electroplating or mining waste streams. VitrokeleTM 573 is another

mercury-binding synthetic biosorbent which is used for the removal of mercury. The biosorbent could be

used over multiple cycles [86]. Another VitrokeleTM product was iron-binding synthetic biosorbent. It

showed good affinity for Fe, but poor affinity for Co and no affinity for Na and Cd.

6. Instrumentation for Biosorption Research

Cation exchange properties of the surfaces of biological materials can be investigated by acid-base or metal

based potentiometric titration [87]. Other than potentiometric methods used in identification of binding

sites are spectroscopic techniques which can be helpful to get information regarding the active sites

involved in binding of pollutants. Among these techniques are infrared absorption spectroscopy or Fourier



Vol. 2, No.6, 2011

transformed infrared spectroscopy (IR or FTIR), scanning electron microscopy (SEM), transmission

electron microscopy (TEM), energy dispersive Xray spectroscopy (EDS), X-ray diffraction (XRD)

analysis, electron spin resonance spectroscopy (ESR), nuclear magnetic resonance (NMR), X-ray

photoelectron spectroscopy (XPS), X-ray absorption spectroscopy (XAS), thermogravimetric analysis

(TGA), and differential scanning calorimetry (DSC). Various analytical techniques used in biosorption

process are summarized in Table 2. The most important of these groups are summarized by Volesky (2007)

including carbonyl (ketone), carboxyl, sulfhydryl(thiol), sulfonate, thioether, amine, amide, imine,

imidazole, phosphonate, phosphodiester [5].

7. Biosorption Experimental Procedures

7.1 Factors influencing batch biosorption

There are several factors which plays a crucial role in the biosorption process. Many factors can affect

biosorption such as

type and nature of the biomass

nature of its application like freely-suspended cells or biomass, immobilized preparations, living

biofilms, etc.

Physical and chemical treatments- boiling, drying, autoclaving, alkali treatment etc.

Growth, nutrition and age of the biomass, due to changes in cell size, wall composition,

extracellular product formation, etc.

Surface area to volume ratio

Biomass concentration [89]

physico-chemical factors- pH, presence of other anions and cations, metal speciation, pollutant

solubility and form, temperature.

The pH influences the magnitude of negative charge on the surface of the material, by either protonation or

deprotonation of metal binding sites [90]. Redox potential is established by oxidation/ reduction reactions

in the environment, particularly in the soils but metabolic activities of microorganisms also play essential

roles in establishing redox potential. Since biosorption is reversible process, decreasing pH would result in

deprotonation [3]. This property is used in regeneration of biosorbents. Combined effects of above

mentioned parameters influences speciation of metals [6].

7.2 Biosorption isotherms

Biosorption isotherms have been classified into six characteristic types. Microporous biosorbents produce

biosorption isotherms of Type 1(which has a convex shape) and it is also associated with monomolecular

layer biosorption. Types II and III depict biosorption for multimoleculer layer formation while Types IV

and V describe the biosorption process of multimolecular layer formation and condensation in pores. Type

VI represents surface phase transition of a monomolecular layer on a homogeneous surface [91]. Type III

has a concave shape whereas II, IV, V and VI are sigmoid shape showing a plateau that is as pressure or

concentration increases, amount adsorbed increases slowly first, sharply and then flattens out. Different

types of isotherms are shown in Figure 2. Isotherm curves can be evaluated by varying the initial solute

concentrations, while fixing the environmental parameters, such as pH, temperature and ionic strength. In

general uptake increases with increase in concentration and will reach saturation at higher concentrations

[1]. Biosorption isotherms may exhibit an irregular pattern due to the complex nature of both the

biosorbents and its varied multiple active sites as well as the complex solutions chemistry of some metallic

compounds [92, 93].

7.3 Batch modeling

Models have an important role in technology transfer from a laboratory to industrial scale [1]. The

biosorption process is very quick and the equilibrium is reached within few minutes. The process is usually

described with either first, second, pseudo-first or pseudo-second order kinetic equations [3]. Biosorption



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has been studied with simplified sorption systems usually containing one heavy metal. Yu and Neretnieks,

1990 thoroughly reviewed model isotherms for single-component adsorption [94]. Some empirical models

for single solute systems are listed in Table 3. There is no critical reason to use more complex models if 2-

parameter models such as Langmuir and Freundlich isotherm can fit the data reasonably well [104]. Since

many industrial wastewaters contain several components to be bound onto the biosorbent, a very judicious

use is necessary for practical applications of effective multicomponent biosorption models [104]. Empirical

models for multi – component adsorption systems are summarized in Table 4.

Equilibrium isotherm models are usually classified into the empirical equations and the mechanistic

models, which are able to explain, represent and predict the experimental behavior.

7.3.1 Emperical modeling: Emperical models are simple mathematical relationship, characterized by a

limited number of adjustable parameters, which are good description of the experimental behavior over a

large range of operating conditions [62]. The most widely accepted and used models in literature are

Langmuir (L type, based on monolayer adsorption of solute) and Freundlich model (F type, developed for

heterogenous surfaces). Beside these, various extended Langmuir models (also called competitive

Langmuir model) or Freundlich type models have been developed to describe two- or multi-metal ions

biosorption system [104].

7.3.2 Mechanistic modeling: Mechanistic models have been proposed to describe solute adsorption onto

the surfaces of biomass [105, 106, 107, 108, and 1]. Although mechanical modeling requires titration or

other biomass characterization data, in addition to the solution chemistry, this approach would be useful for

the understanding and isolation of the operating binding mechanisms, as well as the proper and true

representation of experimental sets [1].

8. Desorption and Regeneration of Biomass

One of the important success of biosorption process in industrial application is recovery of loaded

pollutants from the biosorbent and simultaneous regeration of the biosorbent for reuse. Desorption enables

re-use of the biomass. However, it is desirable that the desorbing agent does not damage or degrade the

biomass to significant level [109]. Desorption is of utmost important when biomass regeration is costly, as

it is possible to decrease the process on a continuous supply of biosorbent. The adsorbate bound onto the

surface of a biosorbent. The adsorbate bound onto the surface of a biosorbent through metabolism-

independent biosorption may be easily desorbed by simple non-destructive physical/ chemical methods

using chemical eluant, but intracellularly bound adsorbate through metabolism dependent bioaccumulation

can be only released by destructive methods like incineration or dissolution into strong acids or alkalis

[110, 111]. Combustion or subsequent removal of metals from ash (Destructive recovery) may also be

possible [9]. To achieve success in desorption process, requires proper selection of eluents which depends

mainly on the type of biosorbent and biosorption mechanisms. It should also be considered that eluent

should not cause any damage to the biomass, should be effective, economical and environmental friendly.

The main focus of attraction in biosorption process is the potential ability of biomass regeneration.

Regeneration of biosorbent is relatively easier in a packed column arrangement with the help of appropriate

eluant.

9. Continuous Biosorption

For evaluating the technical feasibility of a process in real applications, continuous biosorption studies are

very important. There are different types of process configuration, such as

the packed bed column (fixed bed system)

stirred tank reactors

the fluidized bed system

Rotating contactors

Trickle filters

Air-lift reactors



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completely mixed system

Most of these have been utilized in applications that employ living microorganisms for removal of metal

contaminants from complex industrial wastewaters [112, 113]. Of the different column configurations,

packed bed (fixed bed) columns have been established as an effective, economical and most convenient

biosorption process [114]. The packed bed column studies are bestowed with various merits such as high

operational yield and the relative ease of scaling up procedures [101]. A packed bed configuration

comprises a cylindrical column packed firmly with sorbent through which waste water is allowed to flow.

Initially, most of the solute will be sorbed as it is exposed to the fresh biosorbent bed and thus almost zero

concentration would be expected at the column outlet. Theoretically, this is where the highest mass transfer

occurs [115]. Owing to the competitive ion exchange taking place in the column, one or more of the metals

present even at trace levels may overshoot the acceptable limit in the column effluent before the

breakthrough point of the targeted metal.

Other column contactors such as fluidized and continuous stirred tank systems are rarely used for the

purpose of biosorption. Fluidized bed systems, which operate continuously, require high flow rates to keep

the biosorbent particles in suspension. They have low mass transfer limitation relative to packed bed

reactors. The main drawback of fluidized bed reactors is that they can utilize the biosorbent charge to its

maximum potential because their contents are being mixed [116]. Many other bioreactors are also possible,

with biosorbent being utilized in a variety of forms.

10. Scope and Future directions

The overall cost of biosorbent material depends on source and type of biosorbent. Various industrial waste-

biomass types have been investigated for their biosorptive potential which includes yeasts from food and

beverage industry, moulds from food industry, fungi from industrial enzyme producers; bacteria, Bacillus

spp. utilized in amino acid and antibiotic fermentations and Streptomyces noursei from pharmaceutical

industry. These biosorbents are available free of cost since they already possess disposal problems and will

make the process highly economical [4]. Moreover biosorption process can effectively sequester dissolved

metals from very dilute complex solutions with high efficiency thus this process could be effectively used

for the treatment of high volume low concentration complex industrial effluents. It is well known fact that

bioreactors contain metabolically active microbial cells. In such case biosorption contributes as parallel

mechanism together with other metabolically mediated mechanisms. The need of the hour is necessary to

search for better and more selective biosorbents, to elaborate new, universal biosorption models for multi-

metal systems, to make some more market and economic studies to be able to evaluate the real investment

and operation costs of biosorption and bioaccumulation wastewater treatment processes [3]. Since the

ultimate goal of biosorption is to use microbes for cleaning up of metal polluted water, waste streams and

soils, the heterogenous expression of native or genetically modified proteins can prove to be an attractive

solution to improve the metal binding abilities of these organisms. Genetic engineering may further

enhance the potential of robust environmental strains [113]. However the issue of living genetically

modified organisms (LM0) should be addressed prior to field application. Protein engineering may also

conceivably lead to enhanced metal specificity, stability and other useful properties of peptides or other

biopolymers [112]. Although some attempts have been made at the commercialization of biosorption for

wastewater treatment, the progress is very modest considering that there has been more than a decade of

fundamental research [1]. Despite various well known advantages of biosorption processes, the application

of biosoption at commercial level is facing with great challenges.

Common suggestions for future research directions in biosorption include:

Identification of better and more selective biomass with high biosorptive capacity and selectivity.

Improving biomaterials immobilization and optimization of the parameters involved in biosorption

process

To test the performance of biosorbent and to develop more biosorption models

Further assessments of market size and costs of development

Involving molecular biotechnology to elucidate the mechanism of biosorption at molecular level that

will help in construction of engineered organism with higher sorption capacity.

To search methods for regeneration and reuse of spent biosorbent



Vol. 2, No.6, 2011

To analyze the behavior of biosorbents in real wastewaters

To promote research on pilot and full scale biosorption process.

In order to attract more usage of biosorption process, certain strategies should be formulated to

centralize the facilities for accepting the used biosorbent where further processing of biosorbent can be

done to regenerate the biomass and then convert the recovered metal into usable form. This would further

require an interdisciplinary approach for combating the heavy metals load in aqueous streams and

wastewaters. Moreover to promote the application of biosorption process at commercial level, the

biosorption process applications has to be done in conjunction with industrial users/ clients.

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4514–21, 1999.



Vol. 2, No.6, 2011

[108] Haas_J.R., Dichristina_T.J., Wade_R., ―Thermodynamics of U(VI) sorption onto Shewanella

putrefaciens‖, Chem Geol., vol. 180, pp. 33–54, 2001.

[109] Gadd_G.M., White_C., ―The removal of thorium from simulated acid process streams by fungal

biomass: potential for thorium desorption and reuse of biomass and desorbent”, J. Chem Technol

Biotechnol., vol. 55, pp. 39-44, 1992.

[110] Gadd_G.M., ―Heavy metal accumulation by bacteria and other microorganisms‖, Experimentia, vol.

46, pp. 834-840, 1990.

[111] Gadd_G.M., White_C., ―Microbial treatment of metal pollution: a working biotechnology?‖

TIBTECH, vol. 11, pp. 353-9, 1993.

[112] Malik_A., ―Metal bioremediation through growing cells‖, Environ. Int., vol. 30, pp. 261-278, 2003.

[113] Gavrilescu_M., ―Removal of heavy metals from the environment by biosorption‖, Eng. Life Sci., vol.

4, pp. 219-232, 2004.

[114] Chu_K.H., ―Improved fixed bed models for metal biosorption”, Chem. Eng. J., vol. 97, pp. 233–239,

2004.

[115] Dhankhar_R.; Hooda, A., ―Fungal biosorption - an alternative to meet the challenges of heavy metal

pollution in aqueous solutions‖, Environmental Technology, vol. 32, no. 5, pp. 467–491, 2011.

[116] Volesky_B., Naja_G., “Biosorption: Application Strategies”, 16th International Biotechnology

Symposium, S.T.L. Harrison, D.E. Rawlings, J. Petersen, eds. IBS-Compress Co., Cape Town, South

Africa, pp. 531–542, 2005.

Authors Profile

Prof. (Mrs.) Rajesh Dhankhar, Head Department of Environmental Sciences,

Maharshi Dayanand University, Rohtak (India) having research

specialization in the field of Ecotoxicology and Bioremediation. She has

more than 40 publications in esteemed national and international journals.

She is acting as counselor in IGNOU since 1999 and member of UG and PG

Board of studies in various universities. She is an active member of various

Government and Non- Government academic bodies including Haryana

Vidyut Manch and Children Science Congress. Three major projects

sanctioned from UGC, New Delhi are presently under her leadership and is a

resource person in National and International conferences.



Vol. 2, No.6, 2011

Er. Rachna Bhateria Guriyan received M.Sc (Environmental Sciences) and

M.Tech (Environmental Engg. ) degree from Guru Jambheshwar University

of Science and Technology, Hisar, India in 2006 and 2008 respectively.

Beside this the author has also done P.G.D. in Environmental Law. She is

also a Life time member of Society for Science of Climate change and

Sustainable Development and also life member of Association of

Microbiology. She is currently working as an Assistant Professor in

Department of Environmental Sciences, Maharshi Dayanand University,

Rohtak (India). Her major research interests are in Bioremediation and

Environmental Law. She has also received Project entitiled ―Bioremediation

of Chromium (VI) from Electroplating Effluent using bacterial strains‖ from

UGC, New Delhi.


Table 1: Biosorbent uptake of metals by Bacterial Biomass

Metal Biomass type Metal uptake(mg/g) References

Ag Streptomyces noursei 38.4 15

Co Enterobacter cloaceae 4.38 16

Au Bacillus subtilis 79 17

Bacillus subtilis 70 18

Cd Pantoea sp. TEM 18 204.1 19

Enterobacter cloaceae Exopolysaccharide 16 16

Aeromonas caviae 155.3 20

Pseudomonas sp. 278.0 21

Staphylococcus xylosus 250.0 21

Streptomyces pimprina 30.4 22

Streptomyces rimosus 64.9 23

Cr Bacillus coagulans 39.9 24

Bacillus megatarium 30.7 24

Bacillus thuringiensis 83.3 25

Bacillus licheniformis 69.4 26

Zooglia ramigera 2 27

Streptomyces nouresei 1.8 15

Aeromonas caviae 284.4 20

Pseudomonas sp. 95.0 21

Staphylococcus xylosus 143.0 21

Manganese oxidising bacteria

50 28

Cu

Streptomyces noursei sp. 5 15

Bacillus sp 16.3 29

Bacillus firmus 381 30

Bacillus subtilis 20.8 31

Enterobacter cloaceae (Exopolysaccharide) 6.60

16

Enterobacter sp. 32.5 32

Micrococcus luteus 33.5 31

Pseudomonas aeruginosa 23.1 Chang et al., 1997

Pseudomonas cepacia 65.3 33

Pseudomonas putida 96.9 34

Pseudomonas stutzeri 22.9 31

Sphaerotilus natans 60 35

Streptomyces coelicolor 66.7 36

Pantoea sp TEM 18 31.3 19


Thiobacillus thiooxidans 38.54

37

Thiobacillus ferrooxidans 39.8 37

Bacillus subtillis 201 17

Bacillus biomass 107 38

Fe Bacillus licheniformis 29 17

Bacillus subtillis 6 17

Ni Bacillus thuringiensis 45.9 39


Bacillus subtilis (biomass not necessarily in its

natural state)

601 41

Bacillis subtilis (biomass not necessarily in its

natural state)

189 38

Pb Streptomyces longwoodensis 100 42

Streptomyces noursei 55 15

Streptomyces longwoodensis 440 42

Arthrobacter nicotianae 68.8 43

U Bacillus licheniformis 45.9 43

Bacillus megatarium 37.8 43


Corynebacterium equi 21.4 43

Corynebacterium glutamicum 5.9 43

Bacillus sp. 38 44


Nocardia erythropolis 51.2 43

Zoogloea ramigera 49.7 43

Bacillus subtilis (biomass not necessarily in its

natural state)

137 41

Bacillus sp. 3.4 44

Zn Thiobacillus thiooxidans 43.29 37

Thiobacillus ferroxidans 172.4 37

Pseudomonas putida 17.7 45


Streptoverticillium cinnamoneum 21.3 47

Bacillus firmus 418 48

Arthrobacter nicotianae 75.9 43

Bacillus licheniformis 66.1 43

Th Bacillus megaterium 74.0 43


Corynebacterium equi 46.9 43

Corynebacterium glutamicum 36.2 43


Zooglea ramigera 67.8 43

Pd Desulfovibrio desulfuricans 128.2 49

Desulfovibrio 119.8 49



Pt Desulfovibrio 62.5 49




Table 2. Analytical techniques used in biosorption research [88]

Analytical techniques Remarks

Atomic absorption spectroscopy (AAS)

Inductively coupled plasma (ICP)

UV-Vis spectrophotometer

Scanning electron microscope (SEM)

Transmission electron microscope (TEM)

Energy dispersive X-ray spectroscopy

(EDS)

X-ray diffraction (XRD) analysis

Electron spin resonance spectroscopy (ESR)

Nuclear magnetic resonance (NMR)

Fourier transformed infrared spectroscopy

(FT-IR)

Potentiometric titration

X-ray photoelectron spectroscopy (XPS)

X-ray absorption spectroscopy (XAS)

Thermogravimetric analysis (TGA)

Differential scanning calorimetry (DSC)

Determine metal concentration in aqueous phase

Determine metal concentration in aqueous phase

Determine metal or dye concentration in aqueous phase by measuring its

color intensity

Visual confirmation of surface morphology of the biosorbent

Visual confirmation of inner morphology of biomass, especially cells

Element analysis and chemical characterization of metal bound on the

biosorbent

Crystallographic structure and chemical composition of metal

bound on the biosorbent

Determine active sites of the biosorbent



Determine active sites of the biosorbent and its amounts

Determine oxidation state of metal bound on the biosorbent and its ligand

effects

Determine oxidation state of metal bound on biosorbent and its

coordination environment

Characterize thermal stability of the biosorbent

Characterize thermal stability of the biosorbent


Table 3. Frequently used single – component adsorption models

Isotherm type Equations Nomenclature References

Langmuir qe = qmax bCe

1 + bCe

qe is equilibrium metal sorption capacity;

Ce is equilibrium solute concentration in

solution; capacity (monolayer capacity)

and bonding energy of adsorption (or

“affinity”), respectively

[95]

Freundich qe = KFCe1/n KF is a biosorption equilibrium constant,

representative of the sorption capacity

and n is a constant indicative of

biosorption intensity

[96]

Langmuir-

Freundich

qe = qmax bCe1/n

1 + bCe1/n

Assuming that the surface is

homogeneous but that the sorption is a

cooperative process due to adsorbate-

adsorbate interactions.

[97]

BET model

(multilayer

sorption)

qe = BQ0Ce

(Cs-Ce)[1+ (B-1)Ce/Cs]

CS is the saturation concentration of the

adsorbed component; B a constant

indicating the energy of interaction

between solute and adsorbent surface and

Q0 is a constant indicating the amount of

solute adsorbed forming a complete

monolayer.

[98])

Redlich-

Peterson

qe = KRPCe

1 + aRPCeβ

KRP, aRP and β are the Redlich-Peterson

parameters. The exponent β lies between 0 and 1. For β=1 the model converts to the Langmuir form

[99]

Radke-Prausnitz qe = arCep

a + rCep-1

a, r and p are related model constants [100]

Distribution

coefficients

model

qe = KdCe Kd is distribution coefficient [101]

Dubinin-

Radushkevich

Q = QD exp [ -BD ( RT In { 1+ 1 })2 ]

Cf

QD is Dubinin- Radushkevich model

uptake capacity and BD the Dubinin-

Radushkevich model constant; T is

absolute temperature; R the gas constant

(8.314 J/mol K)

[102]

Temkin Q = RT In (aTeCf)

bTe

bTe is the Temkin constant related to heat

of sorption; aTe the Temkin isotherm

constant.

[103]


Table 4. Frequently used multi – component adsorption models

Isotherm type Equations Nomenclature References

Langmuir

(multi-

component)

qei = bi qmaxi cei

N

1 + Σ biCei

i=1

cei and qei are the unadsorbed concentration of

each component at equilibrium and the adsorbed

quantity of each component per g of dried

biomass at equilibrium, respectively; bi and qmaxi

are derived from the corresponding individual

Langmuir isotherm equations.

[96]

Combined

Langmuir-

Freundich

qe i = aiCi 1/n

N

1 + Σ biCei 1/ni

i=1

ai , bi , phase concentration of a single adsorbed

component in equations.

[98]

Competitive

Redlich-

Peterson model

qe = KRPi Cei

N

1 + Σ aRPiCβi

i= 1

KRPi , aRPi and βi are the Redlich-Peterson

parameters derived from the corresponding

individual Redlich-Peterson isotherm equations

[105]

IAST:

Ideal Adsorbed

Solution Theory

1 = Σ Yi

qi qi0

Yi is the solute concentration of component I in

the solid phase, qi0 is the phase concentration of

a single adsorbed component in equations with

Ci0

[101]


Figure 1. Diagram showing Biosorption of metal ion


Figure 2. Types of isotherms


INTERNATIONAL JOURNAL OF ENVIRONMENTAL SCIENCES Volume 1, No 7, 2011

© Copyright 2010 All rights reserved Integrated Publishing Association

Research article ISSN 0976 – 4402

Received on April 2011 Published on August 2011 1884

Strategies for Management of Metal Contaminated Soil Rajesh Dhankhar, Rachna Bhateria Guriyan

Department of Environmental Sciences, M.D.University, Rohtak-124001 (India)

ABSTRACT

Heavy metals are inadvertently released during manufacture of various industrial products. They are serious pollutants due to their toxicity, persistence and bioaccumulation problems. Microorganisms have the potential to alter the reactivity and mobility of metals and thus facilitating the use of bioremediation as a form of treatment for metal contaminated soils. Utilizing microbes for bioremediation possesses various merits such as their natural occurrence, cheap production, easy availability and high selectivity in terms of removal and recovery of specific metals. This paper summarizes the general processes of bioremediation within the soil environment. The effect of environmental factors which governs the rate of biodegradation is addressed together with limitations and potential of ex situ and in situ bioremediation.

Keywords: Bioremediation, heavy metals, microorganisms, biodegradation, soil.

Introduction

Pollution is reduction in the quality of the environment by introduction of impurities. Pollution of environment with heavy metals has become a problem of concern because beside causing specific toxicity symptoms, these metals may also contribute to global warming by destroying the atmospheric ozone layer like atmospheric methane, nitrous oxide and sulphur dioxide because of potentially harmful effects on human and animal health (Meena et al., 2005). Under certain environmental conditions, metals may accumulate to toxic levels and cause ecological damage. Of the important metals mercury, lead, cadmium and chromium (VI) are regarded as toxic; whereas, others, such as copper, nickel, cobalt and zinc are not as toxic but their extensive usage and increasing levels in the environment are of serious concern. Thus metal as a kind of resource is becoming shortage and also brings about serious environmental pollution, threatening human health and ecosystem (Wang and Chen, 2009). The commonly used procedures for removing metal ions from aqueous streams include chemical precipitation, dialysis, ion exchange, reverse osmosis and solvent extraction (Rich and Cherry, 1987; Alloway and Ayres, 1993). These methods are faced with the problems such as high cost, low efficiency, changing soil properties and spreading of contaminants to other places after remediation (Kunito et al., 2001; Huang et al., 2004). Conventional methods are also cost prohibitive having inadequate efficiencies at low metal concentrations, particularly in the range of 1-100 mg/l (Lokeshwari and Joshi, 2009). Since it is impossible to degrade metals by any means, the only way to remove them from environment is to exclude metals from cycling through their concentration, with possible recovery and reuse (Volesky, 1997). This would also reduce the consumption of non-renewable resources (Chojnacka, 2010). To overcome the drawbacks associated with the conventional methods, a new technique bioremediation has been introduced. Bioremediation uses biological agents mainly microorganisms i.e. yeast, fungi or bacteria to clean up contaminated soil and water (Strong and Burgess, 2008). This technology relies on promoting the growth of specific microflora or microbial consortia that are indigenous to the contaminated sites that are able to perform

Strategies for Management of Metal Contaminated Soil

Rajesh Dhankhar, Rachna Bhateria Guriyan International Journal of Environmental Sciences Volume 1 No.7, 2011

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desired activities (Kumar et al., 2011). In bioremediation processes, microorganisms use the contaminants as nutrient or energy sources (Tang et al., 2007). In recent years, applying biotechnology in controlling and removing metal pollution has been paid much attention and gradually becomes hot topic in the field of metal pollution control because of its potential application (Wang and Chen, 2009). Because heavy metals are increasingly found in microbial habitats due to natural and industrial processes, microbes have evolved several mechanisms to tolerate the presence of heavy metals. Bioremediation strategies are based on identification of impediments to the removal, dissipation or stabilization of intolerable levels of pollutants in the environment and provision of necessary conditions to remove those impediments, so natural restoration processes can proceed. Bioremediation has also received considerable attention for the development of an efficient, clean and cheap technology for wastewater treatment at metal concentration as low as 1 mg/l (Lokeshwari and Joshi, 2009). The chief advantage of bioremediation is its reduced cost compared to conventional techniques such as incineration. In addition, bioremediation is often a permanent solution (providing complete transformation of the pollutant to its molecular constituents like carbon dioxide and water) rather than a remediation method that transfers waste from one phase to another. A good number of bioremediation strategies have been explored and successfully implemented. Successful bioremediation strategies are those that are tailored to satisfy specific pollutant, site, public, regulatory, cost and environmental effectiveness considerations. This review provides description of some of the current, most commonly used soil bioremediation strategies.

Fundamental of Bioremediation

Bioremediation is a process in which microorganisms metabolize contaminants either through oxidative or reductive processes. For bioremediation to be effective, microorganisms must enzymatically attack the pollutants and convert them to harmless products (Vidali, 2001). Although microbes cannot degrade heavy metals, they can be used to change the valence states of these metals thus converting them into immobile or less toxic forms. For example microbes can convert mobile hexavalent chromium into immobile and less toxic trivalent chromium. The bioremediation process may be directed to accomplish:

1. Complete oxidation of organic contaminants (termed mineralization),

2. Biotransformation of organic chemicals into smaller less toxic metabolites and

3. Reduction of highly electrophilic halo- and nitro- groups by transferring electron donor (typically a sugar or fatty acid) to the contaminant, resulting in a less toxic compound.

Microbes require carbon as an energy source to sustain the metabolic functions which includes growth and reproduction. The metabolic processes used by bacteria to produce a terminal electron acceptor to enzymatically oxidize the carbon source (organic matter to carbon dioxide)

Organic matter + oxygen + biomass → carbon dioxide + water + ∆ Hf

Where ∆ Hf is the energy generated by the reaction to enhance other metabolic processes including growth and reproduction. Here, oxygen serves as terminal acceptor.



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Bioremediation is the process whereby organic wastes are biologically degraded under controlled conditions to an innocuous state or to levels below concentration limits established by regulatory authorities (Mueller, 1996). During bioremediation, microbes utilize chemical contaminants in the soil as an energy source and, through oxidation-reduction reactions, metabolize the target contaminant into useable energy for microbes. Although a multitude of reactions are used by microbes to degrade and transform pollutants, all energy yielding reactions are oxidation-reduction reactions. Typical electron acceptors are oxygen, nitrates, Fe(III), Sulfate and carbondioxide (Figure 1).

Figure 1: Diagram showing general scheme to biodegrade organic pollutants. In oxidative biodegradation, pollutants are oxidized by external electron acceptors such as oxygen or

sulfate. In reductive biodegradation, electrophilic hydrogen or nitro groups on the pollutanat are reduced by microbes consuming sugars, fatty acids or hydrogen.

In reductive Bioremediation, microbes utilize some easilymetabolized organic electron donor (such as sugar or short fatty acids) and transfer the electron to the pollutant to gain energy By-products (metabolites) released back into the environment are typically in a less toxic form than the parent contaminants. Three primary ingredients for bioremediation are: 1) presence of a contaminant, 2) an electron acceptor, and 3) presence of microorganisms that are capable of degrading the specific contaminant. Generally, a contaminant is more easily and quickly degraded if it is a naturally occurring compound in the environment, or chemically similar to a naturally occurring compound, because microorganisms capable of its biodegradation are more likely to have evolved (State of Mississippi, Department of Environmental Quality, 1998).

Bioremediation technology exploits various naturally occurring mitigation processes: natural attenuation, biostimulation, and bioaugmentation. Bioremediation which occurs without human intervention other than monitoring is often called natural attenuation. This natural attenuation relies on natural conditions and behavior of soil microorganisms that are indigenous to soil. Biostimulation provides nutrients and suitable physiological conditions for the growth of the indigenous microbial populations. This promotes increased metabolic activity, which then degrades the contaminants. Bioaugmentation means introduction of



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specific blends of laboratory cultivated microorganism into a contaminated environment or into a bioreactor to initiate the bioremediation process. Few benefits and limitations associated with bioremediation process are shown in Table 1.

Table 1: Advantages and Disadvantages of Bioremediation Technologies

Technology Examples Benefits Limitations

In situ

In situ bioremediation Biosparging Bioventing Bioaugmentation

Most cost effective Noninvasive Relatively passive Natural attenuation process

Environmental constraints Extended treatment time Monitoring difficulties

Ex situ

Landfarming Composting Biopiles

Cost efficient Low cost Can be done on site

Space requirements Extended treatment time Need to control abiotic loss, mass transfer problem. Difficult to extrapolate from Lab to field operation. Contaminants may be present as solids, liquids and gases.

Bioreactors

Slurry reactors Aqueous reactors

Rapid degradation Kinetic Optimized environmental parameters Residues from this process are harmless such as CO2, H2O and cell biomass Complete destruction of contaminants/pollutants is possible

Soil requires excavation Relatively high cost capital Limited to biodegradable compounds. Highly specific

Bioremediation Treatment Technologies

Field applications of Bioremediation treatment technologies have been broadly divided into two categories based on whether biodegradation is stimulated in-situ or carried out ex-situ in compost heaps or bioreactors (Baker and Herson, 1994; Blackburn and Hafker, 1993).

1. In-situ Bioremediation Techniques

Bioremediation of organic contaminants in in-situ requires stimulation of the degradative activities of endogenous microbial population by the provision of nutrient and/or external electron acceptors. It is safer since it does not require excavation of contaminated soils and does not disturb the natural surroundings of the site. On the other side, the method is time consuming. Microbial activity is affected due to direct exposure to changes in environmental conditions that cannot be controlled. When favourable conditions are not available then their capacity to degrade is reduced. In such cases genetically engineered microorganisms have to



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be used, although stimulating indigenous microorganisms is preferred. In-situ Bioremediation is further categorized into two: Intrinsic bioremediation which deals with stimulation of indigenous or naturally occurring microbial population by feeding them nutrients and oxygen to increase their metabolic activity and Engineered in-situ bioremediation which involves the introduction of certain microorganisms to the site of contamination. Engineered in-situ bioremediation accelerates the degradation process by enhancing the physicochemical conditions to encourage the growth of microorganisms. Environmental factors influencing in-situ bioremediation rates are:

1.1 pH – Majority of bacteria exhibit growth optima at or near neutral pH values. Increase of pH causes deprotonation of metal ions binding sites exposed to cellular surfaces (Chojnacka, 2010). Decreasing pH causes competition between protons and positively charged metal ions. However these rules concern only cations (Naja et al., 2010). In general, an increase in soil pH tends to decrease the availability of Calcium, Magnesium, Sodium, Potassium, Ammonia, Nitrogen and Phosphorus, whereas decrease in soil pH results in decreasing availability of nitrate and chloride (Selatnia et al., 2004a).

1.2 Temperature – Temperature is a major environmental factor influencing in- situ

bioremediation rates. The temperature levels can fluctuate considerably during the course of a bioremediation application, varying on vertical as well as on diurnal and seasonal basis. The vast majority of in-situ bioremediation applications have been carried out under mesophilic conditions (typically between 200C to 400C). Higher temperature usually enhance the bioremediation rates due to increased surface activity and kinetic energy of the solute (Vijayaviraghavan and Yun, 2008).

1.3 Water content – Water availability in soils is an important factor influencing remediation

rates. Water in soils or sediments may not be available to microorganisms because it is either adsorbed to solid substances or tied up as water of hydration to dissolved solutes. Microorganisms generally require water activity (aw) of 0.9-1.0 in order to metabolize and grow (Baker and Herson, 1994). The amount of available oxygen will determine whether the system is aerobic or anaerobic. To increase the oxygen amount in the soil, it is needed to till the land for sparge of air.

1.4 Soil structure - Soil structure controls the effective delivery of air, water and nutrients.

Materials such as gypsum or organic matter can be applied to improve the soil structure. Unfavorable characteristics include fractured rocks, low permeability, complex mineralogy and either water logged or arid conditions.

1.5 Nutrients – Nutrient supplementation is generally practiced both in-situ and ex-situ

bioremediation of soils, sediments, ground and surface water. Carbon is the most basic element of living forms and is needed in greater quantities than other elements. In addition, hydrogen, oxygen and nitrogen constitutes about 95% of weight of the cells. Phosphorus and sulfur contribute about 70% of remainders. The nutritional requirement of carbon to nitrogen ratio is 10:1 and carbon to phosphorus ratio is 30:1 (Vidali, 2001).

1.6 Bioavailability of Pollutants – It is an important factor governing the rate of in-situ

bioremediation. Anthropogenic organic polymers (example polystyrene, PVC etc) are highly recalcitrant because of their insolubility and lack of extracellular microbial enzymes capable of catalyzing depolymerization (Field et al., 1995). Non polymer-degrading bacteria and actinomycetes are however, able to degrade oligomeric



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polystyrene fragments and low molecular weight fragments of lignin resulting from fungal attack on lignin polymer (Jokela, 1993).

1.7 Co-metabolism – Cometabolism is the process where microorganisms involved in the

metabolism of a growth promoting substrate also transform other organic contaminants (cosubstrates) that are not growth supporting if supplied as sole carbon and energy source. Bacterial transformation of trichloroethylene, trans-1,2-dichloroehylene, dichlorodiphenyltrichloroethane (DDT) and Polychlorinated biphenyls (PCBs) provide examples of both aerobic and anaerobic cometabolism biodegradation (Bouwer and Zehnder, 1993).

2. Ex-situ Bioremediation Techniques

Ex-situ Bioremediation techniques are usually aerobic and involve treatment of contaminated soils or sediments using slurry or solid phase systems.

2.1 Slurry-phase bioremediation

is a batch treatment technique in which excavated soils/sediments are mixed with water and treated in reactors vessels or in contained ponds or lagoon (Blackburn and Hafker, 1993; Troy, 1994). Effective bioremediation has been obtained with slurry phase systems for soils and sediments contaminated with wide range of organic compounds, including pesticides, petroleum hydrocarbons, pentachlorophenol, Polychlorinated biphenyls (PCBs), Cresote coal tars, wood-preserving wastes and so forth (Troy, 1994; Yare, 1991). Slurry-phase bioremediation can take place on-site or the soil can be removed and transported to a remote location for treatment (USEPA, 1990). The process generally takes place in a tank or vessel (a "bioreactor"), but can also take place in a lagoon. During treatment, the oxygen and nutrient content, pH, and temperature of the slurry are adjusted and maintained at levels suitable for aerobic microbial growth. When the desired level of treatment has been achieved, the unit is emptied and a second a second volume of soil is treated.

2.2 Solid-phase bioremediation

Includes land farming (soil treatment units), compost heaps and engineered biopiles (Field et al., 1995; Litchfield, 1991). In this system, soil is treated above ground treatment areas equipped with collection systems to prevent any contaminant from escaping the treatment. Moisture, heat, nutrients, or oxygen are controlled to enhance bioremediation for the application of this treatment. These systems are relatively simple to operate and maintain, require large amount of space, and cleanups require more time to complete than slurry-phase processes. Solid-phase bioremediation can be implemented by any of the following ways:

2.3 Contained solid-phase bioremediation,

where the excavated soils are not slurried with water; the contaminated soils are simply blended to achieve a homogeneous texture. Occasionally, textural or bulk amendments, nutrients, moisture, pH adjustment, and microbes are added. The soil is then placed in an enclosed building, vault, tank, or vessel. In addition, since the soil mass is enclosed, rainfall and runoff are eliminated, and volatile organic carbon emissions can be controlled.

2.4 Composting



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This method is similar to contained solid phase bioremediation, but it does not employ added microorganisms if carried out in enclosed vessel. It is usually conducted outdoors rather than in an enclosed space. The two basic types of unenclosed composting are open and static windrow systems. In open windrow systems, the compost is stacked in elongated piles whereas in static windrow systems, the piles are aerated by a forced air system. The waste is not protected from variation in natural environmental conditions, such as rainfall and temperature fluctuations.

2.5 Land farming

involves spreading the contaminated soil in fields or limited treatment beds. The soil is spread in thin lifts up to half inch thick. The soil is tilled periodically thereby providing oxygen. Microorganisms, nutrients, and moisture may also be added. Clay or plastic liners may be installed in the field prior to the placement of the contaminated soil which prevents the leaching of the contaminants into ground water. Treatment is achieved through biodegradation, in combination with aeration and possibly photo-oxidation in sunlight. These processes are most active in warm, moist sunny conditions and is completely diminished or arrested during winter month when temperature is cold and snow covers the ground.

Metal Bioavailability in contaminated soil

The metals in contaminated soils can be divided into two classes: Bioavailable (soluble, nonsorb and mobile) and Non-bioavailable (precipitated, complexed, sorbed and non mobile) (Fellenberg, 2003; Maier et al., 2000). The bioavailability of metals in soil depends on the following factors:

1. Metal Chemistry – Most metals are cationic that means they exhibit a positive charge in their free ionic state. They are most reactive with negative charged surfaces. Negative charge in soil is clay mineral, humic substance and cell surface. Cationic metal sorb to both soil particles and cell surfaces with varying strengths, termed adsorption affinity. For example, common soil cation, Aluminium, binds strongly than calcium and magnesium (Maier et al., 2000).

2. Cation exchange capacity – One of the most important factor affecting metal bioavailability is the soil cation exchange capacity that depends on both the organic matter and clay content of the soil. Cation exchange reflects the capacity of soil to sorb metals. Thus, the toxicity of metals within soils with high cation exchange capacity is often low even at high total metal concentration. In contrast, sandy soil with low cation exchange capacity and therefore low metal capacity, showed decreased microbial activity at comparatively low total metal concentrations, indicative of metal toxicity (Alloway and Ayres, 1993). 3. Redox potential – Metal bioavailability changes in response to changing redox conditions (Fellenberg, 2003). Under oxidizing or aerobic conditions, metals are usually found as soluble cationic forms. In contrast, reduced or anaerobic conditions such as those found in sediments or saturated soils, often was relevant to metal precipitation (Maier et al., 2000). 4. Soil pH level – The effect of pH on metal sorption to soil surface can be found when pH increases. Usually, the electrostatic attraction between metals and soil constituents is enhanced by increased pH. Therefore, at high pH, metals are predominantly found as insoluble metal mineral phosphates and carbonates. When soil pH decreases, soluble metals



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increases. Therefore, in low pH soil commonly find free ionic species or soluble organometals are commonly found (Fellenberg, 2003, Maier et al., 2000).

Role of Microbes in enhancing Bioremediation

Microbes can be isolated from almost any environmental conditions Microbes can adapt and grow at subzero temperatures, as well as extreme heat, desert conditions, in water with an excess of oxygen and in anaerobic conditions, with the presence of hazardous compounds or any waste stream(Kumar et al, 2011). Recent development in the field of environmental biotechnology includes the search for microorganisms as sorbents for heavy metals. Bacteria, fungi, yeast and algae can remove heavy metals and radionuclides from aqueous solution in substantial quantities. To survive under metal stressed condition, bacteria have evolved several types of mechanisms to tolerate the uptake of heavy metal ions. These mechanisms include the efflux of metal ions outside the cell, accumulation and complexation of the metal ions inside the cell and reduction of heavy metal ions to a less toxic state (Nies, 1999). The tested microorganisms can be basically classified into the following categories: bacteria, fungi, yeast, algae, industrial wastes, agricultural wastes and other polysaccharide materials etc. (Vijayaraghavan and Yun, 2008). Potent metal biosorbents under the class of bacteria include genera of Bacillus (Nakajima and Tsuruta, 2004; Tunali, 2006), Pseudomonas (Chang et al., 1997; Uslu and Tanyol, 2006) and Streptomyces (Mameri et al., 1999; Selatnia et al., 2004a) etc. P. aeroginosa was isolated from different heavy metal-polluted environments such as sewage, irrigation and agricultural drainage canals and soil (Shoreit and Soltan, 1992; Soltan, 2001). Therefore, it has been considered as a water quality indicator organism. Pseudomonas plecoglossicida has been proved as a novel organism for the bioremediation of cypermethrin (Boricha and Fulekar, 2009). There are many species of bacteria which are known to reduce chromium such as Pseudomonas aeruginosa (Ganguli and Tripathi, 2002), Bacillus sp. (Camargo et al., 2003; Meghraj et al., 2003), Streptomyces (Amoroso et al., 2001), Pseudomonas fluorescens (Appanna et al., 1996; Khan and Ahmed, 2006). Important fungal biosorbents include Aspergillus (Kapoor and Viraraghavan, 1997; Jianlong et al., 2002; Park et al., 2005) and Penicillium (Niu and Wang ,1993; Tan and Cheng, 2003) etc. Algal divisions include red, green and brown seaweeds; of which brown seaweeds are found to be excellent biosorbents (Davis et al., 2003). This is due to the presence of alginate, which is present in gel form in their cell walls. Davis et al., (2003) reviewed the metal sorbing properties of brown seaweeds and highlighted their biosorption mechanisms. Among micro-organisms, fungal biomass offers the advantages of having high percentage of cell wall material, which shows excellent metal binding properties (Gadd, 1988; Rosenberger, 1975; Paknikar, 1993). Many fungi and yeast have shown an excellent potential of metal biosorption, particularly the genera Rhizopus, Aspergillus, Streptoverticullum and Sacchromyces (Volesky and Tsezos, 1982; Galun et al., 1984; Siegel et al., 1986, Luef et al., 1991; Brady and Duncan, 1993; Puranik and Paknikar, 1997). The identification of microorganisms that can catalyze the reduction of Cr(VI) to Cr(III) has led to the suggestions that bioremediation of Cr(VI)-contaminated effluents would be a low cost, low-technology alternative for presently used methods (Ohtake and Silver, 1994). Bacteria, cyanobacteria and fungi alter the form of occurrence of metal through methylation, chelation, complexation, catalysis or adsorption affecting their bio-availability and movement in the food chain.

Interaction mechanisms between bacteria and metals

The combination of spectroscopic and microscopic methods can be used to characterize the environment of any metal around the cells of bacterial strains. Different spectroscopic and



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microscopic method used to see the interaction of bacteria with metal at molecular state includes

1. Scanning electron microscopy (SEM)

SEM is a technique which images the sample surface by scanning it with a high energy beam of electrons in a raster scan pattern. The electrons interact with the atoms that make up the sample producing signals that contain information about sample’s surface topography, composition and other properties sch as electrical conductivity.

2. Transmission electron microscopy (TEM)

TEM is a useful technique that can help to localize and identify metals deposited within or around microbial cells. Identification of the site of accumulation is important as it can give clues to the biochemical mechanisms driving metal accumulation. In this technique, a beam of electrons is transmitted through an ultra thin specimen as it passes through, is deflected by high mass elements including metals through the electron shells or nuclei. Thus it is possible to visualize metals against faint image of a bacterial cell (Lloyd and Macaskie, 2002).

3. X-ray absorption spectroscopy

It has become the most important method for determining the speciation of environmental pollutants such as uranium, zinc, lead and arsenic, which are often present in complex, multiphase, natural samples at concentration ranging from greater than a weight percent to less than a ppm in a variety of chemical forms (Brown and Sturchio, 2001). This technique has been used to determine the oxidation state (X- ray absorption near edge spectroscopy, XANES) and to identify the number of atoms and their distances in the local structural environment of metals within a variety of microbial samples (Francis et al., 2004).

4. Time resolved induces laser fluorescence spectroscopy (TRLFS)

TRLFS is a very sensitive method which can give insight into complexation reactions, even with poorly defined polyfunctional ligands such as living bacterial cells. This method is inappropriate for metal ions which show no change in their spectroscopic properties with complexation. It is based on the fact that the measured fluorescence lifetime and intensity of the electronic transition of the excited metal ions are dependent on theor molecular environment (Rustenholtz et al., 2001). The use of this technique is studying the interaction of metal ions with bacteria (Moll et al., 2004) is well documented.

5. Electron spin resonance (ESR) spectroscopy

It is a very powerful and sensitive method for the characterization of the electronic structures of materials with unpaired electrons. This technique can be used for the characterization of transition metal ions that are present in the lattice and on the surface, coordinated to lattice oxygen atoms or to extra-lattice ligands. A considerable enhancement in resolution may be obtained by applying modern ESR-related techniques, electron nuclear double resonance (ENDOR) and a pulse variant of CW-ESR, electron spin echo spectroscopy (ESE), which are capable of measuring nuclear magnetic transition frequencies in paramagnetic systems.

6. Fourier transformed infrared spectroscopy (IR or FTIR)



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FT-IR spectroscopy is a rapid nondestructive analytical tool. Fourier transform-infrared (FT-IR) spectroscopy has been used to rapidly and nondestructively analyze bacteria, bacteria-polymer mixtures, digester samples and microbial biofilms. Diffuse reflectance FT-IR (DRIFT) analysis of freeze-dried, powdered samples offered a means of obtaining structural information. Kamnev (2008) used Fourier transform infrared (FTIR) spectroscopy in various modes (transmission, ATR) to analyse compositional and structural changes in whole bacterial cells in response to different stress factors and plant signals.

7. Thermogravimetric analysis (TGA)

TGA is a thermal analysis technique that has been used to measure changes in the weight loss (mass) of sample that is subjected to a steady increase of temperature so as to quantify reactions involving gaseous emissions (Villain et al., 2007; Reis et al., 2007). The materials analyzed by TGA includes polymers, plastics, composites, laminates, adhesives, food, coatings, pharmaceuticals, organic materials, rubber, petroleum, chemicals, explosives and biological samples.

8. Differential scanning calorimetric analysis (DSC)

In DSC analysis, glass transition temperature (Tg) is related to the mobility of the polymeric chains and it determines the transition between the glassy and the rubbery polymeric state (Vilaplana et al., 2007). DSC is used to measure melting point and phase transition of the composites. This technique is also used to characterize metal microbe relationship.

Conclusion

Bioremediation is a rapidly emerging technology for contaminated soil and waste water treatment. Exploitation of the degradative capacities of microorganisms is the fundamental basis of organic pollutant bioremediation. Composting for the bioremediation of contaminated soil has been successfully used to ameliorate soil contaminated with a variety of organic pollutants. Not only have these methods reduces soil associated pollutant concentrations, but they have also improved soil quality through the addition of organic matter. Bioaugmentation treatment method for contaminated soil is suitable method but it is less promising in the commercial application. Successful bioremediation in the field, however, requires a multidisciplinary approach involving hydrologists, chemical, environmental and agricultural engineers as well as ecologists, biochemists, ecologists and geneticists. The development and application of environmentally sound bioremediation technologies depends on integration of these many different disciplines, as well as advances in our understanding of the physicochemical, biological and ecological factors that govern microbial degradation of organic contaminants both in-situ and ex-situ. Though there are many techniques which are in use for bioremediation of contaminated sites but the inexpensive treatment method of such sites is still to be developed.

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