The past decade has shown, in greater or lesser degree, our carelessness and negligence in using our natural resources. The problems associated with contamination of natural resources are prominently increasing in many countries. Contaminated environment generally result from production, use, and disposal of hazardous substances from industrial activities. The problem is worldwide, and the estimated number of contaminated sites is significant. It is now widely recognized that contaminated environment is a potential threat to human health, and its continual discovery over recent years has led to international efforts to remedy many of these sites, to enable the site to be redeveloped for use.

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To “bioremediate”, means to use living things to eliminate environmental contamination such as contaminated soil or groundwater. Some microorganisms that live in soil and groundwater naturally eat certain chemicals that are harmful to people and the environment. The microorganisms are able to change these chemicals into water and harmless gases, such as carbon dioxide. Plants can also be used to clean up soil, water or air; this is called phytoremediation
Bioremediation is an option that offers the possibility to destroy or render harmless various con­taminants using natural biological activity. As such, it uses relatively low-cost, low-technology tech­niques, which generally have a high public acceptance and can often be carried out on site. It will not always be suitable, however, as the range of contaminants on which it is effective is limited, the time scales involved are relatively long, and the residual contaminant levels achievable may not always be appropriate. Although the methodologies employed are not technically complex, considerable experi­ence and expertise may be required to design and implement a successful bioremediation program, due to the need to thoroughly assess a site for suitability and to optimize conditions to achieve a satisfacto­ry result.
Bioremediation has been used at a number of sites worldwide… Here, we intended to assist by providing a straightforward, pragmatic view of the processes involved in bioremediation, the pros and cons of the technique, and the issues to be considered when dealing with a proposal for bioremediation.
HISTORY
Bioremediation has been described as “a treatability technology that uses biological activity to reduce the concentration or toxicity of a pollutant. It commonly uses processes by which microorganisms transform or degrade chemicals in the environment” (King 1). This use of microorganisms (mainly bacteria) to destroy or transform hazardous contaminants is not a new idea. Microorganisms have been used since 600 B.C. by the Romans and others to treat their wastewater. Although this same technology is still usedtoday to treat wastewater it has been expanded to treat an array of other contaminants. In
fact, bioremediation has been used commercially for almost 30 years. The first commercial use of a bioremediation system was in 1972 to clean up a Sun Oil pipeline spill in Ambler, Pennsylvania
CONVENTIONAL STRATEGIES OF REMEDIATION
The conventional techniques used for remediation have been to dig up contaminated soil and remove it to a landfill, or to cap and contain the contaminated areas of a site. The methods have some drawbacks. The first method simply moves the contamination elsewhere and may create significant risks in the excavation, handling, and transport of hazardous material.
Additionally, it is very difficult and increasingly expensive to find new landfill sites for the final disposal of the material. The cap and contain method is only an temporary solution since the contamination remains on site, requiring monitor­ing and maintenance of the isolation barriers long into the future, with all the associated costs and potential liability.
A better approach than these traditional methods is to completely destroy the pollutants if possi­ble, or at least to transform them to innocuous substances. Some technologies that have been used are high-temperature incineration and various types of chemical decomposition (e.g., base-catalyzed dechlorination, UV oxidation).
They can be very effective at reducing levels of a range of contaminants, but have several drawbacks, principally their technological complexity, the cost for small-scale appli­cation, and the lack of public acceptance, especially for incineration that may increase the exposure to contaminants for both the workers at the site and nearby residents.
Conventional ways of Bioremediation
Dig up and remove it to a landfill
Risk of excavation, handling and transport of hazardous material
Very expensive to find another land to finally dispose these materials
Cap and contain the contaminated area.
Maintain it in the same land but isolate it
Only an temporary solution
Requires monitoring and maintenance of isolation barriers for a long time
Better approaches:
Destroy them completely, if possible
Transform them in to harmless substances
Drawbacks
Technological complexity
The cost for small scale application – expensive
Lack of public acceptance – especially in incineration
Incineration generates more toxic compounds
Materials released from imperfect incineration – cause undesirable imbalance in the atmosphere. Ex. Ozone depletion
Fall back on earth and pollute some other environment
Dioxin production due to burning of plastics – leads to cancer
May increase the exposure to contaminants, for both workers and nearby residents
PRINCIPLES OF BIOREMEDIATION
Figure 1: Bioremediation Triangle
There are three essential components needed for bioremediation. These three components are microorganisms, food, and nutrients. These three main components shown in Figure 1 are known as the bioremediation triangle. Microorganisms are found almost everywhere on earth with the exception of active volcanoes. So a lack of food and nutrients are usually the missing ingredients that prevent successful bioremediation. Microorganisms find the food they eat in the soil or water where they live. However, if a contaminant is present it can become an additional food source for the microorganisms. The contaminant serves two useful purposes for the microbes. First, the contaminant provides a source of carbon needed for growth. Second,the microbes obtain energy by breaking chemical bonds and transferring electrons away from the contaminant. This is known as an oxidation-reduction reaction. The contaminant that loses electrons is oxidized and the chemical that gains the electrons(electron acceptor) is reduced. The energy gained from the electron transfer is used along with the carbon and some electrons to produce more cells. Microbes generally use oxygenas an electron acceptor but nitrate, sulfate, iron, and CO2 are also commonly used. The use of oxygen as an electron acceptor is called aerobic respiration. The major byproducts of aerobic respiration are carbon dioxide, water, and an increase in the microbe population. Anaerobic respiration uses nitrate, sulfate, iron, or CO2 as the electron acceptor instead of oxygen. Anaerobic respiration can occur after the oxygen has been depleted by aerobic respiration or where there is not sufficient oxygen in the first place. The process of anaerobic degradation has been ignored for many years. However, recently it has been gaining more attention;
There are also several nutrients that must be accessible to the microorganisms for bioremediation to be successful. These include moisture, nitrogen, phosphorus, and other trace elements. Microorganisms like other organisms need moisture to survive and grow.In addition, microbes depend on the moisture to transport food to them since they do not have mouths. The optimal moisture content for microbes in the vadose zone has been determined to be between 10 and 25% (King 16). Besides moisture, nitrogen (ammonia)and phosphorus (orthophosphate) are two major nutrients needed for the microorganisms. The microorganisms also require minor elements such as sulfur, potassium, magnesium,calcium, manganese, iron, cobalt, copper, nickel, and zinc (King 19). However, these minor elements are usually available in the environment in sufficient amounts where nitrogen and phosphorus may be lacking and need to be added. There are many contaminants susceptible to bioremediation. Petroleum hydrocarbons, in particular, benzene, toluene, ethylbenzene, and xylene (BTEX), the major components of gasoline, have been biodegraded using this technology. In addition, alcohols, ketones, and esters are well established as being biodegradable by microorganisms. Many other contaminants are emerging as treatable using bioremediation such as halogenated aliphatics, halogenated aromatics, polychlorinated biphenyls, and nitroaromatics.
FACTORS AFFECTING BIOREMEDIATION
The factors affecting bioremediation can be divided into following categories.
Microbial factors
Environmental factors
Microbial Factors
Microorganisms can be isolated from almost any environmental conditions. Microbes will 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 on any waste stream. The main requirements are an energy source and a carbon source. Because of the adaptability of microbes and other biological systems, these can be used to degrade or remediate environmental hazards. We can subdivide these microorganisms into the following groups
Aerobic
Anaerobic
Ligninolytic Fungi
Methylotrophs
Aerobic
These microbes have often been reported to degrade pesticides and hydrocarbons, both alkanes and polyaromatic compounds. Many of these bacteria use the contaminant as the sole source of carbon and energy.
Examples of aerobic bacteria recognized for their degradative abilities are Pseudomonas, Alcaligenes, Sphingomonas, Rhodococcus, and Mycobacterium.
Anaerobic
Anaerobic bacteria are not as frequently used as aerobic bacteria. There is an increasing interest in anaerobic bacteria used for bioremediation of polychlorinated biphenyls (PCBs) in river sediments, dechlorination of the solvent trichloroethylene (TCE), and
chloroform.
Ligninolytic fungi
Fungi such as the white rot fungus Phanaerochaete chrysosporium have the
ability to degrade an extremely diverse range of persistent or toxic environmental pollutants. Common substrates used include straw, saw dust, or corn cobs.
Methylotrophs
Aerobic bacteria that grow utilizing methane for carbon and energy. The initial enzyme in the pathway for aerobic degradation, methane monooxygenase, has a broad substrate range and is active against a wide range of compounds, including the chlorinated aliphatics trichloroethylene and 1,2-dichloroethane.
For degradation it is necessary that bacteria and the contaminants be in contact. This is not easily achieved, as neither the microbes nor contaminants are uniformly spread in the soil. Some bacteria are mobile and exhibit a chemotactic response, sensing the contaminant and moving toward it. Other microbes such as fungi grow in a filamentous form toward the contaminant. It is possible to enhance the mobilization of the contaminant utilizing some surfactants such as sodium dodecyl sulphate (SDS)
Microbes are used to degrade gasoline, the most common contaminant of groundwater in the United States. Adding powdered seaweed to DDT-contaminated soil boosts the cleaning activity of DDT-eating microbes. In one test site, 80% of the DDT was removed after six weeks. Microbes and fungi are used in air filters to control odours from sewage treatment plants and in the paint industry. A gene for a protein found in rat livers that binds with toxic metals has been inserted in both tobacco plants and algae. With this gene, the tobacco plant and the algae are able to extract several hundred times more toxic metal compounds from soil or water compared to plants without the gene. One particular microbe degrades polycyclic aromatic hydrocarbons (PAH’s), which are cancer-causing petroleum by-products. The microbes, called simply “sulfate-reducers”, are able to attack PAH’s in the sediment of Boston Harbor where scientists thought the contaminant could not be treated due to lack of oxygen.
Examples of microbes used for bioremediation include:
Deinococcus radiodurans bacteria have been genetically modified to digest solvents and heavy metals, as well as toluene and ionic mercury from highly radioactive nuclear waste.
Geobacter sufurreducens bacteria can turn uranium dissolved in groundwater into a non-soluble, collectable form.
Dehalococcoides ethenogenes bacteria are being used in ten states to clean up chlorinated solvents that have been linked to cancer. The bacteria are naturally found in both soil and water and are able to digest the solvents much faster than using traditional clean-up methods.
Thermus brockianus, found in Yellowstone National Park, produces an enzyme that breaks down hydrogen peroxide 80,000 times faster than current chemicals in use.
Alcaligenes eutrophus, naturally degrades 2,4-D, the third most widely used herbicide in the U.S.
Some contaminants potentially suitable for bioremediation.
Class of contaminants
Specific examples
Aerobic
Anaerobic
Potential sources
Chlorinated solvents
Trichloroethylene
+
Drycleaners
Perchloroethylene
Chemical manufacture
Polychlorinated biphenyls
4-Chlorobiphenyl
+
Electrical manufacturing
4,4
Dichlorobiphenyl
Power station
Railway yards
Chlorinated phenol
Pentachlorophenol
+
Timber treatment
Landfills
“BTEX”
Benzene
+
+
Oil production and storage
Toluene
Gas work sites
Ethylbenzene
Airports
Xylene
Paint manufacture
Port facilities
Railway yards
Chemical manufacture
Polyaromatic hydrocarbons
Naphthalene
+
Oil production and storage
(PAHs)
Antracene
Gas work sites
Fluorene
Coke plants
Pyrene
Engine works
Benzo(a)pyrene
Landfills
Tar production and storage
Boiler ash dump sites
Power stations
Pesticides
Atrazine
+
+
Agriculture
Carbaryl
Timber treatment
Carbofuran
Pesticide manufacture
Coumphos
Recreational areas
ENVIRONMENTAL FACTORS
1. Nutrients
Although the microorganisms are present in contaminated soil, they cannot necessarily be there in the numbers required for bioremediation of the site. Their growth and activity must be stimulated. Biostimulation usually involves the addition of nutrients and oxygen to help indigenous microorgan­isms. These nutrients are the basic building blocks of life and allow microbes to create the necessary enzymes to break down the contaminants. All of them will need nitrogen, phosphorous, and carbon (e.g., see Table below).
Carbon is the most basic element of living forms and is needed in greater quantities than other elements. In addition to hydrogen, oxygen, and nitrogen it constitutes about 95% of the weight of cells.Phosphorous and sulphur contribute with 70% of the remainders. The nutritional requirement of carbon to nitrogen ratio is 10:1, and carbon to phosphorous is 30:1.
3. Environmental requirements
Optimum environmental conditions for the degradation of contaminants are reported in Table below:
Parameters
Condition required for microbial activity
Optimum value for an oil degradation
Soil moisture
25-28% of water holding capacity
30-90%
Soil pH
5.5-8.8
6.5-8.0
Oxygen content
Aerobic, minimum air-filled pore space of 10%
10-40%
Nutrient content
N and p for microbial growth
C:N:P = 100:10:1
Temperature (°C)
15-45
20-30
Contaminants
Not too toxic
Hydrocarbon 5-10% of dry weight of soil
Heavy metals
Total content 2000 ppm
700 ppm
Type of soil
Low clay or silt content
4. Environmental conditions affecting degradation
Microbial growth and activity are readily affected by pH, temperature, and moisture. Although microorganisms have been also isolated in extreme conditions, most of them grow optimally over a nar­row range, so that it is important to achieve optimal conditions.
If the soil has too much acid it is possible to rinse the pH by adding lime. Temperature affects bio­chemical reactions rates, and the rates of many of them double for each 10 °C rise in temperature. Above a certain temperature, however, the cells die. Plastic covering can be used to enhance solar warming in late spring, summer, and autumn. Available water is essential for all the living organisms, and irrigation is needed to achieve the optimal moisture level. The amount of available oxygen will determine whether the system is aerobic or anaerobic. Hydrocarbons are readily degraded under aerobic conditions, whereas chlorurate compounds are degraded only in anaerobic ones. To increase the oxygen amount in the soil it is possible to till or sparge air. In some cases, hydrogen peroxide or magnesium peroxide can be introduced in the environment. Soil structure controls the effective delivery of air, water, and nutrients. To improve soil structure, materials such as gypsum or organic matter can be applied. Low soil permeability can impede move­ment of water, nutrients, and oxygen; hence, soils with low permeability may not be appropriate for in situ clean-up techniques.
STRATEGIES AND TECHNIQUES INVOLVED IN BIOREMEDIATION
Basically two types of techniques are involved in Bioremediation
In situ Bioremediation (at the site)
Ex situ Bioremediation (away from the site)
In situ Bioremediation
In situ techniques are defined as those that are applied to soil and groundwater at the site with minimal disturbance. These techniques are generally the most desirable options due to lower cost and fewer disturbances since they provide the treatment in place avoiding excavation and transport of contaminants. In situ treatment is limited by the depth of the soil that can be effectively treated. In many soils effective oxygen diffusion for desirable rates of bioremediation extend to a range of only a few centimetres to about 30 cm into the soil, although depths of 60 cm and greater have been effectively treated in some cases.
In situ Bioremediation types:
Bioventing is the most common in situ treatment and involves supplying air and nutrients through wells to contaminated soil to stimulate the indigenous bacteria. Bioventing employs low air flow rates and provides only the amount of oxygen necessary for the biodegradation while minimizing volatiliza­tion and release of contaminants to the atmosphere. It works for simple hydrocarbons and can be used where the contamination is deep under the surface.
In situ biodegradation involves supplying oxygen and nutrients by circulating aqueous solutions through contaminated soils to stimulate naturally occurring bacteria to degrade organic contaminants. It can be used for soil and groundwater. Generally, this technique includes conditions such as the infil­tration of water-containing nutrients and oxygen or other electron acceptors for groundwater treatment.
Biosparging involves the injection of air under pressure below the water table to increase groundwater oxygen concentrations and enhance the rate of biological degradation of contam­inants by naturally occurring bacteria. Biosparging increases the mixing in the saturated zone and there­by increases the contact between soil and groundwater. The ease and low cost of installing small-diam­eter air injection points allows considerable flexibility in the design and construction of the system
Bioaugmentation. Bioremediation frequently involves the addition of microorganisms indigenous or exogenous to the contaminated sites. Two factors limit the use of added microbial cultures in a land treatment unit: 1) nonindigenous cultures rarely compete well enough with an indigenous population to develop and sustain useful population levels and 2) most soils with long-term exposure to biodegrad­able waste have indigenous microorganisms that are effective degrades if the land treatment unit is well managed.
Ex situ bioremediation
Ex situ techniques are those that are applied to soil and groundwater at the site which has been removed from the site via excavation (soil) or pumping (water). These techniques involve the excavation or removal of contaminated soil from ground.
Ex situ Bioremediation types:
These techniques involve the excavation or removal of contaminated soil from ground.
Landfarming is a simple technique in which contaminated soil is excavated and spread over a pre­pared bed and periodically tilled until pollutants are degraded. The goal is to stimulate indigenous biodegradative microorganisms and facilitate their aerobic degradation of contaminants. In general, the practice is limited to the treatment of superficial 10-35 cm of soil. Since landfarming has the potential to reduce monitoring and maintenance costs, as well as clean-up liabilities, it has received much atten­tion as a disposal alternative.
Composting is a technique that involves combining contaminated soil with nonhazardous organ­ic amendants such as manure or agricultural wastes. The presence of these organic materials supports the development of a rich microbial population and elevated temperature characteristic of composting.
Biopiles are a hybrid of landfarming and composting. Essentially, engineered cells are con­structed as aerated composted piles. Typically used for treatment of surface contamination with petro­leum hydrocarbons they are a refined version of landfarming that tend to control physical losses of the contaminants by leaching and volatilization. Biopiles provide a favorable environment for indigenous aerobic and anaerobic microorganisms.
Bioreactors Slurry reactors or aqueous reactors are used for ex situ treatment of contaminated soil and water pumped up from a contaminated plume. Bioremediation in reactors involves the pro­cessing of contaminated solid material (soil, sediment, sludge) or water through an engineered con­tainment system. A slurry bioreactor may be defined as a containment vessel and apparatus used to cre­ate a three-phase (solid, liquid, and gas) mixing condition to increase the bioremediation rate of soil-bound and water-soluble pollutants as a water slurry of the contaminated soil and biomass (usually indigenous microorganisms) capable of degrading target contaminants. In general, the rate and extent of biodegradation are greater in a bioreactor system than in situ or in solid-phase systems because the contained environment is more manageable and hence more controllable and predictable. Despite the advantages of reactor systems, there are some disadvantages. The contaminated soil requires pre-treatment (e.g., excavation) or alternatively the contaminant can be stripped from the soil via soil washing or physical extraction (e.g., vacuum extraction) before being placed in a bioreactor.
Monitoring bioremediation
The process of bioremediation can be monitored indirectly by measuring the Oxidation Reduction Potential or redox in soil and groundwater, together with pH, temperature, oxygen content, electron acceptor/donor concentrations, and concentration of breakdown products (e.g. carbon dioxide). This table shows the (decreasing) biological breakdown rate as function of the redox potential.
Process
Reaction
Redox potential (Eh in mV)
Aerobic:
O2 + 4e− + 4H+ → 2H2O
600 ~ 400
Anaerobic:

Denitrification
2NO3− + 10e− + 12H+ → N2 + 6H2O
500 ~ 200
Manganese IV reduction
MnO2 + 2e− + 4H+ → Mn2+ + 2H2O
400 ~ 200
Iron III reduction
Fe(OH)3 + e− + 3H+ → Fe2+ + 3H2O
300 ~ 100
Sulfate reduction
SO42− + 8e− +10 H+ → H2S + 4H2O
0 ~ −150
Fermentation
2CH2O → CO2 + CH4
−150 ~ −220
Types of Bioremediation
Bioremediation techniques can be subdivided into various based on following factors
Based on type of atmosphere in which Bioremediation takes place it can be divided into two types
Engineered Bioremediation
Intrinsic Bioremediation
Based on Type of organism being used for Bioremediation
Mycoremediation
Phytoremediation
ENGINEERED BIOREMEDIATION
Factors effecting engineered bioremediation
Contact between the microbes and the substrate
Proper physical environment
Nutrients
Oxygen
Absence of toxic compounds
Sources of microorganisms
From contaminated field sites(with varying environmental conditions subzero temperatures or extreme heat, desert conditions or in water, with excess of oxygen or in anaerobic conditions, with presence of hazardous compounds or on any waste stream)
From culture collections
Genetically Engineered Microorganisms (GEMs)
Electro kinetically enhanced bioremediation (EEB) is a method of engineered bioremediation of soil contaminated by such organic compounds as solvents and petroleum products. As depicted schematically in the figure, EEB involves the utilization of controlled flows of liquids and gases into and out of the ground via wells, in conjunction with electrokinetic transport of matter through pores in the soil, to provide reagents and nutrients that enhance the natural degradation of contaminants by indigenous and/or introduced microorganisms.
The operational parameters of an EEB setup can be tailored to obtain the desired flows of reagents and nutrients in variably textured and layered soils of variable hydraulic permeability and of moisture content that can range from saturation down to as little as about 7 percent. A major attractive feature of EEB is the ability to control the movements of charged anionic and cationic as well as noncharged chemical species.
The basic components of electrokinetic enhancement of bioremediation are the following:
* Ions are transported by electromigration; that is, with minimum transport of liquid through the soil. The ions of interest include nutrient agents, electron donors (e.g., lactate) or electron acceptors (e.g., nitrate or sulfate) added to the soil. Electromigration is utilized as an efficient mode of electrokinetic transport in vadosezone soils.
* Water in soil is pumped (horizontally or vertically, depending on the positions of electrode wells) by induced electro-osmotic flow. Whereas the hydraulic flow used in older methods decreases with decreasing pore size and is thus not effective for treating tightly packed soil, electro-osmotic flow is less restricted by tight packing. Electro-osmosis is utilized to enhance the transport of both ions and such noncharged particles as micro-organisms, by moving water from anodes (positive electrodes) toward cathodes (negative electrodes).
* Electrophoresis induced in soil under an applied electric field is used to control the transport and/or distribution of micro-organisms throughout the treated soil volume. The beneficial effect of electrophoresis can be augmented or otherwise modified by use of electro-osmotic flushing of the soil.
* The applied electric current can be utilized to heat the soil to the optimum temperature for bioremediation.
* The gaseous and liquid products of electrolysis of water in the soil are removed from electrode wells and mixed and reinjected into the ground as needed to maintain the pH of the soil within a range favorable for bioremediation.
Disadvantages
Mostly GEMs do not work the way we expect:
Lab strains become food source for soil protozoa
Inability of GEMs to contact the compounds to be degraded
Failure of GEMs to survive/compete indigenous microorganisms. Mostly due to lack / decreased activity of House Keeping Genes.
INTRINSIC BIOREMEDIATION
It is a natural attenuation process that leads to the decrease in contaminant levels in a particular environment due to unmanaged physical, chemical and biological processes.
Conversion of environmental pollutants into the harmless forms through the innate capabilities of naturally occurring microbial population is called intrinsic bioremediation. However, there is increasing interest on intrinsic bioremediation for control of all or some of the contamination at waste sites. The intrinsic i.e. inherent capacity of microorganism, to metabolize the contaminants should be tested at laboratory and field levels before use for intrinsic bioremediation. Through site monitoring programmes progress of intrinsic bioremediation should be recorded time to time. The conditions of site that favours intrinsic bioremediation are ground water flow throughout the year, carbonate minerals to buffer acidity produced during biodegradation supply of electron acceptors and nutrients for microbial growth and absence of toxic compounds. The other environmental factors such as pH concentration, temperature and nutrient availability determine whether or not biotransformation takes place. Bioremediation of waste mixtures containing metals such as Hg, Pb, As and cyanide at toxic concentration can create problem (Madsen, l99l).
The ability of surface bacteria to degrade a given mixture of pollutants in ground water is dependent on the type and concentration of compounds, electron acceptor and duration of bacteria exposed to contaminants. Therefore, ability of indigenous bacteria degrading contaminants can be determined in laboratory by plate count and macrocosm studies
Example: Microbes in Hudson River mud developed an ability to partially degrade PCB (Poly Chlorinated Biphenyls)
Process occurs in two steps
Partial dehalogenation of PCBs occurs naturally under anaerobic conditions
Less chlorinated residues
Then mud is aerated to promote the complete degradation
of these less chlorinated residues
MYCOREMEDIATION
Mycoremediation is a form of bioremediation, the process of using fungi to return an environment (usually soil) contaminated by pollutants to a less contaminated state. The term Mycoremediation was coined by Paul Stam