December 14, 2010

Biotechnology In environment Clean up Process - APPSC G1 Mains - Paper 4 - Section 2 - Unit 3

The term "Environment" is defined as our surroundings which includes the abiotic component (the non living) and biotic component (the living) around us. The abiotic environment includes water, air and soil while the biotic environment consists of all living organisms – plants, animals and microorganisms. Environmental pollution broadly refers to the presence of undesirable substances in the environment which are harmful to man and other organisms. In the past decade or two, there has been a significant increase in the levels of environmental pollution mostly due to direct or indirect human activities. The major sources of environmental pollution are –Industries, Agricultural sources (mainly rural area), anthropogenic sources (man related activities mainly in urban areas), biogenic sources etc. The pollutants are chemical, biological and physical in nature. The Chemical pollutants include- gaseous pollutants (hazardous gases like sulfur dioxide, nitrogen oxide), toxic metals, pesticides, herbicides toxins and carcinogens
Etc. The physical pollutants are- heat, sound, radiation, and radioactive substances. The pathogenic organisms and some poisonous and dangerous biological products are the biological pollutants.

Controlling the environmental pollution and the conservation of environment and biodiversity and controlling environmental pollution are the major focus areas of all the countries around the world. In this context the importance and impact of biotechnological approaches and the implications of biotechnology has to be thoroughly evaluated. There have been serious concerns regarding the use of biotechnology products and the impact assessment of these products due to their interaction with the environmental factors.

A lobby of the environmentalists have expressed alarm on the release of genetically engineered organisms in the atmosphere and have stressed on thorough investigation and proper risk assessment of theses organisms before releasing them in to the environment. The effect of the effluents from biotechnological companies is also a cause of concern for everyone. The need of the hour is to have a proper debate on the safety of the use of the biotechnological products.

The efforts are not only on to use biotechnology to protect the environment from pollutionbut also to use it to conserve the natural resources. As we all know that microorganisms are known natural scavengers so the microbial preparations (both natural as well as genetically engineered) can be used to clean up the environmental hazards.

Development of alternate cleaner technologies using biotechnology

Biotechnology is being used to provide alternative cleaner technologies which will help to further reduce the hazardous environmental implications of the traditional technologies. E.g. some Fermentation technologies have some serious environmental implications. Various biotechnological processes have been devised in which all nutrients introduced for fermentation are retained in the final product, which ensures high conversion efficiency and low environmental impact.

In paper industry, the pulp bleaching technologies are being replaced by more environmentally friendly technologies involving biotechnology. The pulp processing helps to remove the lignin without damaging valuable cellulosic fibres but the available techniques suffer from the disadvantages of high costs, high energy use and corrosion. A lignin degrading and modifying enzyme (LDM) was isolated from Phanerochaete chrysosporum and was used, which on one hand, helped to reduce the energy costs and corrosion and on the other hand increased the life of the system. This approach helped in reducing the environmental hazards associated with bleach plant effluents.

In Plastic industry, the conventional technologies use oil based raw materials to extract ethylene and propylene which are converted to alkene oxides and then polymerized to form plastics such as polypropylene and polyethylene. There is always the risk of these raw materials escaping into the atmosphere thereby causing pollution. Using biotechnology, more safer raw materials like sugars (glucose) are being used which are enzymatically or through the direct use of microbes converted into alkene oxides.e.g. Methylococcus capsulatus has been used for converting alkene into alkene oxides.

Integration of biological steps in pulping process leading to lignin degradation


Bioremediation is defined as 'the process of using microorganisms to remove the environmental pollutants where microbes serve as scavengers. The removal of organic wastes by microbes leads to environmental cleanup. The other names/terms used for bioremediation are biotreatment, bioreclamation, and biorestoration. The term "Xenobiotics" (xenos means foreign) refers to the unnatural, foreign and synthetic chemicals such as pesticides, herbicides, refrigerants, solvents and other organic compounds. The microbial degradation of xenobiotics also helps in reducing the environmental pollution.

Pseudomonas which is a soil microorganism effectively degrades xenobiotics. Different strains of Pseudomonas that are capable of detoxifying more than 100 organic compounds (e.g. phenols, biphenyls, organophosphates, naphthalene etc.) have been identified. Some other microbial strains are also known to have the capacity to degrade xenobiotics such as Mycobacterium, Alcaligenes, Norcardia etc.

Factors affecting biodegradation

The factors that affect the biodegradation are: the chemical nature of xenobiotics, the concentration and supply of nutrients and O2, temperature, pH, redox potential and the capability of the individual microorganism. The chemical nature of xenobiotics is very important because it was found out that the presence of halogens e.g. in aromatic compounds inhibits biodegradation. The water soluble compounds are more easily degradable whereas the presence of cyclic ring structure and the length chains or branches decrease the efficiency of biodegradation. The aliphatic compounds are more easily degraded than the aromatic ones.


It is a process by which the microbial activity can be enhanced by increased supply of nutrients or by addition of certain stimulating agents like electron acceptors, surfactants etc.


It is possible to increase biodegradation through manipulation of genes i.e. using genetically engineered microorganisms and by using a range of microorganisms in biodegradation reaction.

Depending on the method followed to clean up the environment, the bioremediation is carried out in two ways:

A) In situ bioremediation - In situ bioremediation involves a direct approach for the microbial degradation of xenobiotics at the site of pollution which could be soil, water etc. The adequate amount of essential nutrients is supplied at the site which promotes the microbial growth at the site itself. The in situ bioremediation is generally used for clean up of oil spillages, beaches etc. There are two types of in situ bioremediation-

1) Intrinsic bioremediation- The microorganisms which are used for biodegradation are tested for the natural capability to bring about biodegradation. So the inherent metabolic ability of the microorganisms to degrade certain pollutants is the intrinsic bioremediation. 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 the duration of bacteria exposed to contamination. Therefore, the ability of indigenous bacteria degrading contaminants can be determined I laboratory by using the techniques of plate count and microcosm studies. The conditions of site that favour 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.

2) Engineered in situ bioremediation- When the bioremediation process is engineered to increase the metabolic degradation efficiency (of pollutants) it is called engineered in situ bioremediation. This is done by supplying sufficient amount of nutrients and oxygen supply, adding electron acceptors and maintaining optimal temperature and pH. This is done to overcome the slow and limited bioremediation capability of microorganisms.


Advantages of in situ bioremediation
a) The method ensures minimal exposure to public or site personnels.
b) There is limited or minimal disruption to the site of bioremediation.
c) Due to these factors it is cost effective.
d) The simultaneous treatment of contaminated soil and water is possible.

Disadvantages of in situ bioremediation
a) The sites are directly exposed to environmental factors like temperature, oxygen supply etc.
b) The seasonal variation of microbial activity exists.
c) Problematic application of treatment additives like nutrients, surfactants, oxygen etc.
d) It is a very tedious and time consuming process.

B) Ex-situ bioremediation - In this the waste and the toxic material is collected from the polluted sites and the selected range of microorganisms carry out the bioremediation at designed place. This process is an improved method over the in situ bioremediation method. On the basis of phases of contaminated materials under treatment ex-situ bioremediation is classified into two : a) Solid phase system and (b) Slurry phase systems.


A) Solid phase treatment- This system includes land treatment and soil piles comprising of organic wastes like leaves, animal manures, agricultural wastes, domestic and industrial wastes, sewage sludge, and municipal solid wastes. The traditional clean-up practice involves the informal processing of the organic materials and production of composts which may be used as soil amendment. Composting is a self heating, substrate-dense, managed microbial system which is used to treat large amount of contaminated solid material. Composting can be done in open system i.e. land treatment and/or in closed treatment system. The hazardous compounds reported to disappear through composting includes aliphatic and aromatic hydrocarbons and certain halogenated compounds. The possible routes leading to the disappearance of hazardous compounds include volatilization, assimilation, adsorption, polymerization and leaching.

B) Slurry phase treatment- This is a triphasic treatment system involving three major components- water, suspended particulate matter and air. Here water serves as suspending medium where nutrients, trace elements, pH adjustment chemicals and desorbed contaminants are dissolved. Suspended particulate matter includes a biologically inert substratum consisting of contaminants and biomass attached to soil matrix or free in suspending medium. The contaminated solid materials, microorganisms and water formulated into slurry are brought within a bioreactor i.e. fermenter. Biologically there are three types of slurry-phase bioreactors: aerated lagoons, low shear airlift reactor, and fluidized-bed soil reactor. The first two types are in use of full scale bioremediation, while the third one is in developmental stage.

Advantages of ex-situ bioremediation
a) As the time required is short, it is a more efficient process.
b) It can be controlled in a much better way.
c) The process can be improved by enrichment with desired and more efficient microorganisms.

Disadvantages of ex-situ bioremediation
a) The sites of pollution remain highly disturbed.
b) Once the process is complete, the degraded waste disposal becomes a major problem.
c) It is a costly process.

Several types of reactions occur during the bioremediation/microbial degradation

a) Aerobic bioremediation- When the biodegradation requires oxygen O2 for the oxidation of organic compounds, it is called aerobic bioremediation. Enzymes like monooxygenases and dioxygenases are involved and act on aliphatic and aromatic compounds.
b) Anaerobic bioremediation-This does not require oxygen O2. the degradation process is slow but more cost effective since continuous supply of oxygen is not required.
c) Sequential bioremediation- Some of the xenobiotic degradation requires both aerobic as well as anaerobic processes which very effectively reduces the toxicity e.g. tetrachloromethane and tetrachloroethane undergo sequential degradation.

Use of genetic engineering and genetic manipulations for more efficient bioremediation

In recent years, efforts have been made to create genetically engineered microorganisms (GEMs) to enhance bioremediation. This is done to overcome some of the limitations and problems in bioremediation. These problems are:

a) Sometimes the growth of microorganisms gets inhibited or reduced by the xenobiotics.
b) No single naturally occurring microorganisms has the capability of degrading all the xenobiotics present in the environmental pollution.
c) The microbial degradation is a very slow process.
d) Sometimes certain xenobiotics get adsorbed on to the particulate matter of soil and thus become unavailable for microbial degradation.

As the majority of genes responsible for the synthesis of enzymes with biodegradation capability are located on the plasmids, the genetic manipulations of plasmids can lead to the creation of new strains of bacteria with different degradative pathways. In 1970s, Chakrabarty and his team of co-workers reported the development of a new strain of bacterium Pseudomonas by manipulations of plasmid transfer which they named as "superbug". This superbug had the capability of degrading a number of hydrocarbons of petroleum simultaneouslysuch as camphor, octane, xylene, naphthalene etc. In 1980, United States granted the patent to this superbug making it the first genetically engineered microorganism to be patented.

In certain cases, the process of plasmid transfer was used. E.g. The bacterium containing CAM (camphor degrading ) plasmid was conjugated with another bacterium with OCT (octane degrading) plasmid. Due to non-compatibility, these plasmids cannot coexist in the same bacterium. However, due to the presence of homologous regions of DNA, recombination occurs between these two plasmids which results in a single CAM-OCT plasmid giving the bacterium the capacity to degrade both camphor as well as octane.

A new strain of Pseudomonas sp. (strain ATCC 1915) has been developed for the degradation of vanillate (which is a waste product from paper industry) and sodium dodecyl sulfate (SDS, a compound used in detergents).

Biotechnological method to reduce atmospheric carbon dioxide (CO2)

Carbon dioxide is the gas that is the main cause of green house effect and rise in the atmospheric temperature. During the past 100-150 years, the level of CO2 has increased about 25% with an increase in the atmospheric temperature by about 0.5% which is a clear indication that CO2 is closely linked with global warming. There is a steady increase in the CO2 content due to continuous addition of CO2 from various sources particularly from industrial processes. It is very clear that the reduction in atmospheric CO2 concentration assumes significance. Biotechnological methods have been used to reduce the atmospheric CO2 content at two levels:
a) Photosynthesis- Plants utilize CO2 during the photosynthesis which reduces the CO2 content in the atmosphere. The equation for photosynthesis is:
6CO2 + 6H2O---------->C 6 H12 O6 + 6O2

The fast growing plants utilize the CO2 more efficiently for photosynthesis. The techniques of micropropagation and synthetic seeds should be used to increase the propagation of such fast growing plants.

Further, the CO2 utilization can be increased by enhancing the rate of photosynthesis. The enzyme ribulose biphosphate carboxylase (RUBP-case) is closely linked with CO2 fixation. The attempts are being made to genetically manipulate this enzyme so that the photosynthetic efficiency is increased.

Some microalgae like Chlorella pyrenodiosa, Spirulina maxima are known to be more efficient than higher plants in utilizing atmospheric CO2 for photosynthesis and generate more O2 than the amount of CO2 consumed.
The growing of these microalgae near the industries and power plants (where the CO2 emission in to atmosphere is very high) will help in the reduction of polluting effects of CO2. Using genetic engineering, attempts are going on to develop new strains of these microalgae that can tolerate high concentrations of CO2. A limited success has already been reported in the mutants of Anacystis nidulans and Oocystis sp.

b) Biological Calcification- Certain deep sea organisms like corals, green and red algae store CO2 through a process of biological calcification. As the CaCO3 gets precipitated, more and more atmospheric CO2 can be utilized for its formation. The process of calcification is as follows:

H2O + CO2---------->H2CO3
H 2CO3 + Ca 2+----------------> CaCO3 + CO2 + H2O

Treatment of sewage using microorganisms

The sewage is defined as the waste water resulting from the various human activities, agriculture and industries and mainly contains organic and inorganic compounds, toxic substances, heavy metals and pathogenic organisms. The sewage is treated to get rid of these undesirable substances by subjecting the organic matter to biodegradation by microorganisms. The biodegradation involves the degradation of organic matter to smaller molecules (CO2, NH3, PO4 etc.) and requires constant supply of oxygen. The process of supplying oxygen is expensive, tedious, and requires a lot of expertise and manpower. These problems are overcome by growing microalgae in the ponds and tanks where sewage treatment is carried out. The algae release the O2 while carrying out the photosynthesis which ensures a continuous supply of oxygen for biodegradation.

The algae are also capable of adsorbing certain heavy toxic metals due to the negative charges on the algal cell surface which can take up the positively charged metals. The algal treatment of sewage also supports fish growth as algae is a good source of food for fishes. The algae used for sewage treatment are Chlorella, Euglene, Chlamydomnas, Scenedesmus, Ulothrix, Thribonima etc.

Role of biotechnology in restoration of degraded lands

The urbanization and increased human activity has led to degradation of habitats. The restoration of the degraded lands can be carried out by using biotechnology which involves the manipulations of biological systems. This restoration could be carried out by the following biotechnological methods:

a) Use of micropropagation and mycorrhiza for reforestation

One of the approaches to tackle this problem is to develop strong and superior species which have the capability to grow well on degraded lands. This can be done by using mass multiplication which involves starting aseptic culture, multiplication of shoot using shoot apical meristems or buds, rooting of in vitro formed shoots, transfer, acclimatization and adaptation of micropropagated plantlets in the field. Using this methodology an estimated 500 million plants of diverse nature have been produced.
Mycorrhizae, which are symbiotic non-pathogenic associations between plant roots and fungi, improves the seedling survival and growth by enhancing uptake of nutrients and water. They also lengthen the root life and provide protection against the pathogens. A list of fungi which can efficiently form mycorrhizae has been prepared. These fungi can be used as inocula which are applied to roots of seedlings, to allow formation of mycorrhizae. The experimental infection of micropropagated plants during rooting increases their survival chances in the field, which is very important in case of plantations on degraded lands.

b) Improvement of soil infertility through the use of nitrogen fixing bacteria, Rhizobium in association with leguminous trees and Frankia in association with non leguminous species.

Biotechnological methods are being developed to help the non-leguminous plants to survive under adverse conditions such as low nutrient supply. There are about 160 species of angiosperms, which are known to form nitrogen fixing root nodules with the actinomycetes bacteria belonging to the genus Frankia which is being used for this purpose. Frankia helps in nitrogen fixation in non-leguminous plant species therefore it can be used for land reclamation through reforestation due to high biomass production
with out the need of expensive nitrogen fertilizers.

c) Development of plants tolerant to abiotic stress which can be grown on degraded lands

The techniques like tissue culture and genetic engineering are being used to develop plants resistant to abiotic stresses e.g. salinity, acidity, aluminium toxicity etc. The cell lines which exhibit resistance to salt stress are selected and then used for plantation on degraded lands. E.g. Brassica spp., Citrus aurantium, Nicotiana tabacum etc. Research is going on to understand the molecular basis of salt tolerance and to isolate genes responsible for this attribute so that salt tolerant plants can be developed using genetic engineering. In vitro selection for tolerance to abiotic stress like aluminium toxicity has been successful in certain plant species e.g. tomato, rice, barley, rice and wheat.
"Triticale" which is a man made synthetic crop has been found suitable for growing on acid soils, dry and sandy soils, on alkaline and calcareous soils and on mineral deficient and high boron soils especially in countries like Kenya, Ethiopia, Ecuador, Mexico, Brazil etc. In China, a number of new stress resistant varieties of rice, wheat and tobacco have been developed using anther culture.

e) Use of selected and engineered microbes for removal and recovery of strategic and precious metals from contaminated degraded lands.

The domestic and industrial effluents often contain harmful heavy metals. These heavy metals cause soil contamination when these effluents are used for irrigation purposes. The biotechnological methods and procedures are being developed to prevent the contamination by these heavy metals and also restore the contaminated soils. This involves the selective use of engineered microbes. Plasmids have been constructed which can enhance the recovery of gold from arsenopyrite ores, by Thiobacillus ferroxidans. Ganoderma lucidum which is a wood rotting macrofungus , is a highly potential biosorbent material for heavy metals and thus can be used to control contamination by heavy metals.

The metal pollution occurs through several processes. As the living organisms including man are constantly exposed to metals, they accumulate by a process referred to as 'bioaccumulation.' The continuous exposure and accumulation of a given metal leads to increase in it's concentration which is referred to as 'biomagnification'. Biomagnification occurs through food chain and the man gets the maximum impact due to it's being on top of the food chain. The 'biomethylation' is carried out by microorganisms in the soil and water and involves the process of transfer of methyl groups from organic compounds to metals.

Some phytoplanktons (plants that float freely on water surface) and some benthics (plants attached to some substratum at the bottom of aquatic bodies) microorganisms can take up the metals from the waste water ponds. These natural bioscavengers not only control the water pollution by absorbing metals but also contributes in the recovery of industrially important metals from the effluents. The microorganisms like algae can absorb metals form the fresh water e.g. Chlorella vulgaris takes up copper, mercury, uranium. Certain fungal species like Rhizopus, Aspergillus, Pencillium, Neurospora are good absorbers of heavy metals like lead, mercury etc. Some of the bacterial species are capable of accumulating metals on cell walls such as E. coli can take up mercury while Bacilus circulans can accumulate copper.
The mechanism of metal scavenging by these microorganisms is very complex and involves multiple steps. Some of the microorganisms bioaccumulate these metals on their cell walls whereas some others have the capacity to transport these metals to intracellular and intercellular free space and cellular organelles. In certain cases some of the metals occur as immobilized metal containing crystals e.g heavy metal complexes of calcium oxalate crystals. Some of the fungal and algal species synthesize metal binding proteins or peptides. 'Phytochelatin' is an ubiquitious metal chelating protein present in all plants and acts as a common buffering molecule for the homeostasis of metals. It is rich in cysteine and can form salt metal complexes through sulfhydral (SH) groups. Due to this property, phytochelatin can be used as a biomarker for metal pollution detection.

The mechanisms involved in the removal of metals by microorganisms are: adsorption, complexation, precipitation and volatilization. The process of adsorption involves the binding of metal ions to the negatively charged cell surfaces of microorganisms. The process of complexation leads to production of organic acids e.g. citric acid, oxalic acid, gluconic acid, lactic acid, malic acid etc. which chelate the metal ions. In precipitation, the metals are precipitated as hydroxides or sulfates by some bacteria such as which produce ammonia, organic bases or H2S.e.g. Desulfovibrio and Desulfotomaculum transform SO4 to H2S which promotes extracellular precipitation of insoluble metal sulfides. Klebsiella aerogenes detoxifies cadmium sulphate which precipitates on cell surface. Volatilization involves bacteria that causes methylation of Hg2+ and converts to dimethyl mercury which is a volatile compound.

Whole cell of Bacillus subtilis have been shown to reduce gold from Au3+ to Au 0 through extracellular enzymatic biotransformation. Under anoxic environment, sulphate- reducing bacteria (Desulfovibrio) oxidize organic matter using sulphate as an electron acceptor. In yeast, Saccharomyces cerevisiae removal of metals is done by their precipitation as sulphides e.g. Cu2+ is precipitated as CuS.

Several technologies for metal removal have been commercialized and employed are given below:
- ATMBIOCLAIMTM process: The advance Mineral Technology (ATM) Inc. (U.S.A.) developed a waste water treatment process with Bacillus sp. immobilized and pre-treated in caustic solution. It is specific for metal cations in the order: Cu2+ > Zn2+ > Cd2+ = Ni2+ > Pb2+.
- AlgaSORBTM process: Biorecovery systems, Inc. (U.S.A) developed this proprietary based material which consists of several types of living and non living algae. The algal cultures are immobilized in silica gel in the form of beads and desorption of metals is carried out.
- Bioremediation of coal wastes through VAM fungi: Selected VAM fungi are introduced through plants in coal mine areas where it was found that VAM fungi improved the growth and survival of desirable re-vegetation species e.g. red maple, maize, alfalfa etc.
- BIO-FIXTM process: The bureau of Mines (U.S.A) developed this process that consists of biomass immobilized in polysulfone. It consists of thermally killed biomass of Sphagnum pat moss, algae, yeast, bacteria and/or aquatic flora. The beads are suitable for practical application in stirred tank reactor, fixed and fluidized-bed columns.

f) Use of biotechnology in the conservation of biodiversity

The extinction of wild species due to the destruction of habitats and ecosystems has raised serious concerns about the biodiversity in general. Biodiversity provides genes from wild species for biotechnology exercises and experiments hence biotechnology and biodiversity are interrelated. Besides taking steps to minimize and regulate the factors responsible for causing loss of biodiversity, efforts are on to develop the techniques of conservation of biodiversity. One of the methods involves the establishment of "gene banks" leading to "in situ conservation and ex situ conservation. The in situ conservation involves the conservation of plants and animals in their natural habitat and ecosystems. The ex situ conservation includes conservation of species away from their habitats. The ex situ conservation uses sample populations and establishes the "gene banks" which includes resource centers, zoos, botanical gardens, national parks, culture collection centers etc.

Biotechnology offers special methods to conserve both animal and plant genetic resources especially in the conservation of endangered plant species. The tissue culture method is being used to multiply an endangered plant species. The method of embryo transfer and artificial insemination is used for the multiplication of endangered animal species.

Use of biotechnology in reducing the use of chemical pesticides, herbicides and fertilizers

A lot of debate is going on the overuse of chemical herbicides, pesticides and fertilizers. They become an environmental hazard because they undergo degradation by microorganisms and ultraviolet light which releases toxic chemicals in the environment. Using biotechnology, bacterial pesticides and viral pesticides are being developed which will help in reducing the use of chemical pesticides. Several companies in USA like Monsanto, Mycogen, Ecogen, Repligen, Zoecon etc are actively involved in the development of biological pesticides. The trials are going on to use the genetically engineered live soil bacteria for coating seeds before planting. Another method being tried is to kill the recombinant bacteria and apply them to the leaves of crop plants. Both these approaches protect the toxin from degradation by microbes or ultraviolet rays once applied to the crop plants.

The company Ecogen Inc. was involved in developing biological pesticides against the two major crop pests budworm and ballworm by transferring a gene from Bacillus thuringiensis (Bt), into either a naturally occurring soil bacterium or in to a strain of Pseudomonas. Bt insecticides are already being marketed for past few years and in future these will be modified using genetic engineering and will be used against a variety of insects.
Genetically engineered insect resistant plants have been successfully produced which will further help in reducing the use of insecticides in the future.

The experiments are going on to develop environmentally safe herbicides. In order to use these herbicides for crop protection programme, genetically engineered herbicide resistant plants have been produced in a variety of crop plants. This will ensure the use of environmentally safer herbicides.


Biofertilizers are also being used in place of chemical fertilizers to further reduce the environmental hazards caused due to chemical fertilizers. The term biofertilizers is used to refer to the nutrient inputs of biological origin to support plant growth which is generally achieved by the addition of microbial inoculants as a source of biofertlizers. Biofertilizers broadly includes the following categories:

A)Symbiotic nitrogen fixers- The diazotrophic microorganisms are the symbiotic nitrogen fixers that serve as biofertilizers e.g. Rhizobium sp., Bradyrhizopium sp.

B) Asymbiotic nitrogen fixers- The asymbiotic nitrogen fixing bacteria can directly convert the gaseous nitrogen to nitrogen rich compounds. On the death of these nitrogen fixers, the soil becomes enriched with nitrogenous compounds thereby serving as biofertilizers e.g. Azobacter sp., Azospirillum sp.

The blue green algae, multiply in the water logging conditions and fixes the nitrogen. They accumulate the biomass which helps in improving the physical properties of the soil. This is useful for reclamation of alkaline soils besides providing partial tolerance to pesticides. The most common blue green algae are Azobacter sp. and Azospirillum sp. Azolla, which is an aquatic fern contains an endophytic cynobacterium Anabaena azollae in the leaf cavities providing symbiotic relationship. Azolla with Anaebaena is useful as biofertilizer.

C) Phosphate solubilising bacteria- Some bacteria like Thiobacillus, Bacillus are capable of converting non-available inorganic phosphorus present in the soil to organic or inorganic form of phosphate. These bacteria can also produce siderophores, which chelates with iron, and makes it unavailable to pathogenic bacteria. Siderophores are iron-binding low molecular weight (400- 1,000 Daltons) peptides synthesized by some soil bacteria.

D) Organic fertilizers- Certain types of organic wastes are used as fertilizers e.g. animal dung (cow dung, elephant dung etc.), urine, urban garbage, sewage, crop residues and oil cakes. All these wastes can be converted in to organic manures.

Advantages of using biofertilizers

- Biofertilizers improve the tolerance of plants against toxic heavy metals.
- It is possible to reclaim saline or alkaline soil by using biofertilizers.
- Use of biofertilizers helps in controlling environmental pollution.
- Fertility of soil is increased year after year.
- Low cost and easy to produce.
- Biofertilizers increase the physico-chemical properties of the soil, soil texture and water holding capacity.

Some of the limitations encountered while using the biofertilizers are that they alone cannot meet the total needs of the plants for nutrient supply and also they do not produce the spectacular results as observed in synthetic fertilizers. It is important to evolve an approach which can maximize the use of biofertilizers and reduce the dependency on the chemical fertilizers in the near future with out affecting the crop productivity. This will help us to solve the environment related problems caused due to overuse of chemical fertilizers.

Use of biotechnology in the removal of Oil and Grease deposits

The oil spills from oil tankers on land surface as well as in seas and oceans are a major environmental hazard. This not only kills the aquatic flora and fauna by destroying the habitat but also creates health problems for the local inhabitants. Traditionally chemical dispersants are being used as remediation efforts. However these chemical dispersants are also toxic in nature and they persist in the environment for a long time. The present techniques of washing the oil off the gravel and cleaning the area of oil spills, is very expensive and time consuming. In order to overcome some of these problems, the oleophilic fertilizers are being developed which allow rapid growth and multiplication of microbes which further leads to the increase in the biodegradation process for removal of oil. In recent years, using genetic engineering, oil utilizing microorganisms have been produced which can grow rapidly on oil. The genetically engineered microbes for cleaning oil spills are mixed with straw. At the site of oil spill, the straw mixed with microbes are scattered over the oil spilled area. The straw soaks the oily water and the microbes break the oil into non-toxic and non polluting materials thereby cleaning up the site.

Some of the oil utilizing microbes can also produce surface active compounds that can emulsify oil in water and thereby removing the oil. A strain of Pseudomonas aeruginosa produces a glycolipid emulsifier that reduces the surface tension of oil-water interface which helps in the removal of oil from water. This microbial emulsifier is nontoxic and biodegradable and has shown promising results in the laboratory experiments.

Some of the microorganisms which are capable of degrading petroleum include pesudomonads, various corynebacteria, mycobacteria and some yeasts. The two methods for bioremediation of oil spills are: a) using a consortium of bacteria, and b) using genetically engineered bacteria/microbial strains. (discussed under the topic of bioremediation) Both bacterial and fungal cultures from the petroleum sludge have been isolated. The fungal culture could degrade 0.4% sludge in 3 weeks. Degradation of petroleum sludge occurred within two weeks when the bacterial culture (Bacillus circulans CI) was used. A significant degradation of petroleum sludge was observed in 10 days when the fungus + B. Circulans and a prepared surfactant were exogenuously added to petroleum sludge.

Use of biotechnology for toxic site reclamation

Generally incineration (drying and then burning to ashes in furnace) or chemical treatment are being used to get rid of toxins and waste from the waste disposal sites. Of late, biotechnological techniques involving biodegradation as an alternative approach is being used. Companies like BioTechnica are working on treating polluted site in situ. However there is a lot of debate over the issue regarding the release of genetically engineered microbes for treatment of toxic sites and the risk involved in the whole procedure. As we know that the released engineered organisms have the capacity to reproduce, spread to sites other then the initial release sites and may undergo mutations. All this can lead to the risk of developing what are described as "super bugs". Some of the companies in US are experimenting and conducting their work in the closed reactors in order to further evaluate the risk assessment and cost effectiveness of this approach.

In order to solve the problem of soil pollution caused due to extensive use of herbicides, pesticides and insecticides, the bioremediation of soil using microorganisms is being carried out. The most common pollutants are: hydrocarbons, chlorinated, solvents, polychlorobiphenyls and metals. The bioremediation of soil involves two processes:

In order to solve the problem of soil pollution caused due to extensive use of herbicides, pesticides and insecticides, the bioremediation of soil using microorganisms is being carried out. The most common pollutants are: hydrocarbons, chlorinated, solvents, polychlorobiphenyls and metals. The bioremediation of soil involves two processes:

a) Biostimulation- Biostimulation involves the stimulaton of microorganisms already present in the soil. This can be done by adding nutrients e.g. nitrogen, phosphorus etc., by supplying co-substrates e.g. methane which can degrade trichloroethylene, or by adding surfactants to disperse the hydrophobic compounds in water.

b) Bioaugmentation- Addition of specific microorganisms to the polluted soil constitutes bioaugmentation. Some of the pollutants like polychlorobiphenyls (PCBs), trinitrotoluene (TNT), polyaromatic hydrocarbons (PAHs) etc are not degraded by only native soil microorganisms so a combination of microorganisms referred to as "consortium" or "cocktail" of microorganisms is added to achieve bioaugmentation.

c) Bioventing- Bioventing involves aerobic biodegradation of pollutants by circulating air through sub-surfaces of soil and is one of the very cost effective and efficient technique used for the bioremediation of petroleum contaminated soils. It is very effectively used for degradation of soluble paraffins, and polyaromatic hydrocarbons.

d) Phytoremediation- Bioremediation by using plants is called phytoremediation. Certain plant species which have the capability to stimulate biodegradation of pollutants (specially near the soil adjacent to roots- rhizophere) are cultivated near the sites of polluted soil. This is a cheap and environmentally friendly process but takes a long time to finish the clean up process.

e) Land farming- Landfarming is a technique for the bioremediation of hydrocarbon contaminated soils. In this the soil is excavated, mixed with microorganisms and nutrients and spread out on a liner just below the polluted soil.

f) Use of slurry-phase bioreactors- In this process, the excavated polluted soil is subjected to bioremediation under optimal controlled conditions in specifically designed bioreactors.

Table showing engineered bacteria used for the degradation of xenobiotics and toxic wastes


Substrate that can be degraded

Pseudomonas capacia

2,4,5- trichoro-phenoxyacetic acid

P. putida & other spp (also E.Coli)

2,2,5-dichloropropionate; mono and dichloroaromatics

Alcaligenes sp.

Dichlorophenoxyacetic acid, mixed chlorophenols; 1,4- dichlorobenzene

Acinetobacter sp.



Substrate that can be degraded

Pseudomonas capacia

2,4,5- trichoro-phenoxyacetic acid`

P. putida & other spp (also E.Coli)

2,2,5-dichloropropionate; mono and dichloroaromatics

Alcaligenes sp.

Dichlorophenoxyacetic acid, mixed chlorophenols; 1,4- dichlorobenzene

Acinetobacter sp.


Use of Biosensors to detect environmental pollutants

Biosensors are biophysical devices which can detect the presence of specific substances e.g. sugars, proteins, hormones, pollutants and a variety of toxins in the environment. They are also capable of measuring the quantities of these specific substances in the environment.

Technically a "Biosensor" is defined as "an analytical device containing an immobilized biological material (which could be an enzyme, or antibody, or nucleic acid, or hormone, or an organelle/whole cell), which can specifically interact with an analyte and produce physical, chemical or electrical signals that can be measured. An analyte is the compound (e.g. glucose, urea, drug, pesticide) whose concentration has to be measured. Biosensors basically involve the quantitative analysis of various substances by converting their biological actions into measurable signals. Generally the performance of the biosensors is mostly dependent on the specificity and sensitivity of the biological reaction, besides the stability of the enzyme.
A biosensor or an enzyme or an antibody is associated with microchip devices which is used for quantitative estimation of the substance. A biosensor equipment has the following components
a) a biological component - enzyme, cell etc,
b) a physical component-a device for measuring the quantity of this product, thus indirectly giving an estimate of the substrate e.g. transducer, amplifier etc.

The biosensors are being used in the area of medicine, industry etc. however their use in environmental monitoring is of great benefit. Special kits have been designed to identify the specific pollutants in the environment. E.g. special cost effective enzymatic tests are available which can detect pesticide contamination in water.

Principle of a biosensor

The biological material in use (e.g. an enzyme) is immobilized by conventional methods like physical or membrane entrapment, non-covalent or covalent binding. A contact is made between the immobilized biological material and the transducer. The analyte binds to the biological material to form a bound analyte which in turn produces the electronic response that can be measured. Sometimes the analyte is converted to a product which could be associated with the release of heat, gas (oxygen), electrons or hydrogen ions. The transducer then converts the product linked changes into electrical signals which can be amplified and measured.

A good example of a biosensor in frequent use is the glucose oxidase enzyme. The enzyme is immobilized on an electrode surface which acts as an electrocatalyst for oxidation of glucose. The biosensor gives reproducible electrical signal for glucose concentrations as low as 0.15 mM.

Another area where biosensors are being used is "Biomonitoring" or "biological monitoring". Biomonitoring is defined as the measurement and assessment of work place agents or their metabolites either in tissues, secreta, excreta, or any combination of these systems in occupationally exposed human subjects. The "Biological effect monitoring" refers to the biological effects of these toxic agents in the workers exposed to these agents. A continuous evaluation of biological monitoring methods is done in order to assess the risk effectiveness of these tests against the various kinds of exposures to toxins. The use of genetic engineering to create organisms specifically designed for bio remediation also has great potential. The bacterium Deinococcus radiodurans , which is the most radioresistant organism known, has been modified to consume and digest toluene and ionic mercury from highly radioactive nuclear waste.

Some of the important biosensors used in environmental pollution monitoring are:

a) Gas biosensors- In order to detect gases such as sulphur dioxide, (SO2), methane, carbon dioxide etc, microbial biosensors have been developed. Thiobacillus-based biosensors can detect the pollutant SO2, whereas methane (CH4) can be detected by immobilized Methalomonas. A particular strain of Pseudomonas is used to monitor carbon dioxide levels.

b) Immunoassay biosensors- Immunoelectrodes as biosensors are used to detect low concentrations of pollutants. Pesticide specific antibodies can detect the presence of low concentrations of triazines, malathion and carbamates, by using immunoassay methods.

c) BOD biosensor- Biological oxygen demand (BOD) is widely used as a test to detect the levels of organic pollution. This requires five days of incubation but a BOD biosensor using the yeast Trichosporon cutaneum with oxygen probe takes only 15 minutes to detect organic pollution.

d) Miscellaneous biosensors- A graphite electrode with Cynobacterium and Synechococcus has been developed to measure the degree of electron transport inhibition during the photosynthesis due to certain pollutants e.g. herbicides. To detect phenol, phenol oxidase enzyme obtained from potatoes and mushrooms is used as a biosensor. Biosensors for the detection of polychlorinated biphenyls (PCBs) and chlorinated hydrocarbons and certain other organic compounds have also been developed. Biosensors employing acetylcholine esterase which can be obtained from bovine RBC can be used for the detection of organophosphorus compounds in water.



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