In the present environment urbanization and highly evolved have led to various significant advancements. However this has given rise to momentous increase in the concentration of toxic pollutants or xenobiotics in the environment. Some of the most dangerous among man made substances are Chlorophenols, Nitrophenols, Benzene, Ethyl Benzene, Toluene, Xyrene, polychlorinated biphenyls and organic solvents. The main sources of these compounds are coal gasification, refinery, petrochemical plants and industries involved in the synthesis of chemicals, pesticides and herbicides. These toxic substances are carcinogenic and mutagenic and their rate of degradation is very slow. They are thus retained in the environment for a long period of time. Thus the removal of these intractable substances is an essential quest in the sustainable management of pollution. In order to make the treatment of organic pollutants more ecofriendly, techniques involving biological methods were introduced. Thus a safe and cost effective technology called bio-remediation was adopted which to a large extent helps in solving the problem of contamination.
Bioremediation: Bioremediation refers to a process which promotes degradation of organic wastes and removal of contaminates from the environment using microorganisms and their enzymes. Majorly bioremediation can be classified into types, namely, in-situ bioremediation which involves bio-augmentation, bio-stimulation and bio-venting and ex-situ bioremediation involving land farming, bio-piles and bioreactors. Many species of microoganisms are able to break down and degrade the contaminants, thus bioremediation techniques empoly microorganisms. These microorganisms have the ability to decompose or transform hazardous substances into less toxic metabolites or they degrade the substances into less toxic end products. This process is called as co-metabolism. In this process transformation contaminants yields little or no benefit to the cell, thus this process is nonbeneficial biotransformation. In the late 1970s and early 1980s, the development of genetic enegineering techniques and intensive study of metabolic potential of microorganisms led to the design of genetically modified organisms. The advancements in the studies has enabled in better understanding of biochemical reactions, many degradative pathways, enzymes and respective genes dealing with degradation of organic substances. This knowledge helps in the generation of engineered microorganisms having new metabolic pathways.
Genetically engineered or modified microorganism: Genetically engineered or modified microorganism is the one whose genetic material has been altered using genetic engineering techniques which are carried out by natural exchange of genetic material between the microorganisms. The techniques which are employed are collectively called as recombinant DNA technology. These genetically engineered microorganisms show enhanced degrading capabilities of wide range of organic and chemical contaminants present in the environment. Molecular biology has helped in creating innovative approaches to reduce the level of toxic organic compounds in the environment by maintaining the ecological status. Gradual studies helped in identification of genes responsible of the degradation specific environmental pollutants. For the degradation of different compounds specific set of plasmids are required. Few categories of plasmids are as follows: OCT plasmid degrades octane, hexane, decane; XYL plasmids degrade xylene and toluenes; CAM plasmid decomposes camphor; while NAH plasmid is found to degrade naphthalene.
In 1981, in USA the first two genetically modified strains of Pseudomonas aeruginosa Pseudomonas putida were patented. They were found to contain genes for degradation of naphthalene, salicylate and camphor. Later on in 1970, genetically engineered microoganisms called super bug was constructed. It had the property of degrading oil by the transfer of plasmids. It could degrade a number of toxic organic chemicals like octane, hexane, xylene, toluence, naphthalene and camphor. Proper idenfification and subsequent manipulation of specific genetic sequences helped in the development of genetically modified microorganisms. These genetically modified microorganisms have the ability to degrade wide range of xenobiotics and they have the potential for bioremediation in the environment. The genetically engineered microorganisms are designed on the basis of information of interaction between the microorganisms and the xenobiotics, the gentic basis of interaction, biochemical mechanisms, operon structure and ecological application. Genetic engineering of endophytic and rhizospheric bacteria for use in plant associated degradation of toxic compounds in soil is considered as one of the most promising new technologies for remediation of contaminated environmental sites. There are three criteria that have been recommended to select a suitable strain for gene recombination to generate the new organism having improved characteristics which are as follows:
1. The strain should be stable after cloning and the target gene should have a high expression.
2. The strain should be tolerant or insensitive to the contaminant and
3. Some microbial strains can survive only specific plant rhizosphere.
Approaches for the construction of genetically engineered microorganism: There are several approaches which are followed for the construction of genetically engineered microorganism for bioremediation application. The first approach involves the proper identification of organisms suitable for the modification with specific relevant genes. As example to explain is, as many microorganisms are well adapted to survive in the soil environment and these microbes may not be able to survive in aquatic environment. Thus genes from aquatic microbes can be used to develop genetically engineered microorganisms by inserting the gene in specific microorganism for bioremediation of aquatic sources. Adoption of such technique reduces the need for supplementation of nutrients into the inoculated environment thus reducing the cost incurred and maintenance required. Second approach involves suitable pathway construction, extension and regulation. The aim of developing a genetically engineered microorganism is to improve the existing catabolic pathways to degrade those compounds which are not degraded by using wild strain. When multiple genes of desired characteristics are introduced in a single microorganism, the genetically modified microorganisms will posses degradation capabilities of different microbial communities due to alteration of gene sequences. Thus it improves the efficiency of catabolic pathways. The third approach involves the modification of enzymes specificity and afinity. During the process of modification of micro-organisms, enzymes that are produced by transcription and translation of specific genes mediate the metabolic pathway. The genetically modified organisms are produced by gene cluster. These gene clusters alter the enzyme activity and substrate specificities, thus improving the transforming and degrading capabilities of the microorganisms. Fourth approach involves proper monitoring and control and bioaffinity, applying sensor applications for chemical sensing toxicity, reduction and end point analysis. In this approach certain gene are introduced in the microorganism which temselves examine the bioremediation and degrading processes. For example, bioluminiscence can be easily detected and this does not require expensive devices or addition of chemical or reagents. Thus genes of bioluminescent organisms can be introduced in desired micro organism. This also helps to understand the spread of microorganisms in polluted area and also indicates the end point of bioremediation process. Construction of strains with broad spectrum of catabolic potential with heavy metal resistant traits make them ideal for bioremediation of polluted environment in both aquatic and terrestrial ecosystems.
The development of genetically engineered microorganism is done with the aim of overcoming the drawbacks of using wild species of microorganisms. Before selecting the strains of microorganisms for bioremediation, all the Xenobiotic and organic waste present in the multicontaminated site should be studied. If the genetically engineered microorganisms are to be used in anaerobic environmental conditions, then these microorganisms are developed by inserting genes for oxygenases during modification by recombinant DNA technology. The genetically engineered microrganisms offer properties of many microorganisms as insertion of many microorganisms as insertion of genes is carried out in a single microorganism. Thus, these microorganisms can successfully be used in bioremediation techniques. An optimal clean-up agent with respect to the genetically modified microorganisms is those which display maximum degrading or catalytic knack with minimal cell mass. It is of utmost importance to ensure that the use of genetically modified microorganisms will not pose as a risk to the environment and human health. Usually microorganisms or Genetically Modified microorganisms (GMM) use the organic waste as the source of carbon, nitrogen and energy. However, they may scatter in an uncontrolled manner and may cause undesirable effects in the environment. Scientists have been developing a novel strategy to construct Suicidal Genetically Engineered Microorganisms (SGEMs) to minimize the hazards that may be caused and to achieve efficient and safe decontamination of the polluted sites.
Conclusion: This technology is very beneficial as it eliminates the microorganisms after degradation of xenobiotics by their autolysis. Thus it reduces the risk to environment and human beings. Nowadays, as the problem of toxic waste accumulation is every increasing, a relatively safe and cost effective technique is necessary to tackle this problem and application of genetically engineered microorganism in bioremediation of toxic waste has proved to meet this need as it follows eco-friendly and human-friendly approaches.