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  Section: General Biotechnology / Biotechnology & Environment
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Environmental Biotechnology

Hazards of Environmental Engineering
The genetically modified microorganisms which are purposefully released into the environment for bioremediation, plant growth stimulation or biocontrol of pathogens and pests of plants and animals either do well as hoped, influence the other harmful microorganisms or themselves changed are still in debate. However, some of performance of GMMs have been discussed in this context There are several types of risks associated with release of GMMs into the environment (Table 21.3).

Table 21.3: Possible types of risks associated with the release of GMMs.




A. Risk for humans, animals, plants

Transfer of drug resistance gene into clinical and/or symbiotic strains,


New forms of known diseases

GMMs useful for food production

Toxins and/or unwanted physiologically active substances in food

B. Risk for environment

GMMs will overgrew the indigenous strain

Disease of biodiversity

Disturbance of ecological balance

Activation of earlier unknown pathogen

Massive transfer of foreign genes into indigenous strains

Formation of new pathogen

C. Social risk

Beneficial usage of the GMMs in industrial countries

Increasing of economical and social differences between industrial and developing countries


GMMs release for military and/ or improper purpose

Industrial countries will use territo­ries of developing countries for field tests of GMMs release

D. Ethical risk

Commercial secrecy concerning information about release of GMMs

Violation of consumers right

Source: Velkov (1996).

Survival of Released Genetically Modified Microorganisms (GMMs) in the Environment
The GMMs after release into the environment are much influenced by multiple factors. Before release GMMs live in a very ideal conditions, but after release their survival is affected by both abiotic and biotic factors. Due to presence of poor quality of available nutrients their growth and multiplication declined. Substrates accessible to microorganisms in open nature are so limiting that within one year a typical soil microbe could go through 1-36 generations of growth. Therefore, under nutrient starvation conditions, non-differentiating microorganisms enter in a dormant viable state. During this course of time certain changes in redox potential, energy status and composition of proteins occur. It has been found that starvation induced proteins (unfoldases and chaperones), that protect non-growing cells of E. coli are produced (Loren and Hengge-Aronis, 1994).

After some days of starvation, microorganisms do not require energy to remain viable but enter into a viable but not culturable (VNC) state. During this period they can remain viable for years, and retain their plasmid. According to physiological survival strategy of non-differentiating bacteria, there is a very high probability of entering of released GMMs into VNC state.





In situ bioremediation



Intrinsic bioremediation



Engineered in situ bioremediation


Ex situ bioremediation



Solid phase system (composting, composting process)



Slurry phase system (aerated laggons, low shear airlift reactor)



Factors affecting slurry phase bioremediation


Bioremediation of hydrocarbon



Use of mixture of bacteria



Use of genetically engineered bacterial strains 


Bioremediation of Industrial wastes



Bioremediation of dyes



Bioremediation of heavy metals



Bioremediation of coal waste through VAM fungi


Bioremediation of xenobiotics



Microbial degradation of xenobiotics



Gene manipulation of pesticide-degrading microorganisms

Utilization of sewage, and agro-wastes


Production of single cell protein


Biogas from sewage


Mushroom production on agro-wastes



Microbial leaching (bioleaching)   


Microorganisms used in leaching


Chemistry of leaching



Direct leaching



Indirect leaching


Leaching process (slope leaching heap leaching in situ leaching)


Examples of bioleaching



Copper leaching



Uranium leaching



Gold and silver leaching



Silica leaching

Hazards of environmental engineering


Survival of released GMMs in the environment



Adaptive mutagenesis in GMMs



Gene transfer from GMMs into other microorganisms



Gene transfer via conjugative transposons



Effect of environmental factors on gene transfer


Ecological impact of GMMs released into the environment



Growth inhibition of natural strains



Growth stimulation of indigenous strains



Replacement of natural strains


Monitoring of GEMs in the environment



Risk assessment of the GEMs released into the environment

In addition to physiological strategy of survival, the GMMs have genetical strategy of survival as discussed below:

Adaptive Mutagenesis in GMMs
The GMMs released into the environment do not grow even in the presence of potential substrates. It seems that the non-growing cells 'adapt' themselves to the potential substrates present in the environment. It may be assumed that in populations of microorganisms growing and persisting in open environment the random mutagenesis is increasing their biodiversity, and adaptive mutagenesis 'adapt' the non-growing cells to the substrates potentially present in the environment. However, if foreign genes from GMMs spread into the indigenous microbes it may result hazardous effects.

The GMMs participate Gene Transfer
There are four ways of gene transfer in microbial ecosystem viz., transformation by free DNAs, transduction by bacteriophage DNAs, conjugation by plasmids, and conjugal transfer by conjugative transposons (for detailed description see A Text Book of Mirobiology by Dubey and Maheshwari, 1999). Conjugal gene transfer is of the most considerable ecological importance. In an experiment the GMMs and natural soil strains were released in sterile soil. Conjugal gene transfer from GMMs to soil strains occurred with a frequency of about 3x10-6-3xl0-5 of transconjugants per donor. The addition of glucose and/or tryptone enhanced up to 2x10-4 (Fray and Day, 1990). However, in non-sterile soil natural microorganisms inhibit conjugative gene transfer. On the other hand in the rhizosphere rich in nutrients, effective gene transfer among Agrobacterium, Pseudomonas and Rhizobium has been observed (Fray and Day, 1990).

There are abundance of plasmids in aquatic microbial systems. In some lake waters up to 46 per cent of heterotrophs contain plasmids, but in river water 10-15 per cent. Majority of these plasmids are conjugative. The frequency of plasmid transfer in aquatic systems varies from 5xl08 to 2.5x10-3 conjugants per donor. Moreover, very effective plasmid transfer was obtained between GMMs and strains of epilithon (aquatic microbial community associated with stones), 10-2 transconjugants per recipient (Fray and Day, 1990).

Gene Transfer via Conjugative Transposons
The conjugative transposons are self transmissible elements that normally are integrated into a chromosome or plasmid but excise themselves and transfer by conjugation to recipients. The conjugative transposons are capable of transferring themselves as well as driving the tramsmission of other elements. For example, conjugative transposons of Bacteroides can mobilize co-resident plasmids either in cis or trans position (Salyers et al., 1995).

The indigenous strains have an efficient capability to transfer their plasmids into GMMs released in soil or aquatic ecosystems.

Effect of Environmental Factors on Gene Transfer
Soil and water polluted with heavy metals and organic xenobiotics are characterized by the presence of many strains with plasmids carrying genes encoding resistance to heavy metals and biodegradation. In such polluted ecosystem selective pressure could be stimulative for conjugative transfer between GMMs and natural strains. However, the risk of spread of foreign genes by conjugation could be reduced if GMMs do not contain any conjugative plasmids (Velkov, 1996).

Ecological Impact of GMMs Released into the Environment
The GMMs released into the environment disturb the ecological balance by inhibiting/ promoting growth of indigenous microflora or replacing the natural strains as discussed below:

Growth Inhibition of Natural Strains
A genetically modified strain of Klebsiella planticola constructed to produce ethanol from organic wastes was found (a) to destroy mycorrhizal fungi, (b) to reduce plant growth, and (c) to increase the population of parasitic nematodes of plants (Stewart-Tull and Sussman, 1992). A genetically modified strain of P. putida pR103 degraded in soil microcosm the herbicide 2,4-D (2,4-dichlorophenoxyacetic acid) and resulted in accumulation of 2,4-DCP (2,4-dichlorophenol) which in turn is toxic metabolite. The 2,4-DCP was more toxic than 2,4-D as the former reduced the rate of CO2 evolution of soil microflora as compared to 2,4-D (Short et al., 1991).

Growth Stimulation of Indigenous Strains
The GMMs strain of Erwinia carotovora affected indigenous bacterial community. The total bacterial density significantly increased. This increase is attributed to an inoculum nutrient effect. The inoculated cells of E. carotovora died and became a source of nutrients for indigenous soil microorganisms (Velkov, 1996). More interesting finding is that the genetically modified P. cepacia released in soil microcosm containing the pollutant increased the taxonomic diversity of soil microorganisms. This is because the intermediate metabolites of genetically modified P. cepacia produced after biodegradation of pollutant which stimulated the growth of static cells. This has resulted in increased taxonomic diversity. Stimulation of plasmid transfer and recombination might cause an increase in genetic diversity.

Surprisingly, Prithviraj and Singh (1997) reported that a strain of Bacillus subtilis used as biocontrol agent, inspite of decreasing the pathogens' inoculum, induced sexual reproduction in Sclerotium rolfsii which might help in survival of the pathogen. Similarly, Trichoderma viride has been found to induce oospore formation in Phytophthora infestans, the causal agent of late blight of potato (Brasier, 1971).

Replacement of Natural Strains
Bolton et al (1992) reported that a strain of Pseudomonas PCI released in a wheat root system replaced efficiently the indigenous pseudomonads in their ecological niche. A genetically modified strain of Agraobacterium tumifaciens, with the deleted genes of tumor formation replaced the initial wild strain and protected plants from tumor formation (Bolton et al., 1992). This ability of GMMs is useful in agricultural biotechnology for protection of plants against pathogens.

Monitoring of GEMs in the Environment
In a microbial community, diverse group of microorganisms are found. It is now possible to monitor individual microbe in a community. Sensitive and selectable trackling tools e.g. ice, lacZY, xylE, lux systems are used in monitoring studies. Nucleic acid hybridization is also useful method where rRNA specific probes can identify the specific microbial strains of interest. Bioluminiscent reporter gene has potential to identify the specific microbe, for example, enzyme luciferase acts on substrate luciferin. In addition, genes capable of giving resistance against certain antibiotics have also been used in environmental monitoring as these detoxify antibiotics such as gentamycin, ampicillin, tetracyclin, kanamycin, steptomycin, etc.

Risk Assessment of the GMMs Released into the Environment
When GMMs are released in the environment, they get dispersed and it becomes difficult to eliminate them. Relatively few small scale field tests have been conducted with genetically modified bacteria (10 strains) and genetically modified viruses (5 strains). Most of the European countries, USA and Japan have Environmental Regulations that ban the deliberate release of GMMs. But the USA has permitted deliberate release of some of the GMMs. No country has defined what steps will be required to obtain permission for commercial use of GMMs for in situ bioremediation.

In July 1995, European parliament passed a resolution saying that "a legally binding international biosafety protocol is necessary". It must be negotiated by States partly on the United Nations' convention on biological diversity. Therefore, resolution of deliberate release of GMMs are being carried out in many developing countries for safe use of GMMs. Otherwise the whole biosphere will be at risk. India has formulated Biosafety Rules and Regulations for GMMs as discussed in Biotechnology : Scope and Importance. For stable development of Indian biotechnology, safety of environmental engineering is very actual.

There are two principal uncertainties of the assessment of risks associated with possible ecological consequences of release of GMMs: (i) total number of hazardous strains present in the environment as only 5 per cent of total species are known and rest of 95 per cent unknown, and (ii) absence of definite criteria for evaluation of similarity between microorganisms. However, estimates of similarity made by different experts may vary drastically. Moreover, there are certain questions unasked which may possibly help in assessment of risk arising from release of GMMs in the environment (Velkov, 1996).


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